In vitro UGT1A1 inhibition by tyrosine kinase inhibitors and association with drug-induced hyperbilirubinemia.

PURPOSE: Hyperbilirubinemia has been observed in patients treated with tyrosine kinase inhibitor (TKI) drugs. Therefore, it would be beneficial to understand whether there is a relationship between inhibition of uridine-5'-diphosphate glucuronosyltransferase (UGT) 1A1 activity and observed bilirubin elevations in TKI drug-treated patients. UGT1A1 is responsible for the glucuronidation of bilirubin which leads to its elimination in the bile. METHODS: To examine this question, an in vitro glucuronidation assay was developed to determine the inhibitory effect of TKI drugs employing human liver microsomes (HLM) with varying UGT1A1 activity. Utilizing ß-estradiol as the UGT1A1 probe substrate, 20 TKI drugs were evaluated at concentrations that represent clinical plasma levels. Adverse event reports were searched to generate an empirical Bayes geometric mean (EGBM) score for clinical hyperbilirubinemia with the TKI drugs. RESULTS: Erlotinib, nilotinib, regorafenib, pazopanib, sorafenib and vemurafenib had IC50 values that were lower than their clinical steady-state Cmax concentrations. These TKI drugs had high incidences of hyperbilirubinemia and higher EBGM scores. The IC50 values and Cmax/IC50 ratios correlated well with EBGM scores for hyperbilirubinemia (P < 0.005). For the TKI drugs with higher incidence of hyperbilirubinemia in Gilbert syndrome patients, who have reduced UGT1A1 activity, six of eight had smaller ratios in the low UGT1A1 activity microsomes than the wild-type microsomes for drugs, indicating greater sensitivity to the drugs in this phenotype. CONCLUSIONS: These results suggest that in vitro UGT1A1 inhibition assays have the potential to predict clinical hyperbilirubinemia.

Internalization and Subcellular Trafficking of Poly-l-lysine Dendrimers Are Impacted by the Site of Fluorophore Conjugation.

Internalization and intracellular trafficking of dendrimer-drug conjugates play an important role in achieving successful drug delivery. In this study, we aimed to elucidate the endocytosis mechanisms and subcellular localization of poly-l-lysine (PLL) dendrimers in Caco-2 cells. We also investigated the impact of fluorophore conjugation on cytotoxicity, uptake, and transepithelial transport. Oregon green 514 (OG) was conjugated to PLL G3 at either the dendrimer periphery or the core. Chemical inhibitors of clathrin-, caveolin-, cholesterol-, and dynamin-mediated endocytosis pathways and macropinocytosis were employed to establish internalization mechanisms, while colocalization with subcellular markers was used to determine dendrimer trafficking. Cell viability, internalization, and uptake were all influenced by the site of fluorophore conjugation. Uptake was found to be highly dependent on cholesterol- and dynamin-mediated endocytosis as well as macropinocytosis. Dendrimers were trafficked to endosomes and lysosomes, and subcellular localization was impacted by the fluorophore conjugation site. The results of this study indicate that PLL dendrimers exploit multiple pathways for cellular entry, and internalization and trafficking can be impacted by conjugation. Therefore, design of dendrimer-drug conjugates requires careful consideration to achieve successful drug delivery.

Intracellular Ca2+ release mediates cationic but not anionic poly(amidoamine) (PAMAM) dendrimer-induced tight junction modulation.

PURPOSE: Poly(amidoamine) (PAMAM) dendrimers show great promise for utilization as oral drug delivery vehicles. These polymers are capable of traversing epithelial barriers, and have been shown to translocate by both transcellular and paracellular routes. While many proof-of-concept studies have shown that PAMAM dendrimers improve intestinal transport, little information exists on the mechanisms of paracellular transport, specifically dendrimer-induced tight junction modulation. METHODS: Using anionic G3.5 and cationic G4 PAMAM dendrimers with known absorption enhancers, we investigated tight junction modulation in Caco-2 monolayers by visualization and mannitol permeability and compared dendrimer-mediated tight junction modulation to that of established permeation enhancers. [(14)C]-Mannitol permeability in the presence and absence of phospholipase C-dependent signaling pathway inhibitors was also examined and indicated that this pathway may mediate dendrimer-induced changes in permeability. RESULTS: Differences between G3.5 and G4 in tight junction protein staining and permeability with inhibitors were evident, suggesting divergent mechanisms were responsible for tight junction modulation. These dissimilarities are further intimated by the intracellular calcium release caused by G4 but not G3.5. Based on our results, it is apparent that the underlying mechanisms of dendrimer permeability are complex, and the complexities are likely a result of the density and sign of the surface charges of PAMAM dendrimers. CONCLUSIONS: The results of this study will have implications on the future use of PAMAM dendrimers for oral drug delivery.

Riboflavin-targeted polymer conjugates for breast tumor delivery.

PURPOSE: In breast cancer, a significant decrease in riboflavin (RF) serum levels and increase in RF carrier protein occurs, indicating a potential role of RF in disease progression. To evaluate RF's ability to serve as a targeting agent, mitomycin C (MMC)-conjugated N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesized and targeted to the RF internalization pathway in human breast cancer cells. METHODS: Competitive uptake studies were used to determine specificity of RF-targeted conjugates, and an MTT assay established the IC50 for the conjugates. Endocytic mechanisms were investigated by confocal microscopy. RESULTS: Studies revealed a high-affinity endocytic mechanism for RF-specific internalization of fluorescently-labeled conjugates in both MCF-7 and SKBR-3 cells, whereas folic acid-mediated endocytosis showed high specificity only in SKBR-3 cells. MMC internalization was significantly higher following nontargeted and RF-targeted MMC-conjugate administration compared to that of free MMC. Cytotoxic analysis illustrated potent IC50 values for RF-targeted MMC conjugates similar to free MMC. Maximum nuclear accumulation of MMC resulted from lysosomal release from RF-targeted and nontargeted MMC-conjugates following 6 h incubations, unlike that of free MMC seen within 10 min. CONCLUSION: Targeting polymer-MMC conjugates to the RF internalization pathway in breast cancer cells enabled an increase in MMC uptake and nuclear localization, resulting in potent cytotoxic activity.