James W. Paxton

Abstract Purposes To develop pH-sensitive liposomes (PSL) containing a high content of gemcitabine; and to investigate whether drug loading (DL) would alter the in vitro and pharmacokinetic properties. Methods PSL with a high DL were obtained using a modified small-volume incubation method. The DL effects on drug release rate and in vitro cytotoxicity of PSL were evaluated using MIA PaCa-2 pancreatic cancer cells and their pharmacokinetics investigated in rats. Results The highest DL of 4.5 ± 0.1% was achieved for gemcitabine in PSL with 145 ± 5 nm diameter. DL did not alter the in vitro release rate from PSL. The IC₅₀ (48 h) of PSL (DL 0.5 and 4.5%) and non pH-sensitive liposomes (NPSL, DL 4.2%) were 1.1 ± 0.1, 0.7 ± 0.1 and 37.0 ± 7.5 μM, respectively. The PSL resulted in a 4.2-fold increase in its elimination half-life (6.2 h) compared to gemcitabine solution (1.4 h) in rats. No significant difference in pharmacokinetic parameters was observed between the two PSL (DL 0.5 and 4.5%). Conclusion The PSL offered advantages over NPSL in restoring the sensitivity of pancreatic cancer cells to gemcitabine without requiring a high DL. DL in the PSL did not alter release rate, cytotoxicity or their long-circulating properties. ᅟ

The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r² = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum. Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.

The in vitro and in vivo properties of PEGylated pH-sensitive liposomes (PSL) prepared by pre- and post-insertion techniques were investigated. A pre-insertion or post-insertion technique was used for PSL PEGylation. For the first time, confocal laser scanning microscopy coupled with a modified calcein self-quench assay was applied to evaluate the endosome escape capability. PSL cellular uptake was evaluated using macrophages and the cytotoxicity using a gemcitabine (model drug)-resistant MIA PaCa-2 cells. The pharmacokinetics of PSL encapsulated gemcitabine was investigated in rats. PEGylation reduced the pH-sensitivity in a concentration-dependent manner (0.5–5% mol). Both PEGylation methods reduced the uptake of PSL by macrophages by over 60%. Cytotoxicity was ranked in the order: post-inserted PSL ≥ pre-inserted PSL > non-PSL > gemcitabine solution, consistent with the confocal microscopic observation and pH-sensitivity. Both pre and post-inserted PSL resulted in significant reductions (p < 0.05) in plasma clearance (58.6 and 38.4 ml/h/kg), increases in the area-under-the-concentration-time curve (56.9 and 87.1 μM· h) and half-life (6.1 and 6.2 h) compared to gemcitabine solution (152.9 ml/h/kg, 22.2 μM· h and 1.4 h). PEGylation by post-insertion offers advantages over pre-insertion to obtain PSL with enhanced pH-sensitivity, more effective intra-cytoplasmic delivery, and a superior pharmacokinetics.

Scope: Hops (Humulus lupulus L.) produce unique prenylflavonoids that exhibit interesting bioactivities. This study investigates the interactions between selected prenylflavonoids and breast cancer resistance protein (BCRP/ABCG2), an efflux transporter important for xenobiotic bioavailability and multidrug resistance (MDR).Methods and results: ABCG2‐inhibitory activity of xanthohumol (XN), isoxanthohumol (IX), 6‐prenylnaringenin (6‐PN), 8‐prenylnaringenin (8‐PN), and 6,8‐diprenylnarigenin (6,8‐diPN) was evaluated using mitoxantrone accumulation and vesicular transport assays. XN, IX, and 8‐PN were tested for a substrate‐type relationship with ABCG2 using ATPase and bidirectional transport assays. The prenylflavonoids exhibited significant ABCG2‐inhibitory activities in mitoxantrone accumulation and vesicular transport assays. In the ATPase assay, XN, IX, and 8‐PN inhibited baseline and sulfasalazine‐stimulated ATPase activities with IC₅₀ of 2.16–27.0 μM. IX and 8‐PNalso displayed bell‐shaped activation curves in Ko143‐suppressed membranes, indicating a substrate‐type relationship. For IX, efflux ratios of 1.25 ± 0.21 and 9.18 ± 0.56 were observed in wild type and ABCG2‐overexpressing MDCKII cell monolayers, respectively. The latter was reduced to 1.25 ± 0.15 in the presence of the ABCG2‐specific inhibitor Ko143, demonstrating an ABCG2‐mediated efflux of IX. Additionally, evidence was shown for the involvement of ABCG2 in the efflux of 8‐PN and/or its sulfate conjugate.Conclusion: Prenylflavonoids are potent inhibitors of ABCG2 and therefore implicated in ABCG2‐mediated food/herb‐drug interactions and MDR. ABCG2‐mediated efflux of prenylflavonoids may represent one mechanism that regulates prenylflavonoid bioavailability.

SN 28049 is a new DNA-binding topoisomerase II poison identified by its curative activity against the murine colon 38 carcinoma. Previous studies showed activity to be associated with selective drug accumulation and retention in tumour tissue. Retention varied widely among different tumours and was related to antitumour activity. We determined whether differences in the uptake and retention of SN 28049 could be observed in vitro. The Co38P and LLTC lines were derived from the murine colon 38 carcinoma and Lewis lung carcinoma (3LL), respectively. The NZM4, NZM10 and NZM52 human melanoma lines, as well as the CCRF/CEM, CEM/VLB100 and CEM/E1000 human leukaemia lines were also utilised. Cell-associated drug was measured by liquid chromatography–mass spectrometry, laser-scanning confocal microscopy and fluorescence microscopy. Data for SN 28049 were compared for four SN 28049 analogues, for the structurally related drug N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (DACA) and for doxorubicin. Cellular uptake of SN 28049 was rapid and associated with increased fluorescence in cytoplasmic vesicles or bodies. SN 28049 uptake after an incubation time of 1 h varied widely with different cell lines (2–98 pmol/10⁶ cells) and did not correlate with growth inhibitory concentrations (IC₅₀ values), which also varied widely (1.2–19 nM). Changes in the length of the N-linked side chain of SN 28049 had large effects on drug uptake by Co38P cells. SN 28049 uptake by CCRF/CEM cells was only slightly affected by the expression of P-glycoprotein (CEM/VLB100) or MRP1 protein (CEM/E1000). As measured by cytoplasmic fluorescence, SN 28049 was taken up rapidly and retained strongly by Co38P cells, DACA was taken up rapidly and retained poorly, and doxorubicin was taken up slowly and retained moderately. The results suggest that SN 28049 is actively transported into cytoplasmic vesicles. While vesicle-associated drug is not important for intrinsic cytotoxicity, it may play a key role as a “slow release” form that modifies pharmacokinetics in multicellular structures such as tumours.

The objective of this study was to develop high-content gemcitabine PEGylated liposomes to reverse gemcitabine resistance in pancreatic tumour cells. The mechanism of drug loading into liposomes was also investigated. To increase the drug entrapment efficiency (EE) and drug loading (DL), a novel passive loading approach named Small Volume Incubation method (SVI) was developed and compared to the reverse phase evaporation (REV) and remote loading methods. The in vitro cytotoxicity was evaluated using MIA PaCa-2 and Panc-1 cell lines. The EE for remote loading was 12.3 ± 0.3%, much lower than expected and a burst release was observed with the resultant liposomes. Using the optimized SVI method, increased EE (37 ± 1%) and DL (4%, w/w) were obtained. The liposomes (200 ± 5 nm) showed minimal drug leakage, good stability, and significant improvement in cytotoxicity to the gemcitabine-resistant pancreatic cancer cell lines. Remote loading was not suitable for loading gemcitabine into liposomes. pK_a > 4.6 for basic drugs and intra-liposomal precipitation of loaded compounds were suggested as an additional requirement to the current criteria for remote loading using ammonium sulphate gradient (pK_a < 11). High DL is essential for liposomes to reverse gemcitabine resistance in pancreatic cell lines.