Comparison of methods for the prediction of human clearance from hepatocyte intrinsic clearance for a set of reference compounds and an external evaluation set.

1. We compared direct scaling, regression model equation and the so-called "Poulin et al." methods to scale clearance (CL) from in vitro intrinsic clearance (CLint) measured in human hepatocytes using two sets of compounds. One reference set comprised of 20 compounds with known elimination pathways and one external evaluation set based on 17 compounds development in Merck (MS). 2. A 90% prospective confidence interval was calculated using the reference set. This interval was found relevant for the regression equation method. The three outliers identified were justified on the basis of their elimination mechanism. 3. The direct scaling method showed a systematic underestimation of clearance in both the reference and evaluation sets. The "Poulin et al." and the regression equation methods showed no obvious bias in either the reference or evaluation sets. 4. The regression model equation was slightly superior to the "Poulin et al." method in the reference set and showed a better absolute average fold error (AAFE) of value 1.3 compared to 1.6. A larger difference was observed in the evaluation set were the regression method and "Poulin et al." resulted in an AAFE of 1.7 and 2.6, respectively (removing the three compounds with known issues mentioned above). A similar pattern was observed for the correlation coefficient. Based on these data we suggest the regression equation method combined with a prospective confidence interval as the first choice for the extrapolation of human in vivo hepatic metabolic clearance from in vitro systems.

In vitro and in vivo drug disposition of cilengitide in animals and human.

Cilengitide is very low permeable (1.0 nm/sec) stable cyclic pentapeptide containing an Arg-Gly-Asp motif responsible for selective binding to αvß3 and αvß5 integrins administered intravenously (i.v.). In vivo studies in the mouse and Cynomolgus monkeys showed the major component in plasma was unchanged drug (>85%). These results, together with the absence of metabolism in vitro and in animals, indicate minimal metabolism in both species. The excretion of [(14)C]-cilengitide showed profound species differences, with a high renal excretion of the parent drug observed in Cynomolgus monkey (50% dose), but not in mouse (7 and 28%: m/f). Consistently fecal (biliary) secretion was high in mouse (87 and 66% dose: m/f) but low in Cynomolgus monkey (36.5%). Human volunteers administrated with [(14)C]-cilengitide showed that most of the dose was recovered in urine as unchanged drug (77.5%, referred to Becker et al. 2015), indicating that the Cynomolgus monkey was the closer species to human. In order to better understand the species difference between human and mouse, the hepatobiliary disposition of [(14)C]-cilengitide was determined in sandwich-cultured hepatocytes. Cilengitide exhibited modest biliary efflux (30-40%) in mouse, while in human hepatocytes this was negligible. Furthermore, it was confirmed that the uptake of cilengitide into human hepatocytes was minor and appeared to be passive. In summary, the extent of renal and biliary secretion of cilengitide appears to be highly species specific and is qualitatively well explained using sandwich hepatocyte culture models.

Variability in P-glycoprotein inhibitory potency (IC50) using various in vitro experimental systems: implications for universal digoxin drug-drug interaction risk assessment decision criteria.

A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.

Application of receiver operating characteristic analysis to refine the prediction of potential digoxin drug interactions.

In the 2012 Food and Drug Administration (FDA) draft guidance on drug-drug interactions (DDIs), a new molecular entity that inhibits P-glycoprotein (P-gp) may need a clinical DDI study with a P-gp substrate such as digoxin when the maximum concentration of inhibitor at steady state divided by IC50 ([I1]/IC50) is ≥0.1 or concentration of inhibitor based on highest approved dose dissolved in 250 ml divide by IC50 ([I2]/IC50) is ≥10. In this article, refined criteria are presented, determined by receiver operating characteristic analysis, using IC50 values generated by 23 laboratories. P-gp probe substrates were digoxin for polarized cell-lines and N-methyl quinidine or vinblastine for P-gp overexpressed vesicles. Inhibition of probe substrate transport was evaluated using 15 known P-gp inhibitors. Importantly, the criteria derived in this article take into account variability in IC50 values. Moreover, they are statistically derived based on the highest degree of accuracy in predicting true positive and true negative digoxin DDI results. The refined criteria of [I1]/IC50 ≥ 0.03 and [I2]/IC50 ≥ 45 and FDA criteria were applied to a test set of 101 in vitro-in vivo digoxin DDI pairs collated from the literature. The number of false negatives (none predicted but DDI observed) were similar, 10 and 12%, whereas the number of false positives (DDI predicted but not observed) substantially decreased from 51 to 40%, relative to the FDA criteria. On the basis of estimated overall variability in IC50 values, a theoretical 95% confidence interval calculation was developed for single laboratory IC50 values, translating into a range of [I1]/IC50 and [I2]/IC50 values. The extent by which this range falls above the criteria is a measure of risk associated with the decision, attributable to variability in IC50 values.

Characterization of the inhibition of breast cancer resistance protein-mediated efflux of mitoxantrone by pharmaceutical excipients.

Previously we showed that some excipients can inhibit breast cancer resistance protein (BCRP/ABCG2) in vitro and in vivo. We then evaluated the reversibility and the mode of BCRP inhibition of excipients, such as Tween 20 and Pluronic P85, by the intracellular mitoxantrone uptake study. To evaluate the reversibility of BCRP inhibitory effects, BCRP expressing cells were preincubated with the excipients and the intracellular mitoxantrone uptake was determined after removing or not removing the excipients. To evaluate the mode of BCRP inhibitory effects, the intracellular mitoxantrone uptake at the different mitoxantrone concentrations in the medium with the excipients was determined. Both Tween 20 and Pluronic P85 increased the mitoxantrone uptake in BCRP expressing cells, but these effects were disappeared when the excipients were removed. Moreover, both excipients increased the uptake at low substrate concentrations. However, at high substrate concentrations, Tween 20 increased the uptake to less extent compared with low substrate concentrations, whereas there was no such effect of Pluronic P85. Taken together, Pluronic P85 and Tween 20 appear to inhibit BCRP-mediated efflux of mitoxantrone reversibly and the inhibition mode of Pluronic P85 may be competitive but not that of Tween 20, which may be mixed type.

Key functions in polymer carriers for intestinal absorption of insulin.

This work aimed to clarify the relationship between polymer function and insulin absorption, and to evaluate the optimized preparative formulation predicted from this relationship. Insulin-loaded polymer (ILP) carrier systems were prepared following a two-factor composite second-order spherical experimental design. To investigate the polymer function, we evaluated its insulin release, bioadhesiveness, and protective effect. Each ILP was administered intestinally, and glucose reduction was monitored as the pharmacological effect. Based on these data, an optimized formulation was predicted and how the polymer function affects insulin absorption was clarified by multivariate spline (MVS) interpolation. A greater pharmacological effect was apparent in ILPs with a smaller particle size and loaded with more insulin. The pharmacological effect predicted by MVS after the administration of ILP made under optimized preparative conditions was almost identical to the observed effect. Moreover, MVS clarified the relationship between the polymer function and the pharmacological effect. These results supported that MVS can be an effective tool with which to approximate the relationship between the function of a dosage form and its absorption, and to explore the optimized preparative conditions.

Effect of excipients on breast cancer resistance protein substrate uptake activity.

Breast cancer resistance protein (BCRP/ABCG2) plays an important role in drug disposition. To examine whether some currently used excipients could inhibit its function, we measured the uptake of [(3)H]mitoxantrone in BCRP-, P-glycoprotein (P-gp)- or green fluorescent protein (GFP)-expressing cells, in the presence or absence of 15 kinds of currently used excipients. Of 15 excipients, five (Cremophor EL, Tween 20, Span 20, Pluronic P85 and Brij 30) increased the uptake of [(3)H]mitoxantrone in BCRP-expressing cells. On the other hand, ten (Cremophor EL, Cremophor RH40, Tween 20, Tween 80, Span 20, Pluronic P85, vitamin E TPGS, Brij 30, Myrj 52 and Gelucire 44/14) significantly increased uptake in P-gp-expressing cells. No significant effects on intracellular ATP levels were observed following treatments with the excipients that inhibited BCRP function. Taken together, this study demonstrated that some excipients might be potent BCRP inhibitors, and there may be differences in the effects of excipients on the functions of BCRP and P-gp.

Improvement of the oral drug absorption of topotecan through the inhibition of intestinal xenobiotic efflux transporter, breast cancer resistance protein, by excipients.

Recently, breast cancer resistance protein (BCRP/ABCG2) has been shown to limit the oral absorption of its substrates in the intestine. The purpose of this study was to examine whether excipients can be used as inhibitors of BCRP, to improve the oral drug absorption of BCRP substrates. In wild-type mice, Pluronic P85 and Tween 20, given orally 15 min before topotecan administration, increased the area under the plasma concentration-time curve (AUC) of topotecan after oral administration (2.0- and 1.8-fold, respectively). In contrast, Pluronic P85 and Tween 20 were less effective (no significant difference) on the AUC of topotecan after oral administration in Bcrp (-/-) mice (1.2- and 1.2-fold, respectively). Pluronic P85 and Tween 20 given orally did not affect significantly the AUC of topotecan after intravenous administration in wild-type and Bcrp (-/-) mice. Moreover, we determined the mucosal-to-serosal absorptive transport of topotecan using everted mouse ileum. Pluronic P85 and Tween 20 significantly increased the intestinal absorption rate of topotecan in everted sacs from wild-type mice whereas, in everted sacs from Bcrp (-/-) mice, the absorption rate was 2.1-fold greater than that in wild-type mice, and these excipients were not significantly effective. There was no significant difference in the intestinal P-glycoprotein (P-gp) expression and serosal-to-mucosal secretory transport of rhodamine 123, a typical P-gp substrate. Taken together, these results suggest that Pluronic P85 and Tween 20 can improve the oral bioavailability of BCRP substrates by inhibiting BCRP function in the small intestine.

Characterization of insulin protection properties of complexation hydrogels in gastric and intestinal enzyme fluids.

The objective of this study was to elucidate the mechanisms contributing to oral bioavailability of insulin by poly(methacrylic acid grafted with poly(ethylene glycol)) (P(MAA-g-EG)) hydrogels using the gastric and intestinal fluids from rats. P(MAA-g-EG) hydrogels successfully protected the incorporated insulin from enzymatic degradation by forming interpolymer complexes in the gastric fluid. The hydrogels also showed the insulin protection ability by itself. In the intestinal fluid, P(MAA-g-EG) hydrogels significantly decreased the insulin degradation rate and calcium ion levels, while protein levels was not changed. Insulin protecting effects were dependent on the fraction of the carboxylic group in the polymer networks. Moreover, the insulin degradation inhibitory effect was significantly correlated with Ca2+ deprivation ability of P(MAA-g-EG) hydrogels in the intestinal fluid, implying that the Ca2+ deprivation ability plays an important role in the inhibition of the intestinal enzyme activities. Insulin-loaded P(MAA-g-EG) (ILPs) hydrogels showed a rapid and almost complete insulin release even in the presence of intestinal proteases. These results suggested that the insulin protection ability of the hydrogels contributed to improve oral insulin absorption and that P(MAA-g-EG) hydrogels can be an excellent carrier for protecting insulin during their transit through the GI tract.

Mucosal insulin delivery systems based on complexation polymer hydrogels: effect of particle size on insulin enteral absorption.

Insulin-loaded polymer (ILP) microparticles composed of poly(methacrylic acid) and poly(ethylene glycol), which have pH-dependent complexation and mucoadhesive properties have been thought to be potential carriers for insulin via an oral route. Nevertheless, further optimization of the polymer delivery system is required to improve clinical application. Therefore, the effect of particle size of the ILP (L-ILP: 180-230 microm, S-ILP: 43-89 microm, SS-ILP: <43 microm) on insulin absorption was studied in the in situ loop system, hypothesizing smaller particle sizes of ILP could induce bigger hypoglycemic effects due to increase mucoadhesive capacity. To verify the hypothesis, the adhesive capacities of differently sized ILPs to the mucosal tissues were evaluated. Additionally, the intestinal site-specificity of ILP for insulin absorption was investigated. Intra- and inter-cellular integrity and/or damage were also examined by lactate dehydrogenase leakage and membrane electrical resistance change to ensure the safety of ILP as a carrier for oral route. As hypothesized, the smaller sized microparticles (SS-ILP) showed a rapid burst-type insulin release and higher insulin absorption compared with the microparticles having larger sizes, resulting in greater hypoglycemic effects without detectable mucosal damage. In fact, SS-ILP demonstrated higher mucoadhesive capacity to the jejunum and the ileum than those of L-ILP. Moreover, SS-ILP's enhancement effect of insulin mucosal absorption showed a site-specificity, demonstrating maximum effect at the ileal segment. These results imply that the particle size and delivery site are very important factors for ILP with respect to increasing the bioavailability of insulin following oral administration.