Pharmacokinetics and pharmacodynamics of intravesical and intravenous TMX-101 and TMX-202 in a F344 rat model.

OBJECTIVES: To evaluate and compare the pharmacokinetic and pharmacodynamic properties of 2 investigational Toll-like receptor 7 agonists, TMX-101, and TMX-202 after intravenous and intravesical administration in a rat model. TLR-7 agonists are successfully used as topical treatment for various (pre)malignant skin lesions and are now under investigation as intravesical therapy for non-muscle-invasive bladder cancer. METHODS: Rats received an intravesical instillation with TMX-101, TMX-202, or vehicle. Additionally 2 groups of rats received an intravenous injection with TMX-101 or TMX-202. Blood sampling was performed at different time points, including pre-exposure and postexposure to determine the plasma concentrations of study drugs for pharmacokinetic and pharmacodynamic analyses and to determine the plasma concentrations of cytokines (IL-2, IL-6, and TNF-α). RESULTS: We observed no signs of toxicity after intravesical or intravenous administration. There was a limited dose dependent systemic uptake of TMX-101 and TMX-202 after intravesical administration. The systemic uptake of TMX-202 after intravesical instillation was 25 times lower compared to TMX-101. CONCLUSIONS: This in vivo study confirms the safety of intravesical TMX-101 and TMX-202 administration, with TMX-202 showing lower systemic uptake. TMX-202 has a larger molecule-mass compared to TMX-101, and it may therefore have a favorable safety profile when treating patients with non-muscle-invasive bladder cancer intravesically.

Delivery of TLR7 agonist to monocytes and dendritic cells by DCIR targeted liposomes induces robust production of anti-cancer cytokines.

Tumor immune escape is today recognized as an important cancer hallmark and is therefore a major focus area in cancer therapy. Monocytes and dendritic cells (DCs), which are central to creating a robust anti-tumor immune response and establishing an anti-tumorigenic microenvironment, are directly targeted by the tumor escape mechanisms to develop immunosuppressive phenotypes. Providing activated monocytes and DCs to the tumor tissue is therefore an attractive way to break the tumor-derived immune suppression and reinstate cancer immune surveillance. To activate monocytes and DCs with high efficiency, we have investigated an immunotherapeutic Toll-like receptor (TLR) agonist delivery system comprising liposomes targeted to the dendritic cell immunoreceptor (DCIR). We formulated the immune stimulating TLR7 agonist TMX-202 in the liposomes and examined the targeting of the liposomes as well as their immune activating potential in blood-derived monocytes, myeloid DCs (mDCs), and plasmacytoid DCs (pDCs). Monocytes and mDCs were targeted with high specificity over lymphocytes, and exhibited potent TLR7-specific secretion of the anti-cancer cytokines IL-12p70, IFN-α 2a, and IFN-γ. This delivery system could be a way to improve cancer treatment either in the form of a vaccine with co-formulated antigen or as an immunotherapeutic vector to boost monocyte and DC activity in combination with other treatment protocols such as chemotherapy or radiotherapy. STATEMENT OF SIGNIFICANCE: Cancer immunotherapy is a powerful new tool in the oncologist's therapeutic arsenal, with our increased knowledge of anti-tumor immunity providing many new targets for intervention. Monocytes and dendritic cells (DCs) are attractive targets for enhancing the anti-tumor immune response, but systemic delivery of immunomodulators has proven to be associated with a high risk of fatal adverse events due to the systemic activation of the immune system. We address this important obstacle by targeting the delivery of an immunomodulator, a Toll-like receptor agonist, to DCs and monocytes in the bloodstream. We thus focus the activation, potentially avoiding the above-mentioned adverse effects, and demonstrate greatly increased ability of the agonist to induce secretion of anti-cancer cytokines.

Monocyte targeting and activation by cationic liposomes formulated with a TLR7 agonist.

OBJECTIVES: Monocytes are one of the major phagocytic cells that patrol for invading pathogens, and upon activation, differentiate into macrophages or antigen-presenting dendritic cells (DCs) capable of migrating to lymph nodes eliciting an adaptive immune response. The key role in regulating adaptive immune responses has drawn attention to modulate monocyte responses therapeutically within cancer, inflammation and infectious diseases. We present a technology for targeting of monocytes and delivery of a toll-like receptor (TLR) agonist in fresh blood using liposomes with a positively charged surface chemistry. METHODS: Liposomes were extruded at 100 nm, incubated with fresh blood, followed by leukocyte analyses by FACS. Liposomes with and without the TLR7 agonist TMX-202 were incubated with fresh blood, and monocyte activation measured by cytokine secretion by ELISA and CD14 and DC-SIGN expression. RESULTS: The liposomes target monocytes specifically over lymphocytes and granulocytes in human whole blood, and show association with 75 - 95% of the monocytes after 1 h incubation. Formulations of TMX-202 in cationic liposomes were potent in targeting and activation of monocytes, with strong induction of IL-6 and IL-12p40, and differentiation into CD14(+) and DC-SIGN+ DCs. CONCLUSION: Our present liposomes selectively target monocytes in fresh blood, enabling delivery of TLR7 agonists to the intracellular TLR7 receptor, with subsequent monocyte activation and boost in secretion of proinflammatory cytokines. We envision this technology as a promising tool in future cancer immunotherapy.

A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects.

BACKGROUND AND PURPOSE: The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and ß is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. EXPERIMENTAL APPROACH: Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. KEY RESULTS: Of the new compounds, O-7460 exhibited the highest potency (IC50 = 690 nM) against the human recombinant DAGLα, and selectivity (IC50 > 10 µM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥ 10 µM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki > 10 µM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 µM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 mg·kg⁻¹, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. CONCLUSIONS AND IMPLICATIONS: O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.

Intracellular delivery of proteins with a new lipid-mediated delivery system.

There are many very effective methods to introduce transcriptionally active DNA into viable cells but approaches to deliver functional proteins are limited. We have developed a lipid-mediated delivery system that can deliver functional proteins or other bioactive molecules into living cells. This delivery system is composed of a new trifluoroacetylated lipopolyamine (TFA-DODAPL) and dioleoyl phosphatidylethanolamine (DOPE). This cationic formulation successfully delivered antibodies, dextran sulfates, phycobiliproteins, albumin, and enzymes (beta-galactosidase and proteases) into the cytoplasm of numerous adherent and suspension cells. Two systems were used to demonstrate that the proteins were delivered in a functionally active form. First, intracellular beta-galactosidase activity was clearly demonstrated within X-gal-stained cells after TFA-DODAPL:DOPE-mediated delivery of the enzyme. Second, the delivery system mediated delivery of several caspases (caspase 3, caspase 8, and granzyme B) into cultured cell lines and primary cells triggering apoptosis. Mechanistic studies showed that up to 100% of the protein mixed with the lipid formulation was captured into a lipid-protein complex, and up to 50% of the input protein associated with cells. This lipid-mediated transport system makes protein delivery into cultured cells as convenient, effective, and reliable as DNA transfection.

Targeting transgene expression for cystic fibrosis gene therapy.

We have developed an expression cassette for cystic fibrosis (CF) gene therapy using control elements from the human cytokeratin 18 gene (KRT18, also known as K18). KRT18 is naturally expressed in a spatial pattern similar to that of CFTR, the gene mutated in CF. We delivered a KRT18-driven lacZ plasmid complexed with cationic liposomes intravenously to mice and examined expression in various tissues. We found expression in nasal and bronchial epithelium, airway submucosal glands, gall bladder, and kidneys. Expression was low in pancreas and gut, and absent from liver and alveolar lung. This is consistent with the expression pattern reported for a K18lacZ transgenic mouse. Following delivery of a cytomegalovirus (CMV) major immediate-early promoter/enhancer-driven lacZ plasmid, we found expression in bronchi, submucosal glands, alveolar cells, liver, and kidney. We did not detect expression in nose, pancreas, gall bladder, or gut. Using fluorescently labeled plasmid delivered by means of liposomes, we identified the liver, alveolar lung, and kidneys as the major plasmid deposition sites. Our data demonstrate that a KRT18-driven expression vector delivered systemically can target gene expression to CF-affected tissues, despite an uneven distribution of plasmid DNA. A KRT18-based vector may be a useful alternative to viral promoter-based vectors in clinical gene therapy trials to treat CF.

Design, characterization and preclinical efficacy of a cationic lipid adjuvant for influenza split vaccine.

We prepared a series of cationic lipid vesicles comprising a cationic cholesterol derivative, DC-Chol with or without a neutral phospholipid, DOPC or DOPE. The vesicles were tested for their ability to bind and adjuvant split inactivated influenza vaccines. We found that DC-Chol-containing liposomes are capable to strongly bind influenza vaccine antigens upon simple mixing with the vaccine. The resulting formulations induced robust anti-influenza immune responses both after s.c. and i.n. administration in BALB/c mice while neutral Cholesterol/DOPC liposomes displayed virtually no stable antigen binding and no adjuvant effect. The parenteral adjuvant effect of DC-Chol on trivalent split influenza vaccines was then confirmed in outbred mice and monkeys. Among the most potent formulations tested, a simple mixture of the vaccine with a microfluidized dispersion of DC-Chol in an aqueous buffer is being considered for further development to produce an improved influenza vaccine.

Membrane perturbation and the mechanism of lipid-mediated transfer of DNA into cells.

Mixtures of cationic lipids and unsaturated phosphatidylethanolamine are used extensively for the intracellular delivery of plasmids and antisense oligodeoxynucleotides (ODN) in vitro. However, the mechanism by which cytoplasmic delivery of these large molecules is achieved remains unclear. The common hypothesis is that phosphatidylethanolamine promotes fusion of lipid/DNA particles with endosomal membranes, but this is inconsistent with several reports that have failed to correlate the fusogenic activity of a wide variety of lipid/DNA particles, measured by lipid mixing techniques, with their transfection activity. To address this issue further we have conducted a detailed analysis of the lipid mixing and DNA transfer activity of two, physically similar but functionally different, lipid/DNA particles composed of equimolar dioleyldimethylammonium chloride (DODAC) and dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC). In combination with DODAC both phospholipids form almost identical lipid/DNA particles, they are endocytosed by cells to the same extent and each undergoes equivalent lipid mixing with cell membranes after uptake. Despite this, DNA transfer is 10- to 100-fold more extensive for lipid/DNA particles containing DOPE. We conclude that lipid mixing between lipid-based delivery systems and endosomal membranes must occur for DNA transfer to occur. However, the potency of different lipid/DNA particles correlates better with the ability of the exogenous lipid to disrupt membrane integrity.

Design of fusogenic liposomes using a poly(ethylene glycol) derivative having amino groups.

As a novel fusogenic liposome, we designed liposomes modified with poly(glycidol) having beta-alanine residues, which is a poly(ethylene glycol) derivative with positively charged groups. The polymer-modified liposomes of egg yolk phosphatidylcholine (EYPC) and dioleoylphosphatidylethanolamine (DOPE) were prepared by reverse phase evaporation. Fusion of the polymer-modified liposomes with anionic liposomes consisting of phosphatidic acid and DOPE was investigated. Fusion ability of the polymer-modified liposomes increased with increasing amount of the polymer fixed on the liposome. Also, inclusion of DOPE was necessary for the generation of the fusion ability of the polymer-modified liposomes. CV1 cells treated with the polymer-modified DOPE/EYPC liposomes containing calcein displayed diffuse fluorescence, suggesting that calcein was introduced into the cytoplasm. In contrast, only punctual fluorescence was observed in the cells treated with the polymer-modified EYPC liposomes containing calcein, indicating that calcein remained in the endosome and/or lysosome. In addition, COS1 cells were transfected efficiently by treatment with the polymer-modified EYPC/DOPE liposomes containing pSV2cat plasmid, whereas the transfection was not induced by treatment with the polymer-modified EYPC liposomes. Close correlation between fusion ability of the polymer-modified liposomes and their ability to deliver their contents to the cytoplasm implies that membrane fusion plays an important role in the liposome-mediated cytoplasmic delivery.

Clinical aspects of intratumoral gene therapy.

One of the major obstacles to the development of gene therapy for cancer is our inability to deliver genes to all targets within the body. Thus, effective methodology does not exist to deliver a gene intravenously with the expectation that it will selectively localize within the target tumor, will not localize in other tissues and will be expressed efficiently. While one can take advantage of tissue-specific promoters to activate the gene only in a given target tissue, only a small fraction of the vector will be taken up in the target tissue and expressed. Consequently, since accessible local or regional tumor masses are a major problem in many cancers, there has been a strong emphasis on clinical trials in intratumoral and peritumoral gene delivery.

Global CNS gene transfer for a childhood neurogenetic enzyme deficiency: Canavan disease.

The neurogenetic prototypic disease on which we chose to test our gene therapy strategy is Canavan disease (CD). CD is an autosomal recessive leukodystrophy associated with spongiform degeneration of the brain. At present the disease is uniformly fatal in affected probands. CD is characterized by mutations in the aspartoacylase (ASPA) gene, resulting in loss of enzyme activity. In this review, recent evidence is summarized on the etiology and possible treatments for CD. In particular, we discuss two gene delivery systems representing recent advances in both viral and liposome technology: a novel cationic liposome-polymer-DNA (LPD) complex, DCChol/DOPE-protamine, as well as recombinant adeno-associated virus (AAV) vectors.