Synthesis and biological evaluation of novel mono- and bivalent ASGP-R-targeted drug-conjugates.

Asialoglycoprotein receptor (ASGP-R) is a promising biological target for drug delivery into hepatoma cells. Nevertheless, there are only few examples of small-molecule conjugates of ASGP-R selective ligand equipped by a therapeutic agent for the treatment of hepatocellular carcinoma (HCC). In the present work, we describe a convenient and versatile synthetic approach to novel mono- and multivalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose and anticancer drug - paclitaxel (PTX). Several molecules have demonstrated high affinity towards ASGP-R and good stability under physiological conditions, significant in vitro anticancer activity comparable to PTX, as well as good internalization via ASGP-R-mediated endocytosis. Therefore, the conjugates with the highest potency can be regarded as a promising therapeutic option against HCC.

Synthesis and biological evaluation of novel doxorubicin-containing ASGP-R-targeted drug-conjugates.

Asialoglycoprotein receptor (ASGP-R) belongs to a wide family of C-type lectins and it is currently regarded as an attractive protein in the field of targeted drug delivery (TDD). It is abundantly expressed in hepatocytes and can be found predominantly on the sinusoidal surface especially of HepG2 cells. Therefore, ASGP-R can be used for the TDD of anticancer therapeutics against HCC and molecular diagnostic tools. To date, a variety of mono- and multivalent selective ASGP-R ligands have been discovered. Although many of these compounds have demonstrated a relatively high binding affinity towards the target, the reported synthetic schemes are not handled, complicated and include many non-trivial steps. In the current study, we describe a convenient and versatile synthetic approach to novel monovalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose fragment as an ASGP-R-recognition "core-head" and well-known nonselective cytostatic - Doxorubicin (Dox). This is the first example of the direct conjugation of a drug molecule to the ASGP-targeted warhead by a really convenient manner via a simple linker sequence. The performed MTS-based biological evaluation in HepG2 cells revealed the novel conjugates as having anticancer activity. Confocal microscopy showed that the molecules readily penetrated HepG2 membrane and were mainly localized within the cytoplasm instead of the nucleus. Per contra, Dox under the same conditions demonstrated good anticancer activity and was predominantly concentrated in the nucleus. Therefore, we speculate that the amide "trigger" that we have used in this study for linker attachment is a sufficiently stable inside the cells to be enzymatically or spontaneously degraded. As a consequence, we did not observe the release of the drug. Ligands containing triggers that are more liable towards endogenous hydrolysis within the tissue of targeting are strongly required.

In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight.

Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.

Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to LDL receptors, leading to their degradation. Genetics studies have shown that loss-of-function mutations in PCSK9 result in reduced plasma LDL cholesterol and decreased risk of coronary heart disease. We aimed to investigate the safety and efficacy of ALN-PCS, a small interfering RNA that inhibits PCSK9 synthesis, in healthy volunteers with raised cholesterol who were not on lipid-lowering treatment. METHODS: We did a randomised, single-blind, placebo-controlled, phase 1 dose-escalation study in healthy adult volunteers with serum LDL cholesterol of 3·00 mmol/L or higher. Participants were randomly assigned in a 3:1 ratio by computer algorithm to receive one dose of intravenous ALN-PCS (with doses ranging from 0·015 to 0·400 mg/kg) or placebo. The primary endpoint was safety and tolerability of ALN-PCS. Secondary endpoints were the pharmacokinetic characteristics of ALN-PCS and its pharmacodynamic effects on PCSK9 and LDL cholesterol. Study participants were masked to treatment assignment. Analysis was per protocol and we used ANCOVA to analyse pharmacodynamic endpoint data. This trial is registered with ClinicalTrials.gov, number NCT01437059. FINDINGS: Of 32 participants, 24 were randomly allocated to receive a single dose of ALN-PCS (0·015 mg/kg [n=3], 0·045 mg/kg [n=3], 0·090 mg/kg [n=3], 0·150 mg/kg [n=3], 0·250 mg/kg [n=6], or 0·400 mg/kg [n=6]) and eight to placebo. The proportions of patients affected by treatment-emergent adverse events were similar in the ALN-PCS and placebo groups (19 [79%] vs seven [88%]). ALN-PCS was rapidly distributed, with peak concentration and area under the curve (0 to last measurement) increasing in a roughly dose-proportional way across the dose range tested. In the group given 0·400 mg/kg of ALN-PCS, treatment resulted in a mean 70% reduction in circulating PCSK9 plasma protein (p<0·0001) and a mean 40% reduction in LDL cholesterol from baseline relative to placebo (p<0·0001). INTERPRETATION: Our results suggest that inhibition of PCSK9 synthesis by RNA interference (RNAi) provides a potentially safe mechanism to reduce LDL cholesterol concentration in healthy individuals with raised cholesterol. These results support the further assessment of ALN-PCS in patients with hypercholesterolaemia, including those being treated with statins. This study is the first to show an RNAi drug being used to affect a clinically validated endpoint (ie, LDL cholesterol) in human beings. FUNDING: Alnylam Pharmaceuticals.

FGF regulates TGF-ß signaling and endothelial-to-mesenchymal transition via control of let-7 miRNA expression.

Maintenance of normal endothelial function is critical to various aspects of blood vessel function, but its regulation is poorly understood. In this study, we show that disruption of baseline fibroblast growth factor (FGF) signaling to the endothelium leads to a dramatic reduction in let-7 miRNA levels that, in turn, increases expression of transforming growth factor (TGF)-ß ligands and receptors and activation of TGF-ß signaling, leading to endothelial-to-mesenchymal transition (Endo-MT). We also find that Endo-MT is an important driver of neointima formation in a murine transplant arteriopathy model and in rejection of human transplant lesions. The decline in endothelial FGF signaling input is due to the appearance of an FGF resistance state that is characterized by inflammation-dependent reduction in expression and activation of key components of the FGF signaling cascade. These results establish FGF signaling as a critical factor in maintenance of endothelial homeostasis and point to an unexpected role of Endo-MT in vascular pathology.

Harnessing a physiologic mechanism for siRNA delivery with mimetic lipoprotein particles.

Therapeutics based on RNA interference (RNAi) have emerged as a potential new class of drugs for treating human disease by silencing the target messenger RNA (mRNA), thereby reducing levels of the corresponding pathogenic protein. The major challenge for RNAi therapeutics is the development of safe delivery vehicles for small interfering RNAs (siRNAs). We previously showed that cholesterol-conjugated siRNAs (chol-siRNA) associate with plasma lipoprotein particles and distribute primarily to the liver after systemic administration to mice. We further demonstrated enhancement of silencing by administration of chol-siRNA pre-associated with isolated high-density lipoprotein (HDL) or low-density lipoprotein (LDL). In this study, we investigated mimetic lipoprotein particle prepared from recombinant apolipoprotein A1 (apoA) and apolipoprotein E3 (apoE) as a delivery vehicle for chol-siRNAs. We show that apoE-containing particle (E-lip) is highly effective in functional delivery of chol-siRNA to mouse liver. E-lip delivery was found to be considerably more potent than apoA-containing particle (A-lip). Furthermore, E-lip-mediated delivery was not significantly affected by high endogenous levels of plasma LDL. These results demonstrate that E-lip has substantial potential as delivery vehicles for lipophilic conjugates of siRNAs.

Notch ligand delta-like 4 blockade attenuates atherosclerosis and metabolic disorders.

Atherosclerosis and insulin resistance are major components of the cardiometabolic syndrome, a global health threat associated with a systemic inflammatory state. Notch signaling regulates tissue development and participates in innate and adaptive immunity in adults. The role of Notch signaling in cardiometabolic inflammation, however, remains obscure. We noted that a high-fat, high-cholesterol diet increased expression of the Notch ligand Delta-like 4 (Dll4) in atheromata and fat tissue in LDL-receptor-deficient mice. Blockade of Dll4-Notch signaling using neutralizing anti-Dll4 antibody attenuated the development of atherosclerosis, diminished plaque calcification, improved insulin resistance, and decreased fat accumulation. These changes were accompanied by decreased macrophage accumulation, diminished expression of monocyte chemoattractant protein-1 (MCP-1), and lower levels of nuclear factor-κB (NF-κB) activation. In vitro cell culture experiments revealed that Dll4-mediated Notch signaling increases MCP-1 expression via NF-κB, providing a possible mechanism for in vivo effects. Furthermore, Dll4 skewed macrophages toward a proinflammatory phenotype ("M1"). These results suggest that Dll4-Notch signaling plays a central role in the shared mechanism for the pathogenesis of cardiometabolic disorders.

Lipophilic siRNAs mediate efficient gene silencing in oligodendrocytes with direct CNS delivery.

Conjugation of small interfering RNA (siRNA) with lipophilic molecules has been demonstrated to enhance cellular uptake in cell culture and to produce efficient endogenous gene silencing in the liver after systemic administration and in neurons after direct local injection. Here, we evaluated the in vivo delivery of siRNAs conjugated with different linkers to cholesterol by targeting CNPase (2'-3'-cyclic nucleotide 3'-phosphodiesterase) in oligodendrocytes. Cholesterol-conjugated siRNAs administered to the rat corpus callosum by intraparenchymal central nervous system (CNS) infusion show improved silencing ability compared with unconjugated siRNA. Furthermore, conjugation of siRNA to cholesterol with a cleavable disulfide linker appears to be beneficial for improving the potency of silencing of CNPase mRNA in oligodendrocytes in vivo. Taken together, these findings indicate that cholesterol-conjugated siRNAs are effective for direct CNS delivery to oligodendrocytes, and that the biocleavable disulfide linker appears to be beneficial for improving the potency of silencing of target mRNA in vivo.

RNA interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy.

We describe the design and characterization of a potent human respiratory syncytial virus (RSV) nucleocapsid gene-specific small interfering RNA (siRNA), ALN-RSV01. In in vitro RSV plaque assays, ALN-RSV01 showed a 50% inhibitory concentration of 0.7 nM. Sequence analysis of primary isolates of RSV showed that the siRNA target site was absolutely conserved in 89/95 isolates, and ALN-RSV01 demonstrated activity against all isolates, including those with single-mismatch mutations. In vivo, intranasal dosing of ALN-RSV01 in a BALB/c mouse model resulted in potent antiviral efficacy, with 2.5- to 3.0-log-unit reductions in RSV lung concentrations being achieved when ALN-RSV01 was administered prophylactically or therapeutically in both single-dose and multidose regimens. The specificity of ALN-RSV01 was demonstrated in vivo by using mismatch controls; and the absence of an immune stimulatory mechanism was demonstrated by showing that nonspecific siRNAs that induce alpha interferon and tumor necrosis factor alpha lack antiviral efficacy, while a chemically modified form of ALN-RSV01 lacking measurable immunostimulatory capacity retained full activity in vivo. Furthermore, an RNA interference mechanism of action was demonstrated by the capture of the site-specific cleavage product of the RSV mRNA via rapid amplification of cDNA ends both in vitro and in vivo. These studies lay a solid foundation for the further investigation of ALN-RSV01 as a novel therapeutic antiviral agent for clinical use by humans.

Direct CNS delivery of siRNA mediates robust silencing in oligodendrocytes.

The most significant challenge remaining in the development of small interfering RNAs (siRNAs) as a new class of therapeutic drugs is successful delivery in vivo. The majority of reported studies describing delivery of siRNA or short hairpin RNA (shRNA) to the central nervous system (CNS) have focused on RNA interference (RNAi) in neurons. Here we show direct CNS delivery of siRNA to a different cell type-oligodendrocytes-using convection-enhanced delivery, and demonstrate robust silencing of an endogenous oligodendrocyte-specific gene, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) with siRNA formulated in saline. The silencing is not sequence-dependent as several different siRNAs are effective in inhibiting target gene expression. Furthermore, we show that CNPase mRNA reduction is dose-dependent, durable for up to 1 week, and mediated by an RNAi mechanism. Increasing the flow rate of siRNA infusion increased the distribution of mRNA suppression to encompass white matter regions distant from the infusion site. Finally, we demonstrate suppression of CNPase mRNA in the nonhuman primate CNS. Taken together, these results show for the first time robust RNAi within oligodendrocytes in vivo and demonstrate the important potential of siRNAs in the treatment of CNS disorders involving oligodendrocyte pathology.

Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.

Psoriasis is a common T cell-mediated autoimmune inflammatory disease. We show that blocking the interaction of alpha1beta1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. Alpha1beta1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. Alpha1beta1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-gamma but not interleukin-4. Blockade of alpha1beta1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-alpha blockers. These results define a crucial role for alpha1beta1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell-extracellular matrix interactions.

Collagen distribution and expression of collagen-binding alpha1beta1 (VLA-1) and alpha2beta1 (VLA-2) integrins on CD4 and CD8 T cells during influenza infection.

Most viral infections occur in extralymphoid tissues, yet the mechanisms that regulate lymphocytes in these environments are poorly understood. One feature common to many extralymphoid environments is an abundance of extracellular matrix. We have studied the expression of two members of the beta(1) integrin family of collagen-binding receptors, alpha(1)beta(1) and alpha(2)beta(1) (CD49a, VLA-1 and CD49b, VLA-2, respectively), on CD4 and CD8 T cells during the response to influenza infection in the lung. Flow cytometry showed that whereas T cells infiltrating the lung and airways can express both CD49a and CD49b, CD49a expression was most strongly associated with the CD8+ subset. Conversely, though fewer CD4+ T cells expressed CD49a, most CD4+ cells in the lung tissue or airways expressed CD49b. This reciprocal pattern suggested that CD4 and CD8 T cells might localize differently within the lung tissue and this was supported by immunofluorescent analysis. CD8+ cells tended to localize in close proximity to the collagen IV-rich basement membranes of either the airways or blood vessels, whereas CD4+ cells tended to localize in the collagen I-rich interstitial spaces, with few in the airways. These observations suggest that CD4 T cell interaction with the tissue microenvironment is distinct from CD8 T cells and support the concept that CD4+ T cells in peripheral tissues are regulated differently than the CD8 subset.