ABSTRACT:Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6␤-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.

No abstract

ABSTRACT:Cultured human hepatocytes are a valuable in vitro system for evaluating new molecular entities as inducers of cytochrome P450 (P450) enzymes. The present study summarizes data obtained from 62 preparations of cultured human hepatocytes that were treated with vehicles (saline or dimethylsulfoxide, 0.1%), ␤-naphthoflavone (33 M), phenobarbital (100 or 250 M), isoniazid (100 M) and/or rifampin (20 or 50 M), and examined for the expression of P450 enzymes based on microsomal activity toward marker substrates, or in the case of CYP2C8, the level of immunoreactive protein. The results show that CYP1A2 activity was markedly induced by ␤-naphthoflavone (on average 13-fold, n ‫؍‬ 28 preparations), and weakly induced by phenobarbital (1.9-fold, n ‫؍‬ 25) and rifampin (2.3-fold, n ‫؍‬ 22); CYP2A6 activity tended to be increased with phenobarbital (n ‫؍‬ 7) and rifampin (n ‫؍‬ 3) treatments, but the effects were not statistically significant; CYP2B6 was induced by phenobarbital (6.5-fold, n ‫؍‬ 13) and rifampin (13-fold, n ‫؍‬ 14); CYP2C8 was induced by phenobarbital (4.0-fold, n ‫؍‬ 4) and rifampin (5.2-fold, n ‫؍‬ 4); CYP2C9 was induced by phenobarbital (1.8-fold, n ‫؍‬ 14) and rifampin (3.5-fold, n ‫؍‬ 10); CYP2C19 was markedly induced by rifampin (37-fold, n ‫؍‬ 10), but relatively modestly by phenobarbital (7-fold, n ‫؍‬ 9); CYP2D6 was not significantly induced by phenobarbital (n ‫؍‬ 5) or rifampin (n ‫؍‬ 5); CYP2E1 was induced by phenobarbital (1.7-fold, n ‫؍‬ 5), rifampin (2.2-fold, n ‫؍‬ 5), and isoniazid (2.3-fold, n ‫؍‬ 5); and, CYP3A4 was induced by phenobarbital (3.3-fold, n ‫؍‬ 42) and rifampin (10-fold, n ‫؍‬ 61), but not by ␤-naphthoflavone. Based on these observations, we generalize that ␤-naphthoflavone induces CYP1A2 and isoniazid induces CYP2E1, whereas rifampin and, to a lesser extent phenobarbital, tend to significantly and consistently induce enzymes of the CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A subfamilies but not the 2D subfamily. Drugs and NMEs5 are often screened for their ability to induce P450 and other drug-metabolizing enzymes with the aim of predicting or explaining drug-drug interactions and pharmacokinetic tolerance.

Nociceptin/OFQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the ORL1/NOP receptor. Nociceptin appears to regulate a host of physiological functions such as biological reactions to stress, anxiety, mood, and drug abuse, in addition to feeding behaviors. To develop tools to study the function of nociceptin and NOP receptor, our research effort sought to identify orally available NOP antagonists. Our effort led to the discovery of a novel chemical series based on the dihydrospiro(piperidine-4,7'-thieno[2,3-c]pyran) scaffold. Herein we show that dihydrospiro(piperidine-4,7'-thieno[2,3-c]pyran)-derived compounds are potent NOP antagonists with high selectivity versus classical opioid receptors (μ, δ, and κ). Moreover, these compounds exhibit sufficient bioavailability to produce a high level of NOP receptor occupancy in the brain following oral administration in rats.

ABSTRACT:In an effort to develop a novel therapeutic agent aimed at addressing the unmet need of patients with osteoarthritis pain, we set out to develop an inhibitor for autotaxin with excellent potency and physical properties to allow for the clinical investigation of autotaxin-induced nociceptive and neuropathic pain. An initial hit identification campaign led to an aminopyrimidine series with an autotaxin IC 50 of 500 nM. X-ray crystallography enabled the optimization to a lead compound that demonstrated favorable potency (IC 50 = 2 nM), PK properties, and a robust PK/PD relationship. KEYWORDS: Autotaxin, tool molecule, osteoarthritis, LPA O steoarthritis (OA) is a highly prevalent disease affecting many adults including more than one out of three individuals aged 65 or older in the United States.1 In addition to significant accompanying pain, OA frequently leads to pronounced disability resulting in the loss of work, hospitalization, and joint replacement procedures.2 Current first-line pharmacological treatment options for OA focus on reducing inflammation and the associated pain. Nonsteroidal antiinflammatory drugs (NSAIDS) and selective COX-2 inhibitors are among the most prescribed medications for OA pain but unfortunately are also frequently accompanied by gastrointestinal, renal, and CV side effects, limiting their use. 3 Recently, the role of lyosophosphatidic acid (LPA) in certain inflammatory conditions has been studied. 4 LPA exists as a number of molecular species that have variable saturated and unsaturated fatty acid chains.5 Signaling of LPA through six GPCRs (LPA Receptors 1−6) has been shown to lead to the upregulation of inflammatory cytokines and matrix metalloproteinases, which contribute to the pathogenesis of OA. 6LPA signaling has also been associated with many other pathologies, such as pulmonary fibrosis and cancer. In vivo, the enzyme autotaxin (ATX), with lyosophosopholipase D activity, is the primary source of extracellular LPA, which results from the cleavage of choline from lysophosphatidylcholine (LPC) (Figure 1). LPA is also produced through action of secreted phospholipases A2 (sPLA2) on phosphatidic acid (PA), although this is believed to be a minor route of extracellular LPA production in vivo. 8,9 Autotaxin is an extracellular, 125 kDa protein that was originally characterized in 1993 by Stracke et al. as a motility stimulating protein. 10 In 2002, Umezu-Goto and co-workers demonstrated that ATX was the same protein as a known lysophopholipase D enzyme, which catalyzed the conversion of LPC to LPA.11 Autotaxin is a multidomain protein with two Nterminal somatomedin B-like domains, a centrally located phosphodiesterase domain, and a catalytically inactive nuclease-like domain on the C-terminal region. It is expressed in four main isoforms (ATXα−δ) with largely unknown differential functionality in vivo.7 The catalytic domain of ATX comprises two zinc ions coordinated with histidine and aspartic acid residues with a threonine alcohol serving as the nucleophile. A large hydro...