Delivery of Therapeutic RNA to the Bone Marrow in Multiple Myeloma Using CD38-Targeted Lipid Nanoparticles.

Multiple myeloma (MM) is a cancer of differentiated plasma cells that occurs in the bone marrow (BM). Despite the recent advancements in drug development, most patients with MM eventually relapse and the disease remains incurable. RNA therapy delivered via lipid nanoparticles (LNPs) has the potential to be a promising cancer treatment, however, its clinical implementation is limited due to inefficient delivery to non-hepatic tissues. Here, targeted (t)LNPs designed for delivery of RNA payload to MM cells are presented. The tLNPs consist of a novel ionizable lipid and are coated with an anti-CD38 antibody (αCD38-tLNPs). To explore their therapeutic potential, it is demonstrated that LNPs encapsulating small interference RNA (siRNA) against cytoskeleton-associated protein 5 (CKAP5) lead to a ≈90% decrease in cell viability of MM cells in vitro. Next, a new xenograft MM mouse model is employed, which clinically resembles the human disease and demonstrates efficient homing of MM cells to the BM. Specific delivery of αCD38-tLNPs to BM-residing and disseminated MM cells and the improvement in therapeutic outcome of MM-bearing mice treated with αCD38-tLNPs-siRNA-CKAP5 are shown. These results underscore the potential of RNA therapeutics for treatment of MM and the importance of developing effective targeted delivery systems and reliable preclinical models.

Therapeutic gene silencing of CKAP5 leads to lethality in genetically unstable cancer cells.

The potential of microtubule-associated protein targets for cancer therapeutics remains largely unexplored due to the lack of target-specific agents. Here, we explored the therapeutic potential of targeting cytoskeleton-associated protein 5 (CKAP5), an important microtubule-associated protein, with CKAP5-targeting siRNAs encapsulated in lipid nanoparticles (LNPs). Our screening of 20 solid cancer cell lines demonstrated selective vulnerability of genetically unstable cancer cell lines in response to CKAP5 silencing. We identified a highly responsive chemo-resistant ovarian cancer cell line, in which CKAP5 silencing led to significant loss in EB1 dynamics during mitosis. Last, we demonstrated the therapeutic potential in an in vivo ovarian cancer model, showing 80% survival rate of siCKAP5 LNPs-treated animals. Together, our results highlight the importance of CKAP5 as a therapeutic target for genetically unstable ovarian cancer and warrants further investigation into its mechanistic aspects.

Dual-Targeted Lipid Nanotherapeutic Boost for Chemo-Immunotherapy of Cancer.

Chemo-immunotherapy is a combination of "standard-of-care" chemotherapy with immunotherapy and it is considered the most advanced therapeutic modality for various types of cancers. However, many cancer patients still poorly respond to current regimen of chemo-immunotherapy and suggest nanotherapeutics as a boosting agent. Recently, heme oxygenase-1 (HO1) is shown to act as an immunotherapeutic molecule in tumor myeloid cells, in addition to general chemoresistance function in cancer cells suggesting that HO1-targeted therapeutics can become a novel, optimal strategy for boosting chemo-immunotherapy in the clinic. Currently the available HO1-inhibitors demonstrate serious adverse effects in clinical use. Herein, tumor myeloid cell- and cancer cell-dual targeted HO1-inhibiting lipid nanotherapeutic boost (T-iLNTB) is developed using RNAi-loaded lipid nanoparticles. T-iLNTB-mediated HO1-inhibition sensitizes cancer cells to "standard-of-care" chemotherapeutics by increasing immunogenic cell death, and directly reprograms tumor myeloid cells with distinguished phenotype. Furthermore, tumor myeloid cell reprogramming by T-iLNTB induces CD8+ cytotoxic T cell recruitment, which drives "Cold-to-Hot" transition and correlates with improved responsiveness to immune checkpoint inhibitor in combination therapy. Finally, ex vivo study proves that HO1-inhibition directly affects tumor macrophage differentiation. This study demonstrates the potential of T-iLNTB as a novel therapeutic modality for boosting chemo-immunotherapy.

Therapeutic inhibitory RNA in head and neck cancer via functional targeted lipid nanoparticles.

Currently there are no specific therapies addressing the distinctive biology of human papillomavirus (HPV)-induced cancer approved for clinical use. Short interfering RNA (siRNA) has much potential for therapeutic manipulation of HPV E6/E7 oncoproteins. Lipid-based nanoparticles (LNPs) can be utilized for systemic transportation and delivery of siRNA at target site. We recently developed a recombinant protein linker that enables uniform conjugation of targeting antibodies to the LNPs. Herein, we demonstrate the therapeutic efficacy of anti-E6/E7 siRNA delivered via targeted LNPs (tLNPs) in a xenograft HPV-positive tumor model. We show that anti-epidermal growth factor receptor (EGFR) antibodies, anchored to the LNPs as targeting moieties, facilitate cargo delivery but also mediate anti-tumor activity. Treatment with siE6 via tLNPs resulted in 50% greater reduction of tumor volume compared to treatment with siControl encapsulated in isoLNPs (coated with isotype control antibodies). We demonstrate superior suppression of HPV oncogenes and higher induction of apoptosis by the tLNPs both in vitro and in vivo. Altogether, the coupling of inhibitory siE6 with anti-EGFR antibodies, that further elicited anti-tumor effects, successfully restricted tumor progression. This system that combines potent siRNA and therapeutically functional tLNPs can be modulated against various cancer models.

Therapeutic Gene Silencing Using Targeted Lipid Nanoparticles in Metastatic Ovarian Cancer.

Ovarian cancer is an aggressive tumor owing to its ability to metastasize from stage II onward. Herein, lipid nanoparticles (LNPs) that encapsulate combination of small interfering RNAs (siRNAs), polo-like kinase-1 (PLK1), and eukaryotic translation-initiation factor 3c (eIF3c), to target different cellular pathways essential for ovarian cancer progression are generated. The LNPs are further modified with hyaluronan (tNPs) to target cluster of differentiation 44 (CD44) expressing cells. Interestingly, hyaluronan-coated LNPs (tNPs) prolong functional activity and reduce growth kinetics of spheroids in in vitro assay as compared to uncoated LNPs (uNPs) due to ≈1500-fold higher expression of CD44. Treatment of 2D and 3D cultured ovarian cancer cells with LNPs encapsulating both siRNAs result in 85% cell death and robust target gene silencing. In advanced orthotopic ovarian cancer model, intraperitoneal administration of LNPs demonstrates CD44 specific tumor targeting of tNPs compared to uNPs and robust gene silencing in tissues involved in ovarian cancer pathophysiology. At very low siRNA dose, enhanced overall survival of 60% for tNPs treated mice is observed compared to 10% and 20% for single siRNA-, eIF3c-tNP, and PLK1-tNP treatment groups, respectively. Overall, LNPs represent promising platform in the treatment of advanced ovarian cancer by improving median- and overall-survival.

An ovarian spheroid based tumor model that represents vascularized tumors and enables the investigation of nanomedicine therapeutics.

The failure of cancer therapies in clinical settings is often attributed to the lack of a relevant tumor model and pathological heterogeneity across tumor types in the clinic. The objective of this study was to develop a robust in vivo tumor model that better represents clinical tumors for the evaluation of anti-cancer therapies. We successfully developed a simple mouse tumor model based on 3D cell culture by injecting a single spheroid and compared it to a tumor model routinely used by injecting cell suspension from 2D monolayer cell culture. We further characterized both tumors with cellular markers for the presence of myofibroblasts, pericytes, endothelial cells and extracellular matrix to understand the role of the tumor microenvironment. We further investigated the effect of chemotherapy (doxorubicin), nanomedicine (Doxil®), biological therapy (Avastin®) and their combination. Our results showed that the substantial blood vasculature in the 3D spheroid model enhances the delivery of Doxil® by 2.5-fold as compared to the 2D model. Taken together, our data suggest that the 3D tumors created by simple subcutaneous spheroid injection represents a robust and more vascular murine tumor model which is a clinically relevant platform to test anti-cancer therapy in solid tumors.

Leukocyte-specific siRNA delivery revealing IRF8 as a potential anti-inflammatory target.

Interferon regulatory factor 8 (IRF8) protein plays a critical role in the differentiation, polarization, and activation of mononuclear phagocytic cells. In light of previous studies, we explored the therapeutic potential of IRF8 inhibition as immunomodulatory therapy for inflammatory bowel disease (IBD). To this end, we utilized siRNA-loaded lipid-based nanoparticles (siLNPs) and demonstrated a ∼90% reduction of IRF8 mRNA levels in vitro (PV < 0.0001), alongside a notable reduction in IRF8 protein. Moreover, silencing IRF8 ex vivo in splenocytes lead to a profound downregulation of IRF8 protein, followed by an immunomodulatory effect, as represented by a decrease in the secretion of TNFα, IL6 and IL12/IL23 (IL12p40) proinflammatory cytokines (PV = 0.0045, 0.0330, <0.0001, respectively). In order to silence IRF8 in vivo, selectively in inflammatory leukocytes, we used siLNPs that were coated with anti-Ly6C antibodies via our recently published ASSET targeting approach. Through this strategy, we have demonstrated a selective binding of the targeted-LNPs (T-LNPs) to Ly6C + inflammatory leukocytes. Finally, an immunomodulatory effect was demonstrated in vivo in an IBD mouse model with a profound decrease of TNFα, IL6, IL12/IL23, and IL1ß pro-inflammatory cytokines (n = 5, PV < 0.0001, <0.0001, <0.0001, 0.02, respectively) and an improvement of colon-morphology as assessed by colon-length measurements and colonoscopy (PV < 0.0001). Overall, using antibody-targeted siLNPs, we showed a notable reduction of IRF8 mRNA and protein and demonstrated a targeted immunomodulation therapeutic effect ex vivo and in vivo, in the DSS colitis model. We claim that a selective silencing of IRF8 in inflammatory leukocytes (such as Ly6C+) may serve as a therapeutic approach for treating inflammatory disorders.

A modular platform for targeted RNAi therapeutics.

Previous studies have identified relevant genes and signalling pathways that are hampered in human disorders as potential candidates for therapeutics. Developing nucleic acid-based tools to manipulate gene expression, such as short interfering RNAs1-3 (siRNAs), opens up opportunities for personalized medicine. Yet, although major progress has been made in developing siRNA targeted delivery carriers, mainly by utilizing monoclonal antibodies (mAbs) for targeting4-8, their clinical translation has not occurred. This is in part because of the massive development and production requirements and the high batch-to-batch variability of current technologies, which rely on chemical conjugation. Here we present a self-assembled modular platform that enables the construction of a theoretically unlimited repertoire of siRNA targeted carriers. The self-assembly of the platform is based on a membrane-anchored lipoprotein that is incorporated into siRNA-loaded lipid nanoparticles that interact with the antibody crystallizable fragment (Fc) domain. We show that a simple switch of eight different mAbs redirects the specific uptake of siRNAs by diverse leukocyte subsets in vivo. The therapeutic potential of the platform is demonstrated in an inflammatory bowel disease model by targeting colon macrophages to reduce inflammatory symptoms, and in a Mantle Cell Lymphoma xenograft model by targeting cancer cells to induce cell death and improve survival. This modular delivery platform represents a milestone in the development of precision medicine.

Comprehensive and Systematic Analysis of the Immunocompatibility of Polyelectrolyte Capsules.

The immunocompability of polyelectrolyte capsules synthesized by layer-by-layer deposition has been investigated. Capsules of different architecture and composed of either non-degradable or biodegradable polymers, with either positively or negatively charged outer surface, and with micrometer size, have been used, and the capsule uptake by different cell lines has been studied and quantified. Immunocompatibility studies were performed with peripheral blood mononuclear cells (PBMCs). Data demonstrate that incubation with capsules, at concentrations relevant for practical applications, did not result in a reduced viability of cells, as it did not show an increased apoptosis. Presence of capsules also did not result in an increased expression of TNF-α, as detected with antibody staining, as well as at mRNA level. It also did not result in increased expression of IL-6, as detected at mRNA level. These results indicate that the polyelectrolyte capsules used in this study are immunocompatible.

Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies.

Despite progress in systemic small interfering RNA (siRNA) delivery to the liver and to solid tumors, systemic siRNA delivery to leukocytes remains challenging. The ability to silence gene expression in leukocytes has great potential for identifying drug targets and for RNAi-based therapy for leukocyte diseases. However, both normal and malignant leukocytes are among the most difficult targets for siRNA delivery as they are resistant to conventional transfection reagents and are dispersed in the body. We used mantle cell lymphoma (MCL) as a prototypic blood cancer for validating a novel siRNA delivery strategy. MCL is an aggressive B-cell lymphoma that overexpresses cyclin D1 with relatively poor prognosis. Down-regulation of cyclin D1 using RNA interference (RNAi) is a potential therapeutic approach to this malignancy. Here, we designed lipid-based nanoparticles (LNPs) coated with anti-CD38 monoclonal antibodies that are specifically taken up by human MCL cells in the bone marrow of xenografted mice. When loaded with siRNAs against cyclin D1, CD38-targeted LNPs induced gene silencing in MCL cells and prolonged survival of tumor-bearing mice with no observed adverse effects. These results highlight the therapeutic potential of cyclin D1 therapy in MCL and present a novel RNAi delivery system that opens new therapeutic opportunities for treating MCL and other B-cell malignancies.

Systemic Gene Silencing in Primary T Lymphocytes Using Targeted Lipid Nanoparticles.

Modulating T cell function by down-regulating specific genes using RNA interference (RNAi) holds tremendous potential in advancing targeted therapies in many immune-related disorders including cancer, inflammation, autoimmunity, and viral infections. Hematopoietic cells, in general, and primary T lymphocytes, in particular, are notoriously hard to transfect with small interfering RNAs (siRNAs). Herein, we describe a novel strategy to specifically deliver siRNAs to murine CD4(+) T cells using targeted lipid nanoparticles (tLNPs). To increase the efficacy of siRNA delivery, these tLNPs have been formulated with several lipids designed to improve the stability and efficacy of siRNA delivery. The tLNPs were surface-functionalized with anti-CD4 monoclonal antibody to permit delivery of the siRNAs specifically to CD4(+) T lymphocytes. Ex vivo, tLNPs demonstrated specificity by targeting only primary CD4(+) T lymphocytes and no other cell types. Systemic intravenous administration of these particles led to efficient binding and uptake into CD4(+) T lymphocytes in several anatomical sites including the spleen, inguinal lymph nodes, blood, and the bone marrow. Silencing by tLNPs occurs in a subset of circulating and resting CD4(+) T lymphocytes. Interestingly, we show that tLNP internalization and not endosome escape is a fundamental event that takes place as early as 1 h after systemic administration and determines tLNPs' efficacy. Taken together, these results suggest that tLNPs may open new avenues for the manipulation of T cell functionality and may help to establish RNAi as a therapeutic modality in leukocyte-associated diseases.

Localized RNAi therapeutics of chemoresistant grade IV glioma using hyaluronan-grafted lipid-based nanoparticles.

Glioblastoma multiforme (GBM) is one of the most infiltrating, aggressive, and poorly treated brain tumors. Progress in genomics and proteomics has paved the way for identifying potential therapeutic targets for treating GBM, yet the vast majority of these leading drug candidates for the treatment of GBM are ineffective, mainly due to restricted passages across the blood-brain barrier. Nanoparticles have been emerged as a promising platform to treat different types of tumors due to their ability to transport drugs to target sites while minimizing adverse effects. Herein, we devised a localized strategy to deliver RNA interference (RNAi) directly to the GBM site using hyaluronan (HA)-grafted lipid-based nanoparticles (LNPs). These LNPs having an ionized lipid were previously shown to be highly effective in delivering small interfering RNAs (siRNAs) into various cell types. LNP's surface was functionalized with hyaluronan (HA), a naturally occurring glycosaminoglycan that specifically binds the CD44 receptor expressed on GBM cells. We found that HA-LNPs can successfully bind to GBM cell lines and primary neurosphers of GBM patients. HA-LNPs loaded with Polo-Like Kinase 1 (PLK1) siRNAs (siPLK1) dramatically reduced the expression of PLK1 mRNA and cumulated in cell death even under shear flow that simulate the flow of the cerebrospinal fluid compared with control groups. Next, a human GBM U87MG orthotopic xenograft model was established by intracranial injection of U87MG cells into nude mice. Convection of Cy3-siRNA entrapped in HA-LNPs was performed, and specific Cy3 uptake was observed in U87MG cells. Moreover, convection of siPLK1 entrapped in HA-LNPs reduced mRNA levels by more than 80% and significantly prolonged survival of treated mice in the orthotopic model. Taken together, our results suggest that RNAi therapeutics could effectively be delivered in a localized manner with HA-coated LNPs and ultimately may become a therapeutic modality for GBM.

Tumor targeting profiling of hyaluronan-coated lipid based-nanoparticles.

Hyaluronan (HA), a naturally occurring high Mw (HMw) glycosaminoglycan, has been shown to play crucial roles in cell growth, embryonic development, healing processes, inflammation, and tumor development and progression. Low Mw (LMw, <10 kDa) HA has been reported to provoke inflammatory responses, such as induction of cytokines, chemokines, reactive nitrogen species and growth factors. Herein, we prepared and characterized two types of HA coated (LMw and HMw) lipid-based targeted and stabilized nanoparticles (tsNPs) and tested their binding to tumor cells expressing the HA receptor (CD44), systemic immunotoxicity, and biodistribution in tumor bearing mice. In vitro, the Mw of the surface anchored HA had a significant influence on the affinity towards CD44 on B16F10 murine melanoma cells. LMw HA-tsNPs exhibited weak binding, while binding of tsNPs coated with HMw HA was characterized by high binding. Both types of tsNPs had no measured effect on cytokine induction in vivo following intravenous administration to healthy C57BL/6 mice suggesting no immune activation. HMw HA-tsNPs showed enhanced circulation time and tumor targeting specificity, mainly by accumulating in the tumor and its vicinity compared with LMw HA-tsNPs. Finally, we show that methotrexate (MTX), a drug commonly used in cancer chemotherapy, entrapped in HMw HA-tsNPs slowly diffused from the particles with a half-life of 13.75 days, and improved the therapeutic outcome in a murine B16F10 melanoma model compared with NPs suggesting an active cellular targeting beyond the Enhanced Permeability and Retention (EPR) effect. Taken together, these findings have major implications for the use of high molecular weight HA in nanomedicine as a selective and safe active cellular targeting moiety.

Hyaluronan grafted lipid-based nanoparticles as RNAi carriers for cancer cells.

RNA interference (RNAi), a natural cellular mechanism for RNA-guided regulation of gene expression could in fact become new therapeutic modality if an appropriate efficient delivery strategy that is also reproducible and safe will be developed. Numerous efforts have been made for the past eight years to address this challenge with only mild success. The majority of these strategies are based on cationic formulations that condense the RNAi payload and deliver it into the cell cytoplasm. However, most of these formulations also evoke adverse effects such as mitochondrial damage, interfering with blood coagulation cascade, induce interferon response, promote cytokine induction and activate the complement. Herein, we present a strategy that is devised from neutral phospholipids and cholesterol that self-assembled into lipid-based nanoparticles (LNPs). These LNPs were then coated with the glycosaminoglycan, hyaluronan (HA). HA-LNPs bound and internalized specifically into cancer cells compared with control, non-coated particles. Next, loaded with siRNAs against the multidrug resistance extrusion pump, p-glycoprotein (P-gp), HA-LNPs efficiently and specifically reduced mRNA and P-gp protein levels compared with control particles and with HA-LNPs loaded with control, non-targeted siRNAs. In addition, no cellular toxicity or cytokine induction was observed when these particles were cultured with human Peripheral Blood Mononuclear Cells (PBMCs). The HA-LNPs may offer an alternative approach to cationic lipid-based formulations for RNAi delivery into cancer cells in an efficient and safe manner.

A ceramide analog inhibits cPLA(2) activity and consequent PGE(2) formation in LPS-stimulated macrophages.

Prostaglandin E(2) (PGE(2)) is an important mediator of the inflammatory response. Phospho-ceramide analogue-1 (PCERA-1), a synthetic phospholipid-like molecule, was previously reported to modulate pro- and anti-inflammatory cytokine production. We show here that PCERA-1 inhibited LPS-stimulated PGE(2) production in RAW264.7 macrophages, without affecting COX-2 expression. Furthermore, PCERA-1 efficiently suppressed arachidonic acid (AA) release in response to LPS. The dephosphorylated derivative of PCERA-1, ceramide analogue-1 (CERA-1), mimicked the inhibitory effect of PCERA-1 on AA release and PGE(2) production in macrophages. Inhibition of PGE(2) production by CERA-1 was completely rescued by addition of exogenous AA. Importantly, PCERA-1 and ceramide-1-phosphate (C1P) stimulated the enzymatic activity of cPLA(2)α in an in vitro assay, whereas CERA-1 and ceramide inhibited both basal and C1P-stimulated cPLA(2)α activity. Collectively, these results indicate that CERA-1 suppresses AA release and subsequent PGE(2) production in LPS-stimulated macrophages by direct interaction with cPLA(2), and suggest that ceramide may similarly counteract C1P effect on cPLA(2) activity in cells. The suppression of PGE(2) production is suggested to contribute to the anti-inflammatory action of PCERA-1.

Synergistic IL-10 induction by LPS and the ceramide-1-phosphate analog PCERA-1 is mediated by the cAMP and p38 MAP kinase pathways.

Expression of the anti-inflammatory cytokine IL-10 can be induced either by TLR agonists such as lipopolysaccharide (LPS), or by various endogenous stimuli, in particular those acting via a cAMP-dependent signaling pathway. We have previously reported that the synthetic phospho-ceramide analogue-1 (PCERA-1) increases cAMP level and subsequently down-regulates production of TNFalpha and up-regulates production of IL-10 in LPS-stimulated macrophages. The objective of this study was to determine the mechanism of activity of PCERA-1 and the role of cAMP in LPS-induced IL-10 production. We show here that PCERA-1 induces IL-10 production in synergism with various TLR agonists in mouse RAW264.7 macrophages. Cooperativity is evident both at the mRNA and protein levels. IL-10 production by LPS and PCERA-1 is mediated by the cAMP pathway and by the p38 MAP kinase. Phosphorylation of p38 is cooperatively accomplished by LPS and PCERA-1 or other cAMP inducers. Furthermore, the activity of PCERA-1 can be partially mimicked by a cell-permeable analog of cAMP, and blocked by the protein kinase A (PKA) inhibitor H89. Finally, in the absence of PCERA-1, the residual IL-10 induction by LPS depends on the basal cAMP level as it can be largely elevated by the phosphodiesterase (PDE)-4 inhibitor rolipram. Our results thus indicate that IL-10 induction by LPS critically depends on basal cAMP level, and that a co-stimulus by a TLR agonist and a cAMP-elevating agent results in synergistic PKA-dependent and p38-dependent IL-10 production.

Modulation of TNFalpha, IL-10 and IL-12p40 levels by a ceramide-1-phosphate analog, PCERA-1, in vivo and ex vivo in primary macrophages.

Phospho-ceramide analog-1 (PCERA-1) has been described as a potent in vivo suppressor of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha), and thus as a putative drug for the treatment of inflammatory diseases. However, the in vivo cell target of PCERA-1 has not been identified, and its in vivo effect on secretion of other relevant cytokines has not been reported. We have previously shown that PCERA-1 suppresses lipopolysaccharide (LPS)-induced TNFalpha production in RAW264.7 macrophages in vitro. We therefore hypothesized that PCERA-1 targets TNFalpha production by primary macrophages. In this study we thus investigated the effect of PCERA-1 on LPS-induced release of TNFalpha, interleukin (IL)-10 and IL-12p40, in vivo, and ex vivo. We found that PCERA-1 suppressed production of the pro-inflammatory cytokines, TNFalpha and IL-12p40, and increased production of the anti-inflammatory cytokine, IL-10, in LPS-challenged mice, and in primary peritoneal macrophages as well as bone marrow-derived macrophages (BMDM) stimulated with LPS and interferon (IFN)-gamma. These activities of PCERA-1 were independent of each other. In contrast, PCREA-1 only slightly affected TNFalpha production in the whole blood assay, where LPS-induced cytokines are mainly produced by monocytes. Moreover, isolated blood monocytes were inert to PCERA-1, but acquired responsiveness to PCERA-1 upon macrophage colony stimulating factor (M-CSF)-induced differentiation into macrophages. Pharmacokinetic analysis in mice showed that while the volume of distribution of PCERA-1 is low, the drug was rapidly exchanged between the peritoneum and the systemic circulation. Together, these results suggest that sensitivity to PCERA-1 increases upon differentiation of blood monocytes into tissue macrophages, and imply a mechanistic role for peritoneal macrophages in the in vivo anti-inflammatory activity of PCERA-1. Finally, we show that the mechanism of activity of PCERA-1 and prostaglandin E2 (PGE2) is distinct, and that PCERA-1 signaling is not mediated by EP2, a PGE2 receptor which is also activated by oxidized phospholipids. The independent and reciprocal modulation of production of TNFalpha and IL-12p40, vs. IL-10, suggests that PCERA-1 may be a candidate drug for the treatment of inflammation-linked diseases.

A ceramide-1-phosphate analogue, PCERA-1, simultaneously suppresses tumour necrosis factor-alpha and induces interleukin-10 production in activated macrophages.

Tight regulation of the production of the key pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) is essential for the prevention of chronic inflammatory diseases. In vivo administration of a synthetic phospholipid, named hereafter phospho-ceramide analogue-1 (PCERA-1), was previously found to suppress lipopolysaccharide (LPS)-induced TNF-alpha blood levels. We therefore investigated the in vitro anti-inflammatory effects of PCERA-1. Here, we show that extracellular PCERA-1 potently suppresses production of the pro-inflammatory cytokine TNF-alpha in RAW264.7 macrophages, and in addition, independently and reciprocally regulates the production of the anti-inflammatory cytokine interleukin-10 (IL-10). Specificity is demonstrated by the inability of the phospholipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) to perform these activities. Similar TNF-alpha suppression and IL-10 induction by PCERA-1 were observed in macrophages when activated by Toll-like receptor 4 (TLR4), TLR2 and TLR7 agonists. Regulation of cytokine production is demonstrated at the mRNA and protein levels. Finally, we show that, while PCERA-1 does not block activation of nuclear factor (NF)-kappaB and mitogen-activated protein kinases by LPS, it elevates the intracellular cAMP level. In conclusion, the anti-inflammatory activity of PCERA-1 seems to be mediated by a cell membrane receptor, upstream of cAMP production, and eventually TNF-alpha suppression and IL-10 induction. Thus, identification of the PCERA-1 receptor may provide new pharmacological means to block inflammation.