Drug efflux by intestinal P-glycoprotein (P-gp) is known to decrease the oral bioavailability of many CYP3A4 substrates. We hypothesized that the interplay occurring between P-gp and CYP3A4 at the apical membrane would increase the opportunity for drug metabolism. To define the roles of P-glycoprotein (P-gp) and CYP3A4 in controlling the extent of intestinal absorption and metabolism, two substrates were tested. The transport, metabolism, and intracellular levels of N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K77, a cysteine protease inhibitor; P-gp and CYP3A4 substrate) and felodipine (CYP3A4 substrate only) were measured across CYP3A4-transfected Caco-2 cells in the presence of an inhibitor of CYP3A4 and P-gp, cyclosporine (CsA), or an inhibitor of P-gp and not CYP3A4, GG918 (N-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine). The extent of metabolism was measured by calculating the extraction ratio (ER) across the cells, while accounting for intracellular changes occurring with P-gp inhibition. The (A)pical to (B)asolateral and B3 A ERs for K77 were 0.33 and 0.06, respectively. These changed with GG918 to 0.14 and 0.12 and with CsA to 0.06 and 0.04. Felodipine ERs were similar in both directions, 0.26 and 0.24 (A3 B and B3 A), and were unchanged in the presence of GG918 but decreased with CsA (0.14 and 0.11). The K77 absorption rate was increased 5 and 4.2-fold in the presence of CsA and GG918, respectively, whereas no change was observed for felodipine absorption. The decreased A3 B ER and increased absorption of K77 with GG918 suggest that P-gp influences the extent of drug metabolism in the intestine via prolonging the access of drugs to CYP3A4 near the apical membrane and decreasing transport across the cells.Intestinal drug metabolism by cytochrome P450 3A (CYP3A) is increasingly being recognized as an important determinant in limiting drug bioavailability. CYP3A4 is the most prominent oxidative cytochrome P450 enzyme present in the human intestine (Watkins et al., 1987;Zhang et al., 1999), where it is localized to the columnar epithelial cells lining the intestinal lumen (Kolars et al., 1994). Despite the lower CYP3A4 content in the intestine relative to the liver, first-pass metabolism in the intestine by CYP3A has conclusively been shown to be important in the disposition of midazolam (Paine et al., 1996) and cyclosporine (CsA) (Kolars et al., 1991) from studies with anhepatic patients. Drug interaction studies performed with grapefruit juice [a suicide inhibitor of intestinal CYP3A (Schmiedlin-Ren et al., 1997a)] have also shown significant increases in the oral bioavailability of many CYP3A4 substrates including felodipine (Edgar et al., 1992).Drug absorption can also be hindered by efflux transporters in the intestine. P-glycoprotein (P-gp) is a plasma membrane-bound drug efflux protein found primarily in drugeliminating organs and presumably functions as a detoxifying transporter because it actively extrud...

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Active ingredient deposition as a whole during foliar spray application was studied by means of microscopic and macroscopic methods. High‐speed photography of the impact of spray droplets showed that rebound from reflective plant surfaces was reduced only with surfactant concentrations well above the critical micelle concentration. These results correlate with laboratory spraying experiments to determine quantitatively the retention of spray solutions. The dynamic interaction between droplet and foliage seemed to be specifically dependent on surfactant structure, concentration and the distribution of isomers or by‐products in the technical product. Rough surfaces with epicuticular wax crystals were difficult to wet; the orientation of the leaf, however, had little influence on retention. Apolar artificial surfaces retained water rather well and are therefore unsuitable as model surfaces for reflective leaves. Measurement of dynamic surface tension at 100 msec gave a satisfactory correlation with retention values in the case of liquids containing surfactant but failed to predict the good adhesion of polyvinyl alcohol solutions.

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1 Clinical studies have shown enhancement of cyclosporine toxicity when co-administered with the immunosuppressant sirolimus. We evaluated the biochemical mechanisms underlying the sirolimus/ cyclosporine interaction on rat brain metabolism using magnetic resonance spectroscopy (MRS) and compared the e ects of sirolimus with those of the structurally related RAD. 2 Two-week-old rats (25 g) were allocated to the following treatment groups (all n=6): I. control, II. cyclosporine (10 mg kg 71 d 71 ), III. sirolimus (3 mg kg 71 d 71 ), IV. RAD (3 mg kg 71 d 71 ), V. cyclosporine+sirolimus and VI. cyclosporine+RAD. Drugs were administered by oral gavage for 6 days. Twelve hours after the last dose, metabolic changes were assessed in brain tissue extracts using multinuclear MRS. 3 Cyclosporine signi®cantly inhibited mitochondrial glucose metabolism (glutamate: 78+6% of control; GABA: 67+12%; NAD + : 76+3%; P50.05), but increased lactate production. Sirolimus and RAD inhibited cytosolic glucose metabolism via lactate production (sirolimus: 81+3% of control, RAD: 69+2%; P50.02). Sirolimus enhanced cyclosporine-induced inhibition of mitochondrial glucose metabolism (glutamate: 60+4%; GABA: 59+8%; NAD + : 45+5%; P50.02 versus cyclosporine alone). Lactate production was signi®cantly reduced. In contrast, RAD antagonized the e ects of cyclosporine (glutamate, GABA, and NAD + , not signi®cantly di erent from controls). 4 The results can partially be explained by pharmacokinetic interactions: co-administration increased the distribution of cyclosporine and sirolimus into brain tissue, while co-administration with RAD decreased cyclosporine brain tissue concentrations. In addition RAD, but not sirolimus, distributed into brain mitochondria. 5 The combination of cyclosporine/RAD compares favourably to cyclosporine/sirolimus in regards to their e ects on brain high-energy metabolism and tissue distribution in the rat.

CYP3A4-transfected Caco-2 cells were used as an in vitro system to predict the importance of drug metabolism and transport on overall drug absorption. We examined the transport and metabolism of two drugs; midazolam, an anesthetic agent and CYP3A4 substrate, and sirolimus, an immunosuppressant and a dual CYP3A4/P-glycoprotein (P-gp) substrate, in the presence of cyclosporine (CsA, a CYP3A4/P-gp inhibitor) or N-{4- [2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918) (an inhibitor of P-gp and not CYP3A4). All major CYP3A4 metabolites were formed in the cells (1-OH Ͼ 4-OH midazolam and 39-O-desmethyl Ͼ 12-OH Ͼ 11-OH sirolimus), consistent with results from human liver microsomes. There was no bidirectional transport of midazolam across CYP3A4-transfected Caco-2 cells, whereas there was a 2.5-fold net efflux of sirolimus (1 M) that disappeared in the presence of CsA or GG918. No change in the absorption rate or extraction ratio (ER) for midazolam was observed when P-gp was inhibited with GG918. Addition of GG918 had a modest impact on the absorption rate and ER for sirolimus (increased 58% and decreased 25%, respectively), whereas a 6.1-fold increase in the absorption rate and a 75% decrease in the ER were found when sirolimus was combined with CsA. Although both midazolam and sirolimus metabolites were preferentially excreted to the apical compartment, only sirolimus metabolites were transported by P-gp as determined from inhibition studies with GG918. Using CYP3A4-transfected Caco-2 cells we determined that, in contrast to P-gp, CYP3A4 is the major factor limiting sirolimus absorption. The integration of CYP3A4 and P-gp into a combined in vitro system was critical to unveil the relative importance of each biochemical barrier.Oral bioavailability and intestinal drug absorption can be significantly limited by metabolizing enzymes and efflux transporters in the gut (Benet et al., 1996b). The most prevalent oxidative drug-metabolizing enzyme present in the intestine is cytochrome P450 3A4 (CYP3A4). Currently, more than 50% of the drugs on the market metabolized by P450 enzymes are metabolized by CYP3A4 (Benet et al., 1996a). Oral absorption of CYP3A4 substrates can also be limited by the multidrug resistance transporter P-glycoprotein (P-gp), because there is extensive substrate overlap between these two proteins (Wacher et al., 1995). P-gp is an ATP-dependent transporter on the apical plasma membrane of enterocytes that functions to limit the entry of drugs into the cell (Ambudkar et al., 1999). We have previously hypothesized that the interplay between CYP3A4 and P-gp in the intestine can serve to enhance drug metabolism and significantly decrease intestinal drug absorption (Cummins et al., 2002).