The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1␣ (MIP-1␣), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K i ranged from 1 nM to 5.5 nM). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca 2؉ mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.It is clear that the inappropriate interaction of immune cells, such as T lymphocytes and monocytes, can lead to extensive inflammation and tissue destruction, which is a hallmark of several autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Immune cells are sent on their destructive journey by chemoattractant molecules known as chemokines, which interact with and signal through specific cell surface chemokine receptors. Chemokine receptors belong to the GPCR 1 superfamily and have been viewed as attractive therapeutic targets by the pharmaceutical industry mainly because of their central role in regulating leukocyte trafficking. The premise that drugs that can inhibit the directed migration and activation of immune cells could be useful therapeutically has prompted the search for specific and highly potent chemokine receptor antagonists.Autoimmune diseases like multiple sclerosis and rheumatoid arthritis are characterized by interactions between invading T lymphocytes and tissue macrophages that result in extensive inflammation, tissue damage, and chronic disease pathologies. Numerous studies have demonstrated CCR1 expression in these cell types, and a variety of evidence provides strong in vivo concept validation for a role of this receptor in animal models of these diseases. For example, Karpus et al. (1, 2) were able to show in a mouse EAE model of multiple sclerosis that antibodies to MIP-1␣ prevented the development of both initial and relapsing paralytic disease as well as infiltration of mononuclear cells into the central nervous system. Treatment wit...

We describe a novel, two-nanoparticle mRNA delivery system and show that it is highly effective as a means of intracellular enzyme replacement therapy (i-ERT) using a murine model of ornithine transcarbamylase deficiency (OTCD). Our Hybrid mRNA Technology delivery system (HMT) comprises an inert lipid nanoparticle that protects the mRNA from nucleases in the blood as it distributes to the liver and a polymer micelle that targets hepatocytes and triggers endosomal release of mRNA. This results in high-level synthesis of the desired protein specifically in the liver. HMT delivery of human OTC mRNA normalizes plasma ammonia and urinary orotic acid levels, and leads to a prolonged survival benefit in the murine OTCD model. HMT represents a unique, non-viral mRNA delivery method that allows multi-dose, systemic administration for treatment of single-gene inherited metabolic diseases.

The CC chemokines macrophage inflammatory protein-1␣ (MIP-1␣) and RANTES (regulated on activation normal T cell expressed) have been implicated in rheumatoid arthritis and multiple sclerosis. Since their effects are mediated through the CCR1 chemokine receptor, we set up a small molecule CCR1 antagonist program to search for inhibitors. Through high capacity screening we discovered a number of 4-hydroxypiperidine compounds with CCR1 antagonist activity and report their synthesis and in vitro pharmacology here. Scatchard analysis of the competition binding data revealed that the compounds had K i values ranging from 40 to 4000 nM. The pharmacological profile of the most potent member of this series, compound 1 (2-2-diphenyl-5-(4-chlorophenyl)piperidin-lyl)valeronitrite), was further evaluated. The directed migration of select populations of leukocytes from the circulation to sites of inflammation is an integral part of the immune response. The chemokines are a diverse group of proteins that play an important role in this process (1). They are classified into two major groups, CXC and CC, based on the position of the first two of their four invariant cysteines (2). Each of the chemokines recognizes and induces the chemotaxis of a particular subset of leukocytes. For example, the CXC chemokines, like IL-8 1 and melonoma growth stimulatory activity, mainly chemoattract and activate neutrophils, in contrast to the CC chemokines, like RANTES and monocyte chemoattractant protein, which preferentially attract T lymphocytes and monocytes and induce their activation by producing changes in cellular morphology, transient increases in cellular calcium concentration, and the up-regulation of surface adhesion proteins.The chemokines produce their biologic effects by interacting with specific receptors on the cell surface of their target cells (3). To date, 14 different chemokine receptors including eight CC chemokine receptors have been identified by cloning (4, 5). All of these receptors are characterized by a heptahelical structure and belong to a superfamily of serpentine receptors that are coupled to guanine nucleotide-binding proteins (G-proteins) (6).Occasionally the immune system can turn upon its host, giving rise to chronic inflammation and disease. Given their important role in this process, chemokines have been implicated in the pathophysiology of autoimmune diseases like multiple sclerosis and rheumatoid arthritis. For example a recent study by Karpus et al. (7) provides strong in vivo concept validation for a role of MIP-1␣ in a mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. These investigators were able to show that antibodies to MIP-1␣ prevented the development of both acute and relapsing paralytic disease as well as infiltration of mononuclear cells into the central nervous system. Treatment with MIP-1␣ antibody was also able to ameliorate the severity of ongoing clinical disease. These results led the authors to conclude that MIP-1␣ plays an important role in this T-cell me...

The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery.

Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.The classic signs of acute cellular rejection during organ transplantation include the infiltration of mononuclear cells into the interstitium (1). This cellular infiltrate consists mainly of T lymphocytes, monocytes, and macrophages that are recruited from the circulation into the transplanted tissue by chemotactic molecules known as chemokines. Chemokines belong to a large family of small (8 -10 kDa) inducible chemotactic cytokines, which are characterized by a distinctive pattern of four conserved cysteine residues (2). Currently over 40 chemokines have been identified and classified into two major groups, CXC and CC, dependent on the number and spacing of the first two conserved cysteine residues. The CXC class members include interleukin (IL-8), 1 melanoma growth stimulatory activity, and neutrophil-activating peptide-2, whereas the CC class includes RANTES, monocyte chemotactic protein-1, and MIP-1␣ (macrophage inflammatory protein-1).A number of studies have provided evidence for a role for RANTES in organ transplant rejection, particularly of the kidney. In a model of reperfusion injury in the rat, RANTES levels were increased over normal levels and remained high for more than a week, correlating with the peak of infiltrating macrophages (3). RANTES protein was detected in infiltrating mononuclear cells, tubular epithelium, and vascular endothelium of renal allograft biopsy specimens from patients with cyclosporin nephrotoxicity but not in normal kidney (1). A recent study suggests that RANTES may play a role in graft atherosclerosis (4). Increased levels of RANTES (both mRNA and protein) were detected in mononuclear cells, myofibroblasts, and endothelial cells of arteries undergoing accelerated atherosclerosis compared with normal coronary arteries. In another recent renal transplant study, the chemokine receptor antagonist Met-RANTES when given with low doses of cyclosporin significantly reduced renal injury including interstitial inflammation mainly by reducing the number of infiltrating monocytes (5).Mechanistically this appeared to be achieved by blocking the firm adhesion of these cells to the inflamed endothelium. In summary, these studies ...

DNA condensation and compaction is induced by a variety of condensing agents such as polycations. The present study analyzed the structure of plasmid DNA (DNA) in the small inner space of reverse micelles formed from nonionic surfactants (isotropic phase). Spectroscopic studies indicated that DNA was dissolved in an organic solvent in the presence of a neutral detergent. Fluorescent quenching of ethidium bromide and of rhodamine covalently attached to DNA suggested that the DNA within neutral, reverse micelles was condensed. Circular dichroism indicated that the DNA structure was C form (member of B family) and not the dehydrated A form. Concordantly, NMR experiments indicated that the reverse micelles contained a pool of free water, even at a ratio of water to surfactant (Wo) of 3.75. Electron microscopic analysis also indicated that the DNA was in a ring-like structure, probably toroids. Atomic force microscopic images also revealed small, compact particles after the condensed DNA structures were preserved using an innovative cross-linking strategy. In the lamellar phase, the DNA was configured in long strands that were 20 nm in diameter. Interestingly, such DNA structures, reminiscent of "nanowires," have apparently not been previously observed.

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Azomethine ylide generation from the oxazolium salt 22 and PhSiH3/CsF affords complex products derived from the initial cycloadduct 23 via three competing pathways. Colorless intermediates 24−27 have been detected, and their oxidation products 10, 28, and 29 have been identified. Nucleophilic activation of the oxazolium salt 22 is reported using the organic-soluble benzyltrimethylammonium cyanide. This variation affords the unstable cycloadduct 32, which undergoes aromatization via a single pathway involving 24 and 27, and DDQ oxidation gives the indoloquinone 10 in an improved 63% yield. Lower yields using PhSiH3/CsF or NaCN activation methods are attributed to the heterogeneous conditions and interception of the azomethine ylide by unreacted oxazolium salt.