Albert Owen

Enhanced intestinal absorption of an RGD peptide from water-in-oil microemulsions of different composition and particle size

Abstract The fibrinogen receptor antagonist SK&F 106760 is a water-soluble tetrapeptide (Mr634) with very low oil/water partitioning and membrane permeability. Thus, the intraduodenal bioavailability of this peptide in the rat from a saline formulation was found to be less than 1%. Upon formulation, however, in w/o microemulsions of different composition and particle size, the intraduodenal bioavailability of SK&F 106760 was increased up to 29% depending on the microemulsion composition. There was no apparent correlation between the particle size of microemulsions (mean droplet diameter of 0.010–1.0 μm) and enhanced absorption. None of the investigated microemulsions induced gross changes in gastrointestinal mucosa at a dosing volume of 3.3 ml/kg. Lipids containing medium-chain fatty acids play a major role on the observed absorption enhancement. These findings further support the use of microemulsion formulations for oral drug/peptide delivery, provided however, that development issues with these systems are properly addressed.

Use of transporter knockdown Caco-2 cells to investigate the in vitro efflux of statin drugs.

The objective of the present study was to determine the efflux transporters responsible for acid and lactone statin drug efflux using transporter knockdown Caco-2 cells. The bidirectional transport was determined in Caco-2 cell monolayers in which the expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), or multidrug resistance associated protein 2 (MRP2) was knocked down by transduction with lentivirus containing human transporter-targeted small hairpin RNAs (shRNAs). Cells transduced with lentivirus containing nontargeted shRNA served as the vector control. Atorvastatin, lovastatin, and rosuvastatin displayed extremely low apical-to-basolateral (A-to-B) transport, which made the Papp,A-B values too unreliable to calculate the efflux ratio. Thus, transport comparisons were performed using the B-to-A permeability (Papp,B-A) values. Presented in the order of vector control, P-gp, BCRP, and MRP2 knockdown Caco-2 cells, the Papp,B-A values (×10−6, cm/s) were 28.1 ± 1.3, 8.6 ± 2.9, 20.3 ± 1.8, and 21.5 ± 1.6 for atorvastatin; 96.1 ± 7.1, 25.3 ± 3.5, 57.3 ± 9.8, and 48.2 ± 2.3 for fluvastatin; and 14.1 ± 1.9, 4.6 ± 1.7, 5.8 ± 0.7, and 6.6 ± 1.8 for rosuvastatin, respectively. Lovastatin and simvastatin showed no efflux in the vector control or knockdown cell monolayers in either lactone or acid forms. Results indicate that atorvastatin, fluvastatin, and rosuvastatin were transported by P-gp, BCRP, and MRP2. On the other hand, neither the lactone nor the resulting acid of lovastatin and simvastatin was transported by P-gp, BCRP, or MRP2. The current study demonstrated that the transporter knockdown Caco-2 cells are useful tools for studying drug-transporter interactions and should help eliminate some of the ambiguity associated with the identification of drug-transporter interactions based on chemical inhibitors alone.

Application and limitation of inhibitors in drug-transporter interactions studies.

The objective of the present study was to investigate the reliability of transporter inhibitors in the elucidation of drug-transporter interactions when multiple transporters are present in a test system. The bidirectional permeabilities of digoxin, estrone-3-sulfate (E3S), and sulfasalazine, substrates of P-gp, BCRP/MRP2 and unspecified efflux transporters, respectively, were examined in Caco-2 and MDR-MDCK cells in the absence and presence of transporter inhibitors: CsA (P-gp), FTC (BCRP) and MK571 (MRP). Digoxin showed significant efflux ratios (ER) in both Caco-2 (ER=17) and MDR-MDCK (ER=120), whereas E3S and sulfasalazine only showed significant efflux in Caco-2 (ER=15 and 88, respectively) but not in MDR-MDCK cells (ER=1.1 and 1.3, respectively). CsA at 10 microM showed complete inhibition of digoxin efflux, partial inhibition of E3S efflux and no effect on sulfasalazine efflux. FTC and MK571 had different inhibitory effects on the efflux of these compounds. The present study shows evidence of the functional expression of multiple efflux transporter systems in Caco-2 cells. Although the use of Caco-2 cells and selected inhibitors of efflux transporters can provide useful mechanistic information on drug-drug interactions involving efflux transporters, the potential cross-reaction of inhibitors with multiple transporters makes it difficult to discern the role of individual transporters in drug transport or drug-drug interactions.

CRYOPRESERVED HUMAN HEPATOCYTES AS ALTERNATIVE IN VITRO MODEL FOR CYTOCHROME P450 INDUCTION STUDIES

SummaryInduction of cytochrome P450 (CYP) by drugs is one of major concerns for drug-drug interactions. Thus, the assessment of CYP induction by novel compounds is a vital component in the drug discovery and development processes. Primary human hepatocytes are the preferred in vitro model for predicting CYP induction in vivo. However, their use is hampered by the erratic supply of human tissue and donor-to-donor variability. Although cryopreserved hepatocytes have been recommended for short-term applications in suspension, their use in studies on induction of enzyme activity has been limited because of poor attachment and response to enzyme inducers. In this study, we report culture conditions that allowed the attachment of cryopreserved human hepatocytes and responsiveness to CYP inducers. We evaluated the inducibility of CYP1A1/2 and CYP3A4 enzymes in cryopreserved hepatocytes from three human donors. Cryopreserved human hepatocytes were cultured in serum-free medium for 4 d. They exhibited normal morphology and measurable viability as evaluated by the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) by cellular dehydrogenases. Treatment with β-naphthoflavone (10 μM) for 3 d increased ethoxyresorufin-O-deethylase activity (CYP1A1/2) by 6- to 11-fold over untreated cultures and increased CYP1A2 messenger ribonucleic acid (mRNA) expression by three- to eightfold. Similarly, treatment of cryopreserved human hepatocytes with rifampicin (25 μM) for 3 d increased testosterone 6β-hydroxylase activity (CYP3A4) by five- to eightfold over untreated cultures and increased CYP3A4 mRNA expression by four- to eightfold. The results suggest that cryopreserved human hepatocytes can be a suitable in vitro model for evaluating xenobiotics as inducers of CYP1A1/2 and CYP3A4 enzymes.

INTER-SPECIES COMPARISON OF 7-HYDROXYCOUMARIN GLUCURONIDATION AND SULFATION IN LIVER S9 FRACTIONS

SummaryUDP glycosyltransferases (UGTs) and sulfotransferases (SULTs) are phase II enzymes that interact with a number of xenobiotics in humans and animals. Species differences in enzymatic characteristics have seldom been investigated. Liver S9 fractions are commonly used for studying phase II metabolism in vitro. The objective of this study was to characterize the UGT and SULT activities in liver S9 fractions from various species including humans, monkeys, dogs, and rats. A single substrate, 7-hydroxycoumarin (7-HC), at several concentrations was incubated at 37° C with the S9 reaction matrices along with necessary cofactors. The rate of formation of two metabolites, 7-HC-glucuronide (7-HC-G) and 7-Km and Vmas values were calculated for each species. For the UGTs, the apparent Km and Vmax for 7-HC-G formation varied greatly among different species, with dog UGTs having both the highest Km and Vmax values. In contrast to UGTs, the Km for 7-HC-S formation showed no significant difference among humans, monkeys, and rats (approximately 3 μM). However, the Km in dog was 8.7 μM. Species differences with respect to phase II metabolism must be carefully considered when selecting an in vitro model system to study various aspects of drug metabolism.

Transport Inhibition of Digoxin Using Several Common P-gp Expressing Cell Lines Is Not Necessarily Reporting Only on Inhibitor Binding to P-gp

We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxins cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable P-gp substrates such as amprenavir, quinidine, ketoconazole and verapamil do not, regardless of whether they actually use the basolateral transporter.

Investigation of the Involvement of P-Glycoprotein and Multidrug Resistance-Associated Protein 2 in the Efflux of Ximelagatran and Its Metabolites by Using Short Hairpin RNA Knockdown in Caco-2 Cells

Liver and bile secretion can be an important first-pass and clearance route for drug compounds and also the site of several drug-drug interactions. In the clinical program for ximelagatran development, an unexpected effect of erythromycin on the pharmacokinetics of the direct thrombin inhibitor ximelagatran and its metabolites was detected. This interaction was believed to be mediated by inhibition of drug transporters, which normally extrude the drug into the bile. Previous Caco-2 cell experiments indicated the involvement of an active efflux mechanism for ximelagatran, hydroxy-melagatran, and melagatran possibly mediated by P-glycoprotein (P-gp). However, the inhibitors used may not have been specific enough and the possibility that transporters other than P-gp were important in the Caco-2 cell assay cannot be excluded. In this study we used RNA interference, a post-transcriptional gene silencing mechanism in which mRNA is degraded in a sequence-specific manner, to specifically knock down P-gp or multidrug resistance-associated protein 2 (MRP2) transporters in Caco-2 cells. The data obtained from bidirectional transport studies in these cells indicate a clear involvement of P-gp but not of MRP2 in the transport of ximelagatran, hydroxy-melagatran, and melagatran across the apical cell membrane. The present study shows that short hairpin RNA Caco-2 cells are a valuable tool to investigate the contribution of specific transporters in the transcellular transport of drug molecules and to predict potential sites of pharmacokinetic interactions. The results also suggest that inhibition of hepatic P-gp is involved in the erythromycin-ximelagatran interaction seen in clinical studies.

Silencing the Breast Cancer Resistance Protein Expression and Function in Caco-2 Cells Using Lentiviral Vector-Based Short Hairpin RNA

A series of stable breast cancer resistance protein (BCRP, ABCG2) knockdown cell lines were produced by transduction of Caco-2 cells with lentiviral vector-based short hairpin RNA (shRNA). Caco-2 cell is a human intestinal-derived cell line widely used to study intestinal drug absorption. Caco-2 expresses three apical drug efflux transporters: BCRP, P-glycoprotein (P-gp; ABCB1), and multidrug resistance protein 2 (MRP2, ABCC2). BCRP and P-gp in particular play a significant role in pharmacokinetics because of their expression at several key interfaces. Overexpression of BCRP in cancer cells may also be a mechanism of tumor resistance to chemotherapeutic drugs. The goal of this study was to engineer and characterize Caco-2 cell clones with stable knockdown of BCRP expression. The shRNA/BCRP lentiviral particles were used to infect a stable clone of Caco-2 cells. Expression of BCRP was monitored using quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence microscopy, and bidirectional transport of probe substrates, estrone-3-sulfate (E3S), and pheophorbide A (PhA). Based on qPCR, expression of BCRP mRNA was knocked down in five clones with a maximum of 97% silencing in clone D. Silencing of BCRP gene expression was maintained for at least 25 passages. Expression of BCRP protein was also reduced significantly. Functionally, BCRP knockdown was reflected in significant reduction of the efflux ratio of E3S and PhA. Clone D in particular should be a useful model for identifying and characterizing P-gp substrates and inhibitors without interference from BCRP and/or MRP2. In addition, it can be used in conjunction with wild-type or vector control Caco-2 cells to identify BCRP substrates.

Application of exogenous mixture of glutathione and stable isotope labeled glutathione for trapping reactive metabolites in cryopreserved human hepatocytes. Detection of the glutathione conjugates using high resolution accurate mass spectrometry.

Metabolites (including reactive metabolites) of troglitazone were generated by incubation with cryopreserved human hepatocytes and trapped in the presence of an exogenous mixture of unlabeled and stable isotope labeled (SIL: [1,2-(13)C, (15)N]-glycine) glutathione (GSH/SIL-GSH). The incubation samples were analyzed using liquid chromatography-high resolution accurate mass spectrometry (LC-HRAMS) implemented on a LTQ Orbitrap mass spectrometer. The GSH conjugates of the reactive metabolites were detected via a characteristic mono-isotopic pattern (peaks separated by 3.0037u). Analysis of the incubation samples led to detection of a number of previously described GSH conjugates, as well as two novel methylated GSH conjugates, which were partially characterized based on accurate mass measurements and MS/MS data. The addition of exogenous GSH led to an increase in the apparent level of detected GSH conjugates. Kinetic isotopic measurements showed that the rates of incorporation of exogenous GSH are conjugate-specific. In conclusion, this approach, based on the use of a mixture of GSH/SIL-GSH, allows facile capture and detection of reactive metabolites in human hepatocytes. Moreover, the data suggest that routine addition of glutathione to the assay medium may be advisable for experiments with cryopreserved hepatocytes.

Evaluation of a Nanoemulsion Formulation Strategy for Oral Bioavailability Enhancement of Danazol in Rats and Dogs

The objective of this study was to determine whether nanoemulsion formulations constitute a viable strategy to improve the oral bioavailability of danazol, a compound whose poor aqueous solubility limits its oral bioavailability. Danazol-containing oil-in-water nanoemulsions (NE) with and without cosurfactants stearylamine (SA) and deoxycholic acid (DCA) were prepared and characterized. Nanoemulsion droplets size ranging from 238 to 344 nm and with surface charges of -24.8 mV (NE), -26.5 mV (NE-DCA), and +27.8 mV (NE-SA) were reproducibly obtained. Oral bioavailability of danazol in nanoemulsions was compared with other vehicles such as PEG400, 1% methylcellulose (MC) in water (1% MC), Labrafil, and a Labrafil/Tween 80 (9:1) mixture, after intragastric administration to rats and after oral administration of NE-SA, a Labrafil solution, or a Danocrine® tablet to dogs. The absolute bioavailability of danazol was 0.6% (PEG400), 1.2% (1% MC), 6.0% (Labrafil), 7.5% (Labrafil/Tween80), 8.1% (NE-DCA), 14.8% (NE), and 17.4% (NE-SA) in rats, and 0.24% (Danocrine), 6.2% (Labrafil), and 58.7% (NE-SA) in dogs. Overall, danazol bioavailability in any nanoemulsion was higher than any other formulation. Danazol bioavailability from NE and NE-SA was 1.8- to 2.2-fold higher than NE-DCA nanoemulsion and could be due to significant difference in droplet size.