The impact of co-administration of ketoconazole and rifampicin on the pharmacokinetics of apremilast in healthy volunteers.

AIMS: Two 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. METHODS: These 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. RESULTS: Ketoconazole 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). CONCLUSION: Ketoconazole 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.

Pharmacological characterization of a novel α2C-adrenoceptor agonist N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1, 4-benzoxazin-6-yl]-N-ethyl-N'-methylurea (compound A).

We define the pharmacological and pharmacokinetic profiles of a novel α(2C)-adrenoceptor agonist, compound A [N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1,4-benzoxazin-6-yl]-N-ethyl-N'-methylurea]. This compound has high affinity (K(i)) for the human α(2C)-adrenoceptor (K(i) = 12 nM), and 190- to 260-fold selectivity over the α(2A)- and α(2B)-adrenoceptor subtypes. In cell-based functional assays, compound A produced good agonist (EC(50) = 166 nM) and efficacy (E(max) = 64%) responses at the α(2C)-adrenoceptor, much lower potency and efficacy at the α(2A)-adrenoceptor (EC(50) = 1525 nM; E(max) = 8%) and α(2B)-adrenoceptor (EC(50) = 5814 nM; E(max) = 21%) subtypes, and low or no affinity and functional activity at the α(1A)-, α(1B)-, and α(1D)-adrenoceptor subtypes. In the human saphenous vein postjunctional α(2C)-adrenoceptor bioassay, compound A functions as a potent agonist (pD(2) = 6.3). In a real-time contraction bioassay of pig nasal mucosa, compound A preferentially constricted the veins (EC(50) = 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 α(2C)-adrenoceptor agonist with desirable in vitro and in vivo pharmacological properties suitable for the treatment of nasal congestion.

Effect of vicriviroc on the QT/corrected QT interval and central nervous system in healthy subjects.

Vicriviroc is a CCR5 antagonist in clinical development for the treatment of HIV-1. Two phase I studies were conducted to assess the safety of vicriviroc. One study characterized the drug's potential to prolong the QT/corrected QT (QTc) interval and to induce arrhythmia. In this partially blind, parallel-group study, 200 healthy subjects aged 18 to 50 years were randomized in equal groups to the following regimens: (i) placebo for 9 days and a single dose of moxifloxacin at 400 mg on day 10, (ii) placebo, (iii) vicriviroc-ritonavir (30 and 100 mg), (iv) vicriviroc-ritonavir (150 and 100 mg), and (v) ritonavir (100 mg). The second study characterized the effects of a range of vicriviroc doses on the central nervous system (CNS). In this third-party-blind, parallel-group study, 30 healthy subjects aged 18 to 48 years were randomized to receive a single dose of either vicriviroc at 200, 250, or 300 mg or placebo, followed by multiple (seven) once-daily doses of either vicriviroc at 150, 200, or 250 mg or placebo, respectively. In the first study, vicriviroc produced no clinically meaningful effect on the QT/QTc interval when administered at a supratherapeutic or therapeutic dose concurrently with ritonavir. In the second study, vicriviroc produced no observable seizure activity, nor was it held to be associated with any clinically relevant changes in brain waveforms in the final consensus of reviewers. These findings showed that vicriviroc produced no clinically relevant QTc prolongation cardiac or epileptogenic effects in healthy individuals at exposures as high as five times those expected for HIV-infected patients receiving therapeutic doses of vicriviroc in a ritonavir-boosted protease inhibitor-containing regimen.

Identification of full, partial and inverse CC chemokine receptor 3 agonists using [35S]GTPgammaS binding.

Study of the CC chemokine receptor 3 (CCR3) has been limited to using radiolabeled agonist chemokines. A small molecule CCR3 antagonist, 2-[(6-amino-2-benzothiazolyl)thio]-N-[1-[(3,4-dichlorylphenyl)methyl]-4-piperidinyl]acetamide, Banyu (I), was tritiated and used for pharmacological studies. Banyu (I) has a K(d) of 5.0+/-0.4 and 4.3+/-1.8 nM on human CCR3 transfectants and eosinophils, and noncompetitively inhibits [125I]eotaxin binding and eotaxin-induced [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding. The proportion of [125I]eotaxin: [3H]Banyu (I) binding sites in eosinophils or transfectants was 35% or 13%, although both binding sites were overexpressed in transfectants. CCR3 spontaneously couples to G-proteins in CCR3 transfectants, demonstrated by changes in basal and eotaxin-induced [35S]GTPgammaS binding under reduced NaCl and GDP concentrations. Consequently, Banyu (I) was identified as an inverse agonist. In contrast, CCL18 and I-TAC (interferon-inducible T cell alpha-chemoattractant) were neutral antagonists, inhibiting eotaxin-induced [35S]GTPgammaS binding, with minimal effect on basal coupling of CCR3 to G proteins. Eotaxin, eotaxin-2 and monocyte chemoattractant protein (MCP)-4 are full agonists inducing [35S]GTPgammaS binding; eotaxin-3, MCP-3, RANTES (regulated on activation normal T cell expressed and secreted), vMIP-I (Kaposi's sarcoma-associated herpesvirus macrophage inflammatory protein-) and vMIP-II are partial agonists, indicating that this is a sensitive method to quantitate agonist efficacy.

The synthesis of substituted bipiperidine amide compounds as CCR3 ligands: antagonists versus agonists.

Structure-activity relationship study of bipiperidine amide 1 has identified the reverse bipiperidine amide 4a as a CC chemokine-3 (CCR3) receptor antagonist. Optimization of the structure-activity relationship of compound 4a has resulted in the identification of a CCR3 antagonist 4i as well as a CCR3 agonist 13.

The synthesis of substituted bipiperidine amide compounds as CCR3 antagonists.

Bipiperidine amide 1 has been identified as a CC chemokine receptor 3 (CCR3) antagonist. Optimization of its structure-activity relationship has resulted in the identification of cis (R,R)-4-[(3,4-dichlorophenyl)methyl]-3-hydroxymethyl-1'(6-quinolinylcarbonyl)-1,4'-bipiperidine 14n, which exhibits potent receptor affinity and inhibition of both calcium flux and eosinophil chemotaxis.

Catalytic activity of human ADAM33.

ADAM33 (a disintegrin and metalloproteinase) is an asthma susceptibility gene recently identified through a genetic study of asthmatic families (van Eerdewegh et al. (2002) Nature 418, 426-430). In order to characterize the catalytic properties of ADAM33, the metalloproteinase domain of human ADAM33 was expressed in Drosophila S2 cells and purified. The N-terminal sequence of the purified metalloproteinase was exclusively (204)EARR, indicating utilization of one of three furin recognition sites. Of many synthetic peptides tested as potential substrates, four peptides derived from beta-amyloid precursor protein (APP), Kit-ligand-1 (KL-1), tumor necrosis factor-related activation-induced cytokine, and insulin B chain were cleaved by ADAM33; mutation at the catalytic site, E346A, inactivated catalytic activity. Cleavage of APP occurred at His(14)/Gln(15), not at the alpha-secretase site and was inefficient (k(cat)/K(m) (1.6 +/- 0.3) x 10(2) m(-1) s(-1)). Cleavage of a juxtamembrane KL-1 peptide occurred at a site used physiologically with a similar efficiency. Mutagenesis of KL-1 peptide substrate indicated that the P3, P2, P1, and P3' residues were critical for activity. In a transfected cell-based sheddase assay, ADAM33 functioned as a negative regulator of APP shedding and mediated some constitutive shedding of KL-1, which was not regulated by phorbol 12-myristate 13-acetate activation. ADAM33 activity was sensitive to several hydroxamate inhibitors (IK682, K(i) = 23 +/- 7 nm) and to tissue inhibitors of metalloproteinase (TIMPs). Activity was inhibited moderately by TIMP-3 and TIMP-4 and weakly inhibited by TIMP-2 but not by TIMP-1, a profile distinct from other ADAMs. The identification of ADAM33 peptide substrates, cellular activity, and a distinct inhibitor profile provide the basis for further functional studies of ADAM33.

Human ADAM33 messenger RNA expression profile and post-transcriptional regulation.

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.

Mouse ADAM33: two splice variants differ in protein maturation and localization.

We compared the tissue mRNA prevalence and protein maturation of two splice variants of mouse ADAM33, a metalloprotease implicated in airway hyperresponsiveness. These variant cDNAs, designated 914 (alpha) and 906 (beta), encode membrane-bound forms that differ primarily in 26 residues (exon 17) between the cysteine-rich and epidermal growth factor-like domains. Proteins of approximately 120 and 103 kD, detectable by anti-ADAM33 antibodies, were expressed in 914-transfected HEK293 cells. The time-dependent appearance of the approximately 100-kD form and its inhibition by a peptidyl chloromethylketone, or the calcium ionophore, A23187, indicated that this was mature ADAM33, which was processed by a furin-like convertase. One form, approximately 110 kD, was detected in 906-transfected cell lysates. Trypsin and biotinylation treatment of transfected cells demonstrated that all of the mature approximately 100-kD, a minority of the approximately 120-kD pro-form, and none of the 906-expressed 110-kD form localized to the cell surface. The mature form was resistant to endoglycosidase H(f). The approximately 110-kD form was endoglycosidase H(f)-sensitive, indicating retention proximal to the trans-Golgi, consistent with a lack of maturation. Quantitation of transcripts demonstrated that those containing exon 17 predominate, whereas those lacking exon 17 are negligible in the mouse lung, although detectable at low levels in mouse testis, heart, and brain. Thus, potential dominant-negative effects exerted by the nonprocessed 906-encoded beta splice variant are unlikely to occur in mouse lung.

Microarray profile of differentially expressed genes in a monkey model of allergic asthma.

BACKGROUND: Inhalation of Ascaris suum antigen by allergic monkeys causes an immediate bronchoconstriction and delayed allergic reaction, including a pulmonary inflammatory infiltrate. To identify genes involved in this process, the gene-expression pattern of allergic monkey lungs was profiled by microarrays. Monkeys were challenged by inhalation of A. suum antigen or given interleukin-4 (IL-4) treatment; lung tissue was collected at 4, 18 or 24 h after antigen challenge or 24 h after IL-4. Each challenged monkey lung was compared to a pool of normal, unchallenged monkey lungs. RESULTS: Of the approximately 40,000 cDNAs represented on the microarray, expression levels of 169 changed by more than 2.5-fold in at least one of the pairwise probe comparisons; these cDNAs encoded 149 genes, of which two thirds are known genes. The largest number of regulated genes was observed 4 h after challenge. Confirmation of differential expression in the original tissue was obtained for 95% of a set of these genes using real-time PCR. Cluster analysis revealed at least five groups of genes with unique expression patterns. One cluster contained genes for several chemokine mediators including eotaxin, PARC, MCP-1 and MCP-3. Genes involved in tissue remodeling and antioxidant responses were also identified as regulated by antigen and IL-4 or by antigen only. CONCLUSION: This study provides a large-scale profile of gene expression in the primate lung following allergen or IL-4 challenge. It shows that microarrays, with real-time PCR, are a powerful tool for identifying and validating differentially expressed genes in a disease model.

Human ADAM33: protein maturation and localization.

ADAM33 (a disintegrin and metalloprotease) was recently found to be a novel asthma susceptibility gene. Domain-specific antibodies were used to study its expression and processing. When the pro-domain and catalytic domain were expressed by a stable-transfected cell line, the pro-domain was removed by cleavage within a putative furin cleavage site. The catalytic domain was active in an alpha(2)-macroglobulin complex formation assay and mutation of the catalytic site glutamic acid (E346A) eliminated activity. In transient transfections using the full-length protein, a pro-form and mature form were detectable and alternate glycosylation was demonstrated at sites within the catalytic domain. ADAM33 was detected on the cell surface, with the majority of protein detected intracellularly. The E346A mutation had no significant effect on protein processing. Endogenous ADAM33 was detected in bronchus tissue, bronchial smooth muscle cells, and MRC-5 fibroblasts, consistent with a role in the pathophysiology of asthma.