Prediction of human hepatic clearance from in vivo animal experiments and in vitro metabolic studies with liver microsomes from animals and humans. (25/379)

We investigated the quantitative prediction of human hepatic metabolic clearance from in vitro experiments focusing on cytochrome P450 metabolism with eight model compounds, FK1052, FK480, zolpidem, omeprazole, nicardipine, nilvadipine, diazepam, and diltiazem. For the compounds, in vivo human hepatic extraction ratios ranged widely from 0.03 to 0.87. In vitro and in vivo hepatic intrinsic clearance (CL(int)) values for each compound were measured and calculated in rats and/or dogs and humans. CL(int,in vitro) was determined from a substrate disappearance rate at 1 microM in hepatic microsomes, which was a useful method. CL(int,in vivo) was calculated from in vivo pharmacokinetic data using three frequent mathematical models (the well stirred, parallel-tube, and dispersion models). The human scaling factor values (CL(int,in vivo)/CL(int,in vitro)) showed marked difference among the model compounds (0.3-26.6-fold). On the other hand, most of the animal scaling factors were within 2-fold of the values in humans, suggesting that scaling factor values were similar in the different animal species. When human CL(int,in vitro) values were compared with the actual CL(int,in vivo), correlation was not necessarily good. By contrast, using human CL(int,in vitro) corrected with the rat and/or dog scaling factors yielded better predictions of CL(int,in vivo) that were mostly within 2-fold of the actual values. Furthermore, successful predictions of human CL(oral) and hepatic extraction ratio (E(H)) were obtained by use of the human CL(int,in vitro) corrected with animal scaling factors. The new variant method is a simple one, incorporating additional information from animal studies and providing a more reliable prediction of human hepatic clearance.  (+info)

Specific ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in human colorectal cancer cells. (26/379)

The peripheral benzodiazepine receptor (PBR) has been implicated in growth control of various tumour models. Although colorectal cancers were found to overexpress PBR, the functional role of PBR in colorectal cancer growth has not been addressed to date. Using primary cell cultures of human colorectal cancers and the human colorectal carcinoma cell lines HT29, LS174T, and Colo320 DM we studied the involvement of PBR in the growth control and apoptosis of colorectal cancers. Both mRNA and protein expression of PBR were detected by RT-PCR and flow cytometry. Using confocal laser scanning microscopy and immunohistochemistry the PBR was localized in the mitochondria. The specific PBR ligands FGIN-1-27, PK 11195, or Ro5-4864 inhibited cell proliferation dose-dependently. FGIN-1-27 decreased the mitochondrial membrane potential, which indicates an early event in apoptosis. Furthermore, FGIN-1-27, PK 11195 or Ro5-4864 increased caspase-3 activity. In addition to their apoptosis-inducing effects, PBR ligands induced cell cycle arrest in the G(1)/G(0)-phase. Thus, our data demonstrate a functional involvement of PBR in colorectal cancer growth and qualify the PBR as a possible target for innovative therapeutic approaches in colorectal cancer.  (+info)

Role of the histidine residue at position 105 in the human alpha 5 containing GABA(A) receptor on the affinity and efficacy of benzodiazepine site ligands. (27/379)

1. A histidine residue in the N-terminal extracellular region of alpha 1,2,3,5 subunits of the human GABA(A) receptor, which is replaced by an arginine in alpha 4 and alpha 6 subunits, is a major determinant for high affinity binding of classical benzodiazepine (BZ)-site ligands. The effect of mutating this histidine at position 105 in the alpha 5 subunit to an arginine (alpha 5H105R) on BZ-site pharmacology has been investigated using radioligand binding on HEK293 and L(tk-) cells and two electrode voltage clamp recording on Xenopus oocytes in which GABA(A) receptors of subtypes alpha 5, alpha 5H105R, alpha 4 and alpha 6 were co-expressed with beta 3 gamma 2s. 2. The classical BZs, diazepam and flunitrazepam (full agonists on the alpha 5 receptor) showed negligible affinity and therefore negligible efficacy on alpha 5H105R receptors. The beta-carbolines DMCM and beta CCE (inverse agonists on the alpha 5 receptor) retained some affinity but did not exhibit inverse agonist efficacy at alpha 5H105R receptors. Therefore, the alpha 5H105R mutation confers an alpha 4/alpha 6-like pharmacology to the classical BZs and beta-carbolines. 3. Ro15-4513, flumazenil, bretazenil and FG8094, which share a common imidazobenzodiazepine core structure, retained high affinity and were higher efficacy agonists on alpha 5H105R receptors than would be predicted from an alpha 4/alpha 6 pharmacological profile. This effect was antagonized by DMCM, which competes for the BZ-site and therefore is likely to be mediated via the BZ-site. 4. These data indicate that the conserved histidine residue in the alpha subunit is not only a key determinant in the affinity of BZ-site ligands on alpha 5 containing GABA(A) receptors, but also influences ligand efficacy.  (+info)

Peripheral benzodiazepine receptor ligands reverse apoptosis resistance of cancer cells in vitro and in vivo. (28/379)

The mitochondrial peripheral benzodiazepine receptor (mPBR) is involved in a functional structure designated as the permeability transition pore, which controls apoptosis. Binding of Fas/APO-1/CD95 triggers a prototypic apoptosis-inducing pathway. Using four different human tumor cell lines (T-cell Jurkat, neuroblastoma SHEP, osteosarcoma 143N2, and glioblastoma SNB79 cell lines), all of which express CD95 and mPBR, we investigated the potential role of mPBR ligands in CD95-induced apoptosis. We show that, in vitro, the three mPBR ligands tested (RO5-4864, PK11195, and diazepam) enhanced apoptosis induced by anti-CD95 antibody in Jurkat cells, as demonstrated by mitochondrial transmembrane potential drop and DNA fragmentation. In contrast, RO5-4864, but not PK11195 or diazepam, enhanced anti-CD95 apoptosis in all other cell lines. These effects were obtained in Bcl-2-overexpressing SHEP cell lines, but not in Bcl-X(L) SHEP cell lines. Enhancement of anti-CD95 antibody-induced apoptosis by RO5-4864 was characterized by an increased mitochondrial release of cytochrome c and Smac/DIABLO proteins and an enhanced activation of caspases 9 and 3, suggesting a mitochondrion-dependent mechanism. Preincubation of cells with the different mPBR ligands or anti-CD95 did not affect the levels of expression of either mPBR or CD95. In vivo, we found that the RO5-4864 mPBR ligand significantly increased the growth inhibition induced by two chemotherapeutic agents, etoposide and ifosfamide, using two human small cell lung cancers xenografted into nude mice. Peripheral benzodiazepine receptor ligands may therefore act as chemosensitizing agents for the treatment of human neoplasms.  (+info)

Uric acid is a genuine metabolite of Penicillium cyclopium and stimulates the expression of alkaloid biosynthesis in this fungus. (29/379)

On searching for endogenous, low-molecular-weight effectors of benzodiazepine alkaloid biosynthesis in Penicillium cyclopium uric acid was isolated from ethanolic or autoclaved mycelial extracts of this fungus. The isolation was based on a three-step high-pressure liquid chromatography procedure guided by a microplate bioassay, and uric acid was identified by mass spectrometry and the uricase reaction. Conidiospore suspensions that were treated with this compound during the early phase of outgrowth developed emerged cultures with an enhanced rate of alkaloid production. Uric acid treatment did not increase the in vitro measurable activity of the rate-limiting biosynthetic enzyme, cyclopeptine synthetase. However, these cultures displayed a reduced rate of uptake of the alkaloid precursor L-phenylalanine into the vacuoles of the hyphal cells as assayed in situ. It is suggested that the depressed capacity of vacuolar uptake caused by the contact of outgrowing spores with uric acid liberated from hyphal cells results in an enhanced availability of the precursor L-phenylalanine in the cytoplasm and thus accounts at least in part for the increase in alkaloid production.  (+info)

Antiarrhythmic efficacy of combined I(Ks) and beta-adrenergic receptor blockade. (30/379)

Suppression of malignant ventricular arrhythmias by selective blockade of the cardiac slowly activating delayed rectifier current (I(Ks)) has been demonstrated with the benzodiazepine L-768673 [(R)-2-(2,4-trifluoromethyl-phenyl)-N-[2-oxo-5-phenyl-1-(2,2,2-trifluoro-ethyl)-2 ,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl]acetamide] in canine models of recent and healed myocardial infarction. The present study extends the initial antiarrhythmic assessment of I(Ks) blockade by demonstrating prevention of ischemic malignant arrhythmias in dogs with recent (8.0 +/- 0.4 days) anterior myocardial infarction with the coadministration of a subeffective dose of L-768673 and a subeffective, minimally beta-adrenergic blocking dose of timolol. Administered individually, neither 0.3 microg/kg i.v. L-768673 nor 1.0 microg/kg i.v. timolol prevented the induction of ventricular tachyarrhythmia (VT) by programmed ventricular stimulation (PVS) or the development of malignant ventricular arrhythmia in response to acute coronary artery thrombosis. In contrast, coadministration of 0.3 microg/kg i.v. L-768673 + 1.0 microg/kg i.v. timolol suppressed the induction of VT by PVS (8/10, 80% rendered noninducible versus 1/10, 10% noninducible in vehicle group; p < 0.01) and prevented the development of acute ischemic lethal arrhythmias (3/10, 30% incidence versus 8/10, 80% incidence in vehicle group; p < 0.05). Concomitant administration of low-dose L-768673 + timolol produced modest increases in QTc and paced QT intervals (4.5 +/- 1.2 and 5.5 +/- 1.4%; both p < 0.01), increases in noninfarct zone relative and effective refractory periods (7.0 +/- 1.7 and 12.3 +/- 3.0%; both p < 0.01), and lesser increases in infarct zone relative and effective refractory periods (5.3 +/- 1.6 and 5.8 +/- 1.4%; both p < 0.01). These findings suggest that concomitant low-dose I(Ks) and beta-adrenergic blockade may constitute a potential pharmacologic strategy for prevention of malignant ischemic ventricular arrhythmias.  (+info)

L-365,260 reversed effect of sincalide against morphine on electrical and mechanical activities of rat duodenum in vitro. (31/379)

AIM: To study the antagonism of sincalide (CCK-8) to the effect of morphine and its mechanism. METHODS: The electrical and mechanical activities of rat duodenum in vitro were recorded simultaneously. RESULTS: Acetylcholine (ACh, 300 nmol/L) increased the spike potential amplitude (SPA) and the number (SPN) of rat duodenum in vitro, followed by an increase of duodenal contraction amplitudes (CA). The SPA, SPN, and CA of duodenum in vitro were not obviously affected by injection of morphine (330 nmol/L), but it could selectively inhibit the potentiation of ACh. After administration of CCK-8 (0.7 nmol/L), the SPA, SPN, and CA of duodenal segment did not exhibit obvious changes. But CCK-8 could selectively antagonize the effects of morphine, ie, the SPA and SPN were increased again, followed by an increase of CA. CCK-B receptor antagonist L-365,260 (30 nmol/L) reversed the antagonism of CCK-8 to the effect of morphine. CONCLUSION: CCK-8 could selectively antagonize the effect of morphine which inhibited the potentiation of ACh on duodenal activities in vitro. The antagonistic effect of CCK-8 on morphine was mainly mediated by CCK-B receptor.  (+info)

The integrin alphavbeta3 is a receptor for the latency-associated peptides of transforming growth factors beta1 and beta3. (32/379)

The integrins alpha(v)beta(1), alpha(v)beta(5), alpha(v)beta(6) and alpha(v)beta(8) have all recently been shown to interact with the RGD motif of the latency-associated peptide (LAPbeta(1)) of transforming growth factor beta(1) (TGFbeta(1)), with binding to alpha(v)beta(6) and alpha(v)beta(8) leading to TGFbeta(1) activation. Previously it has been suggested that the remaining alpha(v) integrin, alpha(v)beta(3,) does not interact with LAPbeta(1). However, here we show clearly that alpha(v)beta(3) does indeed interact with the LAPbeta(1) RGD motif. This interaction is similar to other alpha(v)beta(3) ligands in terms of the cations required for adhesion, the concentrations of LAPbeta(1) required for binding and the ability of a small-molecule inhibitor of alpha(v)beta(3), SB223245, to block the interaction. Using glutathione S-transferase fusion proteins we have mapped a minimal integrin-binding loop in LAPbeta(1) and then used this approach to probe the integrin-binding properties of the equivalent loops in LAPbeta(2) and LAPbeta(3). We show that the RGD motif of LAPbeta(3) also interacts with alpha(v)beta(3), in addition to alpha(v)beta(6), alpha(v)beta(1) and alpha(v)beta(5), whereas the corresponding loop in LAPbeta(2) does not interact with these integrins. These observations therefore correct a previously reported inaccuracy in the literature. Furthermore, they are important as they link alpha(v)beta(3) and TGFbeta, which may have implications in cancer and a number of inflammatory and fibrotic diseases where expression of both proteins has been documented.  (+info)