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The concept of triggered drug release offers a possibility to overcome the toxic side effects of chemotherapeutics in cancer treatment by reducing systemic exposure to the active drug. In the present work, the concept foresees the use of the extracellular enzyme MMP9 as an enzymatic trigger for drug release in the proximity of tumor cells.A paclitaxel-hemisuccinate-peptide conjugate as a building block for self-assembling nanoparticles was synthesized using standard conjugation approaches. The building block was purified via preparative HPLC and analyzed by LC-MS. Nanoparticles were formed using the nanoprecipitation method and characterized. For selection of a suitable in vitro model system, common bioanalytical methods were used to determine mRNA expression, enzyme amount, and activity of MMP9.The MMP9-labile prodrug was synthesized and characterized. Nanoparticles were formed out of MMP9-labile conjugate-building blocks. The nanoparticle's diameter averaged at around 120 nm and presented a spherical shape. LN-18 cells, a glioblastoma multiforme derived cell line, were chosen as an in vitro model based on findings in cancer tissue and cell line characterization. The prodrug showed cytotoxicity in LN-18 cells, which was reduced by addition of an MMP9 inhibitor.taken together, we confirmed increased MMP9 in several cancer tissues (cervical, esophageal, lung, and brain) compared to healthy tissue and showed the effectiveness of MMP9-labile prodrug in in vitro tests.

The organic anion transporting polypeptide (OATP) 2B1 is ubiquitously expressed and known to facilitate cellular entry. It is widely accepted that transport proteins play a pivotal role in pharmacokinetics. Consequently, testing for interaction with drug transporters became an important part in the assessment of new molecular entities in order to predict and prevent drug-drug interactions. Recently, competitive counterflow (CCF), an indirect method allowing the identification of substrates, was successfully applied to the organic cation transporter 2. It was the aim of this study to test whether CCF can be used to identify substrates of OATP2B1. A protocol for CCF experiments using estrone 3-sulfate (E1S) as the driven compound in expression-verified MDCKII-OATP2B1 cells was established. The protocol was tested using a substance library, which was prior screened for inhibition of OATP2B1-mediated transport accounting for both E1S-binding sites. In CCF experiments, all previously reported OATP2B1 substrates significantly reduced the amount of E1S in equilibrium, classifying them as substrates. In addition, we identified and verified novel substrates of OATP2B1, namely, astemizole and domperidone. Results of the CCF were complemented with cytotoxicity assays or cell-based reporter gene assays to validate the finding of etoposide and teniposide or hyperforin being substrates of OATP2B1, respectively. Our study indicates that the method of CCF can be used to identify substrates of OATP2B1, irrespective, whether interacting with binding site A or A and B, but is limited by solubility issues or the amount of transporter that is expressed in the used cellular system.

Drug uptake transporters are now increasingly recognized as clinically relevant determinants of variable drug responsiveness and unexpected drug-drug interactions. Emerging evidence strongly suggests members of the organic anion transporting polypeptide (OATP) family appear to be particularly important to the disposition of many drugs in clinical use today. Specifically, the liver-enriched OATP1B subfamily members OATP1B1 and OATP1B3 exhibit broad substrate specificity and the ability to transport drugs which are ligands for xenobiotic sensing nuclear receptors such as the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). Accordingly, OATP1B transporters may indirectly regulate expression of drug metabolism genes via modulation of the intracellular concentration of PXR and CAR ligands. Moreover, a number of functionally important single nucleotide polymorphisms (SNPs) in OATP1B transporters have been described. In this review, a brief summary of known SNPs in PXR and CAR will be followed by an in-depth outline of OATP1B1 and OATP1B3 transporters particularly in relation to the known SNPs in these OATPs and the interplay between OATP1B transporters with PXR and CAR, both in vitro and in vivo.