O-P05 Gemcitabine and paclitaxel loaded microbubbles for the treatment of pancreatic cancer

Background Ultrasound targeted microbubble destruction (UTMD) has emerged as an effective strategy for the delivery of drug payloads to solid tumours. However, loading a single microbubble (MB) formulation with two drug payloads is challenging and often involves several manipulations post-MB preparation to enable attachment of drug payloads which can be cumbersome and generally results in low / inconsistent drug loading. Here we report a one-step synthesis of a gemcitabine-functionalised phospholipid and its subsequent incorporation into a stable MB formulation co-loaded with paclitaxel (PTX). The efficacy of the MB conjugate was determined in a Panc-1 spheroid model and ectopic BxPC-3 tumour model of pancreatic cancer. Methods Gemcitabine-modified phospholipid (Lipid-Gem MB) was prepared from 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) though a transphosphatidylation reaction using gemcitabine (Gem) as the acceptor alcohol. Lipid-Gem MB and Lipid-Gem-PTX MB were prepared from Lipid-Gem MB and/or PTX using a standard thin-film hydration technique followed by sonication in the presence of PFB gas. In vitro efficacy of Lipid-Gem MB and Lipid-Gem-PTX MB were determined in Panc-1 spheroids using an MTT assay. The in vivo effectiveness was determined in BxPC-3 tumour bearing mice following IV administration of either Lipid-Gem MB or Lipid-Gem-PTX MB plus ultrasound (US). Free Gem, free Gem + PTX and untreated mice were used for comparative purposes. Results Spheroids treated with Lipid-Gem MB +US or Lipid-Gem-PTX MB +US were significantly reduced relative to spheroids treated with US alone (p = 0.033 and p = 0.0031 respectively) or with the respective MB formulation alone (i.e. no US) (p = 0.0336 and p = 0.0037 respectively). Furthermore, cell viability for spheroids treated with Lipid-Gem-PTX MB +US was significantly reduced compared with spheroids treated with Lipid-Gem MB +US (p = 0.0077) (Figure a). Mice treated with Lipid-Gem MB +US or Lipid-Gem-PTX MB +US showed an average change in tumour volume of + 7 ± 7% and -10 ± 10 % respectively compared with +45 ±10% and +30 ± 10% for free gem and free gem + PTX respectively (Figure b). Conclusions A Gem-modified lipid was succesfully synthesised using a single step reaction and was subsequently incorporated into MBs containging PTX, eliminating the need for cumbersome drug conjgation methods. UTMD mediated treatment of Panc-1 spheroids and BxPC-3 tumours demonstrated the efficacy and tolerability of the formulations. Given that all components of this formulation are already clinicaly apporved, UTMD using Lipid-Gem-PTX MB offers a promising alternative to existing treatments

Polar Head Modified Phospholipids by Phospholipase D-Catalyzed Transformations of Natural Phosphatidylcholine for Targeted Applications: An Overview

This review describes the use of phospholipase D (PLD) to perform the transphosphatidylation of the most common natural phospholipid (PL), phosphatidylcholine (PC) to obtain polar head modified phospholipids with real targeted applications. The introduction of different polar heads with distinctive physical and chemical properties such as charge, polarity and dimensions allows the obtainment of very different PLs, which can be exploited in very diverse fields of application. Moreover, the inclusions of a bioactive moiety in the PL polar head constitutes a powerful tool for the stabilization and administration of active ingredients. The use of this biocatalytic approach allows the preparation of compounds which cannot be easily obtained by classical chemical methods, by using mild and green reaction conditions. PLD is a very versatile enzyme, able to catalyze both the hydrolysis of PC to choline and phosphatidic acid (PA), and the transphosphatidylation reaction in the presence of an appropriate alcohol. The yield of production of the desired product and the ratio with the collateral PA formation is highly dependent on parameters such as the nature and concentration of the alcohol and the enzymatic source. The application of PLD catalyzed transformations for the production of a great number of PLs with important uses in medical, nutraceutical and cosmetic sectors will be discussed in this work.

A real-time, click chemistry imaging approach reveals stimulus-specific subcellular locations of phospholipase D activity

The fidelity of signal transduction requires spatiotemporal control of the production of signaling agents. Phosphatidic acid (PA) is a pleiotropic lipid second messenger whose modes of action differ based on upstream stimulus, biosynthetic source, and site of production. How cells regulate the local production of PA to effect diverse signaling outcomes remains elusive. Unlike other second messengers, sites of PA biosynthesis cannot be accurately visualized with subcellular precision. Here, we describe a rapid, chemoenzymatic approach for imaging physiological PA production by phospholipase D (PLD) enzymes. Our method capitalizes on the remarkable discovery that bulky, hydrophilic trans-cyclooctene–containing primary alcohols can supplant water as the nucleophile in the PLD active site in a transphosphatidylation reaction of PLD’s lipid substrate, phosphatidylcholine. The resultant trans-cyclooctene–containing lipids are tagged with a fluorogenic tetrazine reagent via a no-rinse, inverse electron-demand Diels–Alder (IEDDA) reaction, enabling their immediate visualization by confocal microscopy in real time. Strikingly, the fluorescent reporter lipids initially produced at the plasma membrane (PM) induced by phorbol ester stimulation of PLD were rapidly internalized via apparent nonvesicular pathways rather than endocytosis, suggesting applications of this activity-based imaging toolset for probing mechanisms of intracellular phospholipid transport. By instead focusing on the initial 10 s of the IEDDA reaction, we precisely pinpointed the subcellular locations of endogenous PLD activity as elicited by physiological agonists of G protein-coupled receptor and receptor tyrosine kinase signaling. These tools hold promise to shed light on both lipid trafficking pathways and physiological and pathological effects of localized PLD signaling.

PLD pathway involved in carbachol-induced Cl− secretion: possible role of TNF-α

In a previous study, it was found that exposure to tumor necrosis factor-α (TNF-α) potentiated the electrophysiological response to carbachol in a time-dependent and cycloheximide-sensitive manner. It was deduced that the potentiation could be due to protein kinase C activity because of increased 1,2-diacylglycerol. It was also observed that propranolol could decrease the electrophysiological response to carbachol (Oprins JC, Meijer HP, and Groot JA. Am J Physiol Cell Physiol 278: C463–C472, 2000). The aim of the present study was to investigate whether the phospholipase D (PLD) pathway plays a role in the carbachol response and the potentiating effect of TNF-α. The transphosphatidylation reaction in the presence of the primary alcohol 1-butanol [leading to stable phosphatidylbutanol (Pbut) formation] was used to measure activity of PLD. The phosphatidic acid (PA) levels were also measured. Muscarinic stimulation resulted in an increased formation of Pbut and PA. TNF-α decreased levels of PA.

Role of Phospholipase D in Pasteurella haemolytica Leukotoxin-Induced Increase in Phospholipase A2 Activity in Bovine Neutrophils

ABSTRACT The effects of Pasteurella haemolytica leukotoxin (LKT) on the activity of phospholipase D (PLD) and the regulatory interaction between PLD and phospholipase A2 (PLA2) were investigated in assays using isolated bovine neutrophils labeled with tritiated phospholipid substrates of the two enzymes. Exposure of [3H]lysophosphatidylcholine-labeled neutrophils to LKT caused concentration- and time-dependent production of phosphatidic acid (PA), the product of PLD. LKT-induced generation of PA was dependent on extracellular calcium. Both production of PA and metabolism of [3H]-arachidonate ([3H]AA)-labeled phospholipids by PLA2 were inhibited when ethanol was used to promote the alternative PLD-mediated transphosphatidylation reaction, resulting in the production of phosphatidylethanol rather than PA. The role of PA in regulation of PLA2 activity was then confirmed by means of an add-back experiment, whereby addition of PA in the presence of ethanol restored PLA2-mediated release of radioactivity from neutrophil membranes. Considering the involvement of chemotactic phospholipase products in the pathogenesis of pneumonic pasteurellosis, development and use of anti-inflammatory agents that inhibit LKT-induced activation of PLD and PLA2 may improve therapeutic management of the disease.

Accumulation of phosphatidylalcohol in cultured cells: use of subcellular fractionation to investigate phospholipase D activity during signal transduction

Phosphatidylalcohol accumulates as a product of a phospholipase D (PLD)-catalysed transphosphatidylation reaction in cells incubated in the presence of a primary alcohol. In the presence of ethanol the phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated the accumulation of [3H]phosphatidylethanol (PEth) in HeLa cells prelabelled with [3H]palmitic acid. Radioactivity associated with PEth increased linearly during a 30 min incubation, indicating that a sustained activation of PLD is caused by PMA in these cells. This was accompanied by the membrane association of protein kinase C-alpha (PKC-alpha), the PKC isoform that recent studies indicate is involved in the activation of PLD. In similar experiments, the neuropeptide bradykinin stimulated an accumulation of PEth in 3T3 Li cells. The radioactivity associated with PEth increased to a maximal level at 30 s and plateaued after this time, suggesting that bradykinin induces only a transient activation of PLD in these cells. This is consistent with the effects of bradykinin on PKC-alpha, which underwent a rapid and transient association with cell membranes. The subcellular localization of PEth was examined using the technique of subcellular fractionation on Percoll density gradients to isolate organelle-enriched fractions from HeLa and 3T3 Li cells. An accumulation of [3H]PEth was measured in the plasma-membrane (PM)-enriched fractions of both HeLa and 3T3 Li cells after incubation with PMA and bradykinin respectively. This was accompanied by a time-dependent accumulation of [3H]PEth in the combined mitochondrial and endoplasmic reticulum (MER)-enriched fractions of both cell lines. PMA was also found to cause translocation of PKC-alpha to both the PM- and MER-enriched fractions in HeLa cells. However, bradykinin stimulated the translocation of PKC-alpha to the PM-enriched fractions only of 3T3 Li cells. The results show that PLD activation leads to the accumulation of PEth in both the PM and MER fractions. We therefore propose that either bradykinin activates a PM-associated PLD and the PLD reaction product is rapidly translocated to other membrane systems or it activates an MER-associated PLD by a mechanism that does not involve PKC-alpha.

Cholinergic activation of phospholipase D in lacrimal gland acini is independent of protein kinase C and calcium

To determine if rat lacrimal gland acini contain phospholipase D (PLD) activity, we took advantage of PLD's unique ability, in the presence of ethanol, to catalyze a transphosphatidylation reaction to produce phosphatidylethanol (PEth). Lacrimal gland acini were labeled for 3 h with [14C]stearic acid, preincubated for 20 min in the presence of 2% ethanol, and incubated for 20 min with or without agonists. Total cellular lipids were then extracted and analyzed by thin-layer chromatography, and the radioactivity was determined by liquid scintillation counting. Carbachol (1 mM), a cholinergic agonist, stimulated the production of both [14C]PEth and [14C]phosphatidic acid ([14C]PA) twofold. This effect was completely blocked by the muscarinic antagonist atropine (10 microM). [14C]PEth accumulation was also stimulated twofold by the active phorbol esters, 4 beta-phorbol 12-myristate 13-acetate and 4 beta-phorbol 12,13-dibutyrate at 1 microM. Ionomycin (1 microM), a Ca2+ ionophore, also stimulated the production of [14C]PEth twofold. In contrast to carbachol, neither phorbol esters nor ionomycin stimulated [14C]PA production. Neither [14C]PEth nor [14C]PA production was altered by epinephrine (1 mM), a nonselective adrenergic agonist, or phenylephrine (0.1 and 1 mM), a specific alpha 1-adrenergic agonist. We concluded that PLD activity, modulated by muscarinic receptors, protein kinase C, and Ca2+, but not by adrenergic receptors, is present in rat lacrimal gland acini. We also concluded that cholinergic activation of PLD appears to be independent of PKC and Ca2+.