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Purpose: Vincristine is known to interfere with OATP-mediated uptake of other compounds, hinting that vincristine itself could be a substrate of OATP transporters. The present study therefore aimed to investigate the role of OATP transporters in the hepatocellular disposition of vincristine., Methods: Vincristine uptake was studied in suspended rat and human hepatocytes as well as OATP-transfected Chinese hamster ovary (CHO) cells in the absence and presence of OATP transporter inhibitors. Membrane vesicles containing MDR1 or MRP1/2/3 were used to directly assess the role of these efflux transporters in vincristine disposition., Results: Uptake in suspended rat hepatocytes was temperature-dependent and could be inhibited by a range of OATP inhibitors. Furthermore, the MRP-inhibitor benzbromarone, but none of the tested MDR1 inhibitors, reduced vincristine efflux in rat and human suspended hepatocytes. OATP1B1-, OATP1B3- and OATP2B1- transfected CHO cells showed significantly increased vincristine uptake as compared to wild-type cells. Moreover, uptake in OATP-transfected CHO cells was reduced by OATP inhibitors. However, uptake studies in suspended human hepatocytes showed that only 10% of the total vincristine uptake process could be attributed to OATP-mediated transport. Studies with transporter-expressing membrane vesicles confirmed vincristine as an MDR1 substrate, while MRP1/2/3-mediated transport of vincristine could not be observed with this model system., Conclusions: Our findings show the involvement of OATP transporters in the disposition of vincristine in rat and human hepatocytes. However, in both species, hepatic uptake is overshadowed by a benzbromarone-sensitive efflux mechanism, possibly MRP3.

Purpose: OCT1/3 (Organic Cation Transporter-1 and -3; SLC22A1/3) are transmembrane proteins localized at the basolateral membrane of hepatocytes. They mediate the uptake of cationic endogenous compounds and/or xenobiotics. The present study was set up to verify whether the previously observed variability in OCT activity in hepatocytes may be explained by inter-individual differences in OCT1/3 mRNA levels or OCT1 genotype., Methods: Twenty-seven batches of cryopreserved human hepatocytes (male and female, age 24-88 y) were characterized for OCT activity, normalized OCT1/3 mRNA expression, and OCT1 genetic mutation. ASP + (4-[4-(dimethylamino)styryl]-N-methylpyridinium iodide) was used as probe substrate., Results: ASP + uptake ranged between 75 ± 61 and 2531 ± 202 pmol/(min × million cells). The relative OCT1 and OCT3 mRNA expression ranged between 0.007-0.46 and 0.0002-0.005, respectively. The presence of one or two nonfunctional SLC22A1 alleles was observed in 13 batches and these exhibited significant (p = 0.04) association with OCT1 and OCT3 mRNA expression. However, direct association between genotype and OCT activity could not be established., Conclusion: mRNA levels and genotype of OCT only partially explain inter-individual variability in OCT-mediated transport. Our findings illustrate the necessity of in vitro transporter activity profiling for better understanding of inter-individual drug disposition behavior.

Purpose: To classify the crystallization behavior of amorphous active pharmaceutical ingredients (API) exposed to aqueous environments., Methods: A set of approximately 50 chemically and physically diverse active pharmaceutical ingredients (APIs) was selected for this study. Two experimental setups were employed to characterize the crystallization behavior of the amorphous API in an aqueous environment. For the first approach, precipitation, as evidenced by the development of turbidity, was induced using the solvent shift method, by mixing concentrated API solutions in DMSO with an aqueous buffer in a capillary. Subsequently, crystallization was monitored in situ over time using synchrotron radiation (simultaneous SAXS/WAXS beamline 12-ID-B at the Advanced Photon Source, Argonne National Laboratories, Argonne, IL). In the second approach, amorphous films were prepared by melt quenching; after adding buffer, crystallization was monitored with time using polarized light microscopy., Results: In general, the crystallization behavior of a given compound was similar irrespective of the experimental method employed. However, the crystallization behavior among different compounds varied significantly, ranging from immediate and complete crystallization to no observable crystallization over biorelevant time scales. Comparison of the observed behavior with previous studies of crystallization tendency in non-aqueous environments revealed that the crystallization tendency of individual APIs was somewhat similar regardless of the crystallization environment., Conclusions: API properties, rather than the method by which amorphous materials are generated, tend to dictate crystallization behavior in aqueous media.

Purpose: To explore the effect of the nutritional state on the solubilizing properties of human intestinal fluids (HIF) on a time-after-food administration basis., Methods: HIF were collected in fractions of 30 min from five volunteers in the fasted, fed and fat-enriched fed state. In vitro solubility of five BCS class II drugs (danazol, diazepam, nifedipine, ketoconazole, indomethacin) was assessed in the intestinal fractions and simulated intestinal fluids., Results: Solubilities in intestinal fractions were characterized by high time- and subject-dependent variability. For the non-ionized drugs, solubility in early intestinal fractions was higher in both fed states compared to the fasted state, and in the fat-enriched fed state compared to the fed state. Solubility in simulated intestinal fluids did not sufficiently predict the solubilizing capacity of the early postprandial phase. Solubility in HIF was shown to be determined by a complex interplay of various intraluminal parameters. For the ionized drugs, pH played a significant role for indomethacin (R (2) = 0.86); for the partly ionized ketoconazole other intraluminal parameters were also important., Conclusions: Solubilizing capacity of HIF in the fed state is strongly time-dependent. Intraluminal dissolution may, therefore, vary with drug arrival time in the small intestine and constitute a source of variability in intestinal drug absorption.

Purpose: The purpose of this study was to explore the feasibility of linking the pharmacokinetic profile of a drug with its gastrointestinal behavior by simultaneously monitoring plasma and intraluminal drug concentrations. Fosamprenavir, a phosphate ester prodrug of the poorly water-soluble HIV-inhibitor amprenavir, was selected as model compound., Methods: A single tablet of fosamprenavir (Telzir) was administered to 5 volunteers in the fasted and fed state (simulated by intake of a nutritional drink). Gastric and duodenal fluids were aspirated in function of time and characterized with respect to the concentration of (fos)amprenavir, inorganic phosphate and pH. In parallel, blood samples were collected and analyzed for amprenavir., Results: The observed plasma concentration-time profiles suggested a food-induced delay in the absorption of amprenavir: in the fed state, mean tmax increased by more than 150 min compared to the fasted state. A similar delay was seen in the duodenal appearance of fosamprenavir (concentrations in mM-range) and, after dephosphorylation, amprenavir (concentrations below 160 microM). This observation could be related to the behavior of fosamprenavir in the stomach. In the fasted state, gastric dissolution of fosamprenavir started immediately, resulting in a Cmax of 4 +/- 2 mM after 43 +/- 15 min; however, in the fed state, the fosamprenavir concentration remained below 20 microM for the first 90 min after drug intake. The postponed gastric dissolution may be attributed to a food-induced delay in tablet disintegration., Conclusion: For the first time, the pharmacokinetic profile of a drug was monitored in parallel with its gastrointestinal concentrations. The observed food effect in the plasma concentration-time profile of amprenavir after intake of its phosphate ester prodrug could be related to a food-induced delay in gastric dissolution of fosamprenavir.

Purpose: To test two in vitro human nasal epithelial cell culture systems for their ability to screen the effects of pharmaceutical compounds on ciliary beating., Methods: Human nasal epithelial cells were cultured as monolayer and in a sequential monolayer-suspension culture with in vitro ciliogenesis. The influence of reference cilio-stimulatory compounds (glycocholate, isoprenaline), reference cilio-inhibitory compounds (chlorocresol, diphenhydramine) and pH on ciliary beating was investigated using computerized microscope photometry., Results: Sodium glycocholate (0.5% w/v) maximally and reversibly increased CBF of the cells in both culture systems by 26 +/- 4% (monolayer) and 18 +/- 6% (suspension). Similarly, isoprenaline (10(-3) M) maximally, but irreversibly increased CBF of the cells by 14 +/-3% (monolayer) and 17 +/- 4% (suspension). Chlorocresol (0.005% w/ v) reversibly reduced the CBF of the cells by 50 +/- 6% (monolayer) and 34 +/- 4% (suspension); at a higher concentration (0.1% w/v) it resulted in instantaneous and irreversible ciliostasis. Diphenhydramine (0.1% w/v) reversibly reduced CBF in both culture systems by 45 +/- 13% (monolayer) and 69 +/- 5% (suspension); irreversible cilio-stasis occurred in less than 2 minutes in both culture systems upon cell exposure to diphenhydramine (1.0% w/v). In the monolayer culture system, CBF was stable only within the physiological pH range of 6.5-8.0; ciliary beating in the suspension culture remained stable within a pH range of 4.0-10.0., Conclusions: Both cell culture systems are suitable for screening the effects of pharmaceutical compounds on ciliary beating. Especially the sequential monolayer-suspension culture appears to be very promising as ciliary activity can be preserved for as long as 6 months.