Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells

The coordination of the functional activities of intestinal CYP3A4 and P-gp in limiting the absorption of xenobiotics in Caco-2 cells was investigated. Growing Caco-2 cells were exposed to increasing concentrations of doxorubicin (1-2 μM) in plastic flasks to encourage a subpopulation of cells, that displayed an intrinsically higher multidrug resistance (mdr) phenotype than the parent cells, to survive and grow. Doxorubicin-exposed (hereinafter referred to as type I cells) and nonexposed Caco-2 cells (parent cells) on collagen-coated inserts were also treated with either 0 (control) or 0.25 μM 1α,25-dihydroxyvitamin D3 to promote cellular CYP3A4 expression. Increased P-gp protein expression, as detected by Western blotting, was noted in type I cells (213±54.35%) compared to that of parent cells (100±6.05%). Furthermore, they retained significantly less [3H]vincristine sulphate (p<0.05), a P-gp substrate, after efflux (272.89±11.86 fmol/mg protein) than the parent cells (381.39±61.82 fmol/mg protein). The expression of CYP3A4 in parental cells after 1α,25-dihydroxyvitamin D3 treatment was quantified to be 76.2±7.6 pmol/mg protein and comparable with that found in human jejunal enterocytes (70.0±20.0 pmol/mg protein). Type I cells, however, expressed a very low quantity of CYP3A4 both before and after the treatment that was beyond the minimum detection limit of Western blotting. Functionally, the rates of 1-hydroxylation of midazolam by CYP3A for both cell types ranged from 257.0±20.0 to 1057.0±46.0 pmol/min/mg protein. Type I cells, although having a higher P-gp expression and activity comparatively, metabolized midazolam less extensively than the parent cells. The results suggested that there were noncoordinated functional activities of intestinal CYP3A4 and P-gp in Caco-2 cells, although they both functioned independently to minimize intestinal epithelial absorption of xenobiotics. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association.

Pulse compression applied to the detection of F.S.K. and P.S.K. signals

The matched filter detector is well known as the optimum detector for use in communication, as well as in radar systems for signals corrupted by Additive White Gaussian Noise (A.W.G.N.). Non-coherent F.S.K. and differentially coherent P.S.K. (D.P.S.K.) detection schemes, which employ a new approach in realizing the matched filter processor, are investigated. The new approach utilizes pulse compression techniques, well known in radar systems, to facilitate the implementation of the matched filter in the form of the Pulse Compressor Matched Filter (P.C.M.F.). Both detection schemes feature a mixer- P.C.M.F. Compound as their predetector processor. The Compound is utilized to convert F.S.K. modulation into pulse position modulation, and P.S.K. modulation into pulse polarity modulation. The mechanisms of both detection schemes are studied through examining the properties of the Autocorrelation function (A.C.F.) at the output of the P.C.M.F.. The effects produced by time delay, and carrier interference on the output A.C.F. are determined. Work related to the F.S.K. detection scheme is mostly confined to verifying its validity, whereas the D.P.S.K. detection scheme has not been reported before. Consequently, an experimental system was constructed, which utilized combined hardware and software, and operated under the supervision of a microprocessor system. The experimental system was used to develop error-rate models for both detection schemes under investigation. Performances of both F. S. K. and D.P. S. K. detection schemes were established in the presence of A. W. G. N. , practical imperfections, time delay, and carrier interference. The results highlight the candidacy of both detection schemes for use in the field of digital data communication and, in particular, the D.P.S.K. detection scheme, which performed very close to optimum in a background of A.W.G.N.

Methodology for development of a physiological model incorporating CYP3A and P-glycoprotein for the prediction of intestinal drug absorption

The small intestine poses a major barrier to the efficient absorption of orally administered therapeutics. Intestinal epithelial cells are an extremely important site for extrahepatic clearance, primarily due to prominent P-glycoprotein-mediated active efflux and the presence of cytochrome P450s. We describe a physiologically based pharmacokinetic model which incorporates geometric variations, pH alterations and descriptions of the abundance and distribution of cytochrome 3A and P-glycoprotein along the length of the small intestine. Simulations using preclinical in vitro data for model drugs were performed to establish the influence of P-glycoprotein efflux, cytochrome 3A metabolism and passive permeability on drug available for absorption within the enterocytes. The fraction of drug escaping the enterocyte (F(G)) for 10 cytochrome 3A substrates with a range of intrinsic metabolic clearances were simulated. Following incorporation of P-glycoprotein in vitro efflux ratios all predicted F(G) values were within 20% of observed in vivo F(G). The presence of P-glycoprotein increased the level of cytochrome 3A drug metabolism by up to 12-fold in the distal intestine. F(G) was highly sensitive to changes in intrinsic metabolic clearance but less sensitive to changes in intestinal drug permeability. The model will be valuable for quantifying aspects of intestinal drug absorption and distribution.

Intestinal absorption barriers as modelled by p-glycoprotein and cytochrome p450 a34 in caco2 cells

The passage number and origin of two populations of Caco-2 cells influence their enterocyte-like characteristics. Caco-2 cells of passage number >90 from Novartis pharmaceutical company possess higher levels of expression of alkaline phosphatase and P-glycoprotein and a greater cellular uptake of Gly-1.-Pro than those of passage number <40 from the American Type Tissue Culture collection. High P-gp expressing Caco-2 cells have been developed through stepwise selection of the cells with doxonibicin. This newly-developed cell line (hereafter referred to as Type I) possesses approximately twice as much P-gp protein than non-exposed cells, restricts the transepithelial transport of vincristine in the apical-to-basolateral direction whilst facilitating its transport in the reverse direction and accumulates less vincristine than non-exposed cells. There is no apparent evidence of the co-existence of the multidrug resistance protein (MIT) in Type I cells to account for the above-listed observations. Stopping the exposure for more than 28 days decreases the P-gp protein expression in previously doxorubicin-exposed Type I Caco-2 cells and reduces the magnitude of vincristine transepithelial fluxes in both directions to the levels that are almost similar to those of non-exposed cells. Exposing Caco-2 cells to 0.25 JAM la, 25-dihydroxyvitamin D3 induces their expression of cytochrome P450 3A4 protein to the level that is equivalent to that from isolated human jejunal cells. Under the same treatment, doxorubiein-exposed (Type I) cells metabolise naidazolam poorly and less extensively compared to non-exposed cells, suggesting that there is no such co-regulation of P-gp and CYP3A4 in Caco-2 cells. However, there is evidence which suggests CYP3A metabolises mida_zolam into 1- and 4-hydroxymidazolam, the latter may possibly be a P-gp substrate and is transported extracellularly by P-gp, supporting the hypothesis of P-gp-CYP3A4 synergistic roles in keeping xenobiotics out of the body. Doxoru.bicin-exposed (Type I) cells are less effective in translocating L-proline and glycyl-L-proline across the cell mono layers.

A novel multilocus sequence typing scheme for the opportunistic pathogen Propionibacterium acnes and characterisation of type 1 cell surface-associated antigens

We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 64?% of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.