Leveraging immune memory against measles virus as an antitumor strategy in a preclinical model of aggressive squamous cell carcinoma.

Viral antigens are among the strongest elicitors of immune responses. A significant proportion of the human population already carries pre-existing immunity against several childhood viruses, which could potentially be leveraged to fight cancer. We sought to provide proof of concept in mouse models that a pre-existing measles virus (MeV) immunity can be redirected to inhibit tumor growth by directly forcing expression of cognate antigens in the tumor. To this end, we designed DNA vaccines against known MeV cytotoxic and helper T epitopes, and administered these intradermally to mice that were subsequently challenged with syngeneic squamous cancer cells engineered to either express the cognate antigens or not. Alternatively, established wild-type tumors in vaccinated animals were treated intratumorally with in vitro transcribed mRNA encoding the cognate epitopes. Vaccination generated MeV cytotoxic T lymphocyte (CTL) immunity in mice as demonstrated by enhanced interferon gamma production, antigen-specific T cell proliferation, and CTL-mediated specific killing of antigen-pulsed target cells. When challenged with syngeneic tumor cells engineered to express the cognate antigens, 77% of MeV-vaccinated mice rejected the tumor versus 21% in control cohorts. Antitumor responses were largely dependent on the presence of CD8+ cells. Significant protection was observed even when only 25% of the tumor bulk expressed cognate antigens. We therefore tested the strategy therapeutically, allowing tumors to develop in vaccinated mice before intratumoral injection with Viromer nanoparticles complexed with mRNA encoding the cognate antigens. Treatment significantly enhanced overall survival compared with controls, including complete tumor regression in 25% of mice. Our results indicate that redirecting pre-existing viral immunity to fight cancer is a viable alternative that could meaningfully complement current cancer immune therapies such as personalized cancer vaccines and checkpoint inhibitor blockade.

Viromers as carriers for mRNA-mediated expression of therapeutic molecules under inflammatory conditions.

Therapeutic mRNA delivery has been described for several treatment options, such as vaccination and cancer immunotherapy. However, mRNA delivery has to be accompanied by the development and testing of suitable carrier materials due to the instability of mRNAs in human body fluids. In the present study, we investigated the ability of recently developed Viromers to deliver mRNAs in a classical inflammatory setting. We tested mRNAs coding for active components of preclinical (7ND) and approved (sTNF-RII) biologics, in vitro and in vivo. 7ND is an established blocker of the CCR2 axis, whereas sTNF-RII is the active component of the approved drug Etanercept. Viromer/mRNA complexes were transfected into murine macrophages in vitro. Protein expression was analysed using Luciferase reporter expression and mainly identified in spleen, blood and bone marrow in vivo. 7ND-mRNA delivery led to efficient blockage of monocytes infiltration in thioglycolate-induced peritonitis in mice, underlining the ability of Viromers to deliver a therapeutic mRNA cargo without overt toxicity. Therefore, we propose Viromer-based mRNA delivery as a suitable option for the treatment of inflammatory disorders beyond infusion of biological molecules.

Development and antitumor activity of a BCL-2 targeted single-stranded DNA oligonucleotide.

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.

An ion switch regulates fusion of charged membranes.

Here we identify the recruitment of solvent ions to lipid membranes as the dominant regulator of lipid phase behavior. Our data demonstrate that binding of counterions to charged lipids promotes the formation of lamellar membranes, whereas their absence can induce fusion. The mechanism applies to anionic and cationic liposomes, as well as the recently introduced amphoteric liposomes. In the latter, an additional pH-dependent lipid salt formation between anionic and cationic lipids must occur, as indicated by the depletion of membrane-bound ions in a zone around pH 5. Amphoteric liposomes fuse under these conditions but form lamellar structures at both lower and higher pH values. The integration of these observations into the classic lipid shape theory yielded a quantitative link between lipid and solvent composition and the physical state of the lipid assembly. The key parameter of the new model, κ(pH), describes the membrane phase behavior of charged membranes in response to their ion loading in a quantitative way.

Fusion-relevant changes in lipid shape of hydrated cholesterol hemisuccinate induced by pH and counterion species.

Cholesterol hemisuccinate (CHEMS) is a protonable lipid that is frequently used for the construction of pH-responsive delivery systems. Such systems have a stable, lamellar phase at pH 7, but can form a fusogenic, hexagonal phase at pH 5. This behavior can be explained by binding or release of counterions from the solvent and the related variations of the effective size of the polar lipid part. Here, we use MD simulations to study the ion recruitment to neutral or anionic bilayers of CHEMS in water. For deprotonated (anionic) CHEMS, we observed an almost complete decoration of the bilayer with sodium, potassium, or argininium cations, which challenges the previous hypothesis that the stability of bilayers from anionic CHEMS results from the electrostatic repulsion between the charged head groups. Protonated (neutral) bilayers of CHEMS did not bind sodium or potassium, but did adsorb argininium cations. Whereas the headgroup of protonated CHEMS is bent and strongly tilted away from the bilayer normal, the headgroup of the deprotonated CHEMS is found to become outstretched and significantly less tilted arising from the adsorption of the counterions. The tilt reduction is most pronounced upon adsorption of arginine which also leads to an increase in the otherwise constant area per lipid. In general, the cation binding to the deprotonated CHEMS acts to increase the effective headgroup volume. This change in the lipid shape may be one possible fact explaining the hexagonal-lamellar phase transitions for CHEMS known from experiments.

Liposomal encapsulation enhances and prolongs the anti-inflammatory effects of water-soluble dexamethasone phosphate in experimental adjuvant arthritis.

INTRODUCTION: The objective of this study was to evaluate the efficacy of intravenous (i.v.) injection of liposomally encapsulated dexamethasone phosphate (DxM-P) in comparison to free DxM-P in rats with established adjuvant arthritis (AA). This study focused on polyethylene glycol (PEG)-free liposomes, to minimize known allergic reactions caused by neutral PEG-modified (PEG-ylated) liposomes. METHODS: Efficacy was assessed clinically and histologically using standard scores. Non-specific and specific immune parameters were monitored. Activation of peritoneal macrophages was analyzed via cytokine profiling. Pharmacokinetics/biodistribution of DxM in plasma, synovial membrane, spleen and liver were assessed via mass spectrometry. RESULTS: Liposomal DxM-P (3 × 1 mg/kg body weight; administered intravenously (i.v.) on Days 14, 15 and 16 of AA) suppressed established AA, including histological signs, erythrocyte sedimentation rate, white blood cell count, circulating anti-mycobacterial IgG, and production of interleukin-1beta (IL-1ß) and IL-6 by peritoneal macrophages. The suppression was strong and long-lasting. The clinical effects of liposomal DxM-P were dose-dependent for dosages between 0.01 and 1.0 mg/kg. Single administration of 1 mg/kg liposomal DxM-P and 3 × 1 mg/kg of free DxM-P showed comparable effects consisting of a partial and transient suppression. Moreover, the effects of medium-dose liposomal DxM-P (3 × 0.1 mg/kg) were equal (in the short term) or superior (in the long term) to those of high-dose free DxM-P (3 × 1 mg/kg), suggesting a potential dose reduction by a factor between 3 and 10 by liposomal encapsulation. For at least 48 hours after the last injection, the liposomal drug achieved significantly higher levels in plasma, synovial membrane, spleen and liver than the free drug. CONCLUSIONS: This new PEG-free formulation of macrophage-targeting liposomal DxM-P considerably reduces the dose and/or frequency required to treat AA, with a potential to enhance or prolong therapeutic efficacy and limit side-effects also in the therapy of rheumatoid arthritis. Depot and/or recirculation effects in plasma, inflamed joint, liver, and spleen may contribute to this superiority of liposomally encapsulated DxM-P.

Separating therapeutic efficacy from glucocorticoid side-effects in rodent arthritis using novel, liposomal delivery of dexamethasone phosphate: long-term suppression of arthritis facilitates interval treatment.

INTRODUCTION: Glucocorticoids have extensively been used in the treatment of rheumatoid arthritis and other inflammatory diseases. However, their side-effects remain the major limitation in clinical use and an improved therapeutic index is needed. METHODS: Therapeutic efficacy and persistence of free and liposomal dexamethasone phosphate (DXM-P) were determined in mouse collagen-induced arthritis. For regimens with equal therapeutic benefit, the side-effect profiles were analysed over time with respect to collagen breakdown, suppression of the hypothalamus-pituitary-adrenal (HPA) axis, changes in blood glucose levels and the haematological profile. In addition, the presence of drug was monitored in plasma. RESULTS: Liposomal DXM-P, but not free drug, resulted in a persistent anti-inflammatory effect. Comparable clinical benefit was achieved with a single administration of 4 mg/kg liposomal DXM-P or daily administrations of 1.6 mg/kg free drug for at least 7 days. For the liposomal form, but not for the free form, we observed a limitation of the suppression of the HPA axis in time and an absence of the drug-induced gluconeogenesis. CONCLUSIONS: Liposomal DXM-P, but not free DXM-P, achieves therapeutic persistence in mouse collagen-induced arthritis, which results in drug-free periods of therapeutic benefit. The physical absence of drug after day 2 is associated with a reduction of the typical glucocorticoid side-effects profile. Liposomal DXM-P thereby has an improved therapeutic window.

Amphoteric liposomes enable systemic antigen-presenting cell-directed delivery of CD40 antisense and are therapeutically effective in experimental arthritis.

OBJECTIVE: Mediation of RNA interference by oligonucleotides constitutes a powerful approach for the silencing of genes involved in the pathogenesis of inflammatory disease, but in vivo application of this technique requires effective delivery to immune cells and/or sites of inflammation. The aim of the present study was to develop a new carrier system to mediate systemic administration of oligonucleotides to rheumatoid arthritis (RA) joints, and to develop an antisense oligonucleotide (ASO)-based approach to interfere with CD40-CD154 interactions in an experimental model of RA. METHODS: A novel liposomal carrier with amphoteric properties, termed Nov038, was developed and assessed for its ability to systemically deliver an ASO directed against CD40 (CD40-ASO). Male DBA/1 mice with collagen-induced arthritis were treated with Nov038-encapsulated CD40-ASO, and the effects of treatment on various parameters of disease activity, including clinical score, paw swelling, lymph node responses, and inflammatory cytokine production in the joints, were assessed. RESULTS: Nov038 was well tolerated, devoid of immune-stimulatory effects, and efficacious in mediating systemic oligonucleotide delivery to sites of inflammation. In mice with collagen-induced arthritis, Nov038 enabled the therapeutic administration of CD40-ASO and improved established disease, while unassisted CD40-ASO was ineffective, and anti-tumor necrosis factor alpha (anti-TNFalpha) treatment was less effective in this model. Nov038/CD40-ASO efficacy was attributed to its tropism for monocyte/macrophages and myeloid dendritic cells (DCs), resulting in rapid down-regulation of CD40, inhibition of DC antigen presentation, and reduction in collagen-specific T cell responses, as well as decreased levels of TNFalpha, interleukin-6 (IL-6), and IL-17 in arthritic joints. CONCLUSION: Amphoteric liposomes represent a novel carrier concept for systemic and antigen-presenting cell-targeted oligonucleotide delivery with clinical applicability and numerous potential applications, including target validation in vivo and inflammatory disease therapeutics. Moreover, Nov038/CD40-ASO constitutes a potent alternative to monoclonal antibody-based approaches for interfering with CD40-CD40L interactions.

Innovative liposomes as a transfollicular drug delivery system: penetration into porcine hair follicles.

Liposomes had been widely used for drug delivery in the past. In this study, five different liposomes were used as a follicular delivery system in pig ear skin. The liposomes mainly differed in their sphere diameter, lipid composition, and surface charge. A novel class of liposomes being amphoteric in their charge behavior are compared to established anionic and cationic liposomes. Two different fluorescent dyes, hydrophilic carboxyfluoresceine or lipophilic curcumin, were enclosed in the liposomes and used as model drugs. The fluorescent dyes were also applied in a standard formulation for reference. The penetration depth of the dyes was measured by laser scanning microscopy in histological sections. One hour, 3, 5, and 7 days after application, biopsies were taken and the penetration depth into the hair follicle was measured in longitudinal sections. The liposomes showed a higher penetration depth compared to the standard formulation. The relative penetration depth of the dyes, applied in the standard formulation, averaged 30% of the full follicle length during the whole observation period, whereas the liposomal formulations penetrated considerably deeper into the hair follicles. Amphoteric and cationic liposomes reached an average relative penetration depth of approximately 70% of the full hair follicle length.

9th International drug delivery technologies & deal-making summit.

SRI's 9th International drug delivery summit analysed the current position of drug delivery technologies within the pharmaceutical industry. The classical position of drug delivery as a tool in life cycle management is currently expanding into the primary formulations or even into enabling positions. Using drug delivery as a platform for drug development or redevelopment was recognised as a trend in the industry which is supported by the rapidly growing portion of drug delivery enhanced products already sold as well as by industry initiatives supporting future growth of the sector.

In vitro folding, functional characterization, and disulfide pattern of the extracellular domain of human GLP-1 receptor.

The N-terminal, extracellular domain of the receptor for glucagon-like peptide 1 (GLP-1 receptor) was expressed at a high level in E. coli and isolated as inclusion bodies. Renaturation with concomitant disulfide bond formation was achieved from guanidinium-solubilized material. A soluble and active fraction of the protein was isolated by ion exchange chromatography and gel filtration. Complex formation with GLP-1 was shown by cross-linking experiments, surface plasmon resonance measurements, and isothermal titration calorimetry. The existence of disulfide bridges in the N-terminal receptor fragment was proven after digestion of the protein with pepsin. Further analysis revealed a disulfide-binding pattern with links between cysteines 46 and 71, 62 and 104, and between 85 and 126.