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Arrowhead Publishes Primate Data on Co-Delivery Approach for Cholesterol-Conjugated siRNAs

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Investigators from Arrowhead Research this week published data on a novel strategy of administering a cholesterol-conjugated siRNA payload independently from its delivery vehicle, showing that the co-delivery method can achieve significant gene target knockdown in non-human primates.

The approach, which is being employed with the still-preclinical hepatitis B therapy ARC-520, was initially developed by Roche researchers as they improved the dynamic polyconjugate, or DPC, delivery molecules that were acquired through the company's purchase of Mirus Bio in 2008. Roche sold off its RNA drug assets to Arrowhead about a year ago as part of a broader restructuring GSN 10/27/2011).

In their original form, DPCs were composed of an endosomolytic polymer linked to an siRNA, which was then modified with carboxy dimethylmaleic anhydride derivatives containing polyethylene glycol and a tissue-specific targeting ligand. After the DPC was taken into a cell via endocytosis, the decreasing pH of the maturing endosome unmasked the polymer, which disrupted the endosomal membrane and released the siRNA into the cytoplasm.

As reported by Gene Silencing News, the Roche researchers — now with Arrowhead — found that the DPCs and siRNAs didn't need to be joined as long as they were both targeted to the same cell type. In addition to simplifying drug manufacturing, the co-delivery approach enabled Arrowhead to explore different types of endosomolytic polymers with the DPCs, eventually settling on a peptide with improved pharmacokinetics.

Earlier this month, Arrowhead presented data on the co-delivery approach in two different mouse models of HBV. They showed that ARC-520, which comprises cholesterol-conjugated siRNAs against the virus and DPCs incorporating the new peptide, could achieve multi-log reductions in circulating viral antigens, as well as reduced levels of RNA and DNA replicative intermediates in the liver (GSN 11/8/2012).

This week, Arrowhead published data in Nucleic Acid Therapeutics that extend the drug-delivery findings to non-human primates.

While the use of cholesterol with siRNAs has long been an established method of directing the RNAi agents to the liver while improving their pharmacokinetics (GSN 11/12/2004), “this delivery strategy was highly inefficient, as multiple injections of large doses of cholesterol-siRNA targeting apolipoprotein B were required to achieve just 50 percent target gene knockdown” in rodents, the Arrowhead researchers wrote in the paper. “Although no adverse effects were associated with such a dosing regimen, the low potency ... made it impractical for use in clinical settings.”

Reasoning that the poor efficiency of this method was, at least in part, due to inefficient endosomal escape, the Arrowhead team looked to test whether their co-delivery method could overcome this issue.

In the paper, Arrowhead researchers co-injected into rhesus monkeys cholesterol-conjugated siRNAs against the liver-expressed gene apolipoprotein B, along with the original hepatocyte-targeted DPC formulation developed by Mirus.

They found that a single 2 mg/kg dose of the apoB siRNA co-injected with the DPCs led to a steady reduction in serum apoB protein levels, with a maximum of 78 percent reduction relative to pre-dose levels after 14 days. The maximal reduction was observed until day 30.

“Serum apoB reduction was accompanied by reductions in serum cholesterol and triglyceride levels, as would be expected in animals with lowered apoB levels,” they wrote. “A corresponding reduction in LDL was also observed, again consistent with apoB gene silencing.” No toxicity was observed.

The study, the Arrowhead team wrote, demonstrates that the in vivo efficacy of cholesterol-conjugated siRNA in hepatocytes can be “dramatically improved” by the co-injection of a targeted endosomolytic agent.

“This delivery strategy also enabled, for the first time to our knowledge, the demonstration of [cholesterol-conjugated siRNA]-mediated gene knockdown in non-human primates,” they added.

The investigators noted that the co-delivery strategy is dependent on the use of liver-targeted DPCs, and that the siRNAs must be conjugated to cholesterol to facilitate their uptake into hepatocytes, although the means by which this process occurs is not entirely clear.

Regardless, confocal data indicate that the siRNAs and DPCs end up in the same endosome, an effect not dependent on interactions between the two prior to injection or in the bloodstream before contact with the target cell.

Experiments have shown that the two do not form complexes when mixed together, they wrote in Nucleic Acid Therapeutics, and that target gene knockdown can occur even if they are injected up to two hours apart.

“The simplicity of the formulation and efficacy of this mode of siRNA delivery should prove beneficial in the use of siRNA as a therapeutic,” the researchers concluded.

This is, indeed, something that Arrowhead is banking on.

The company made headlines when its subsidiary Calando Pharmaceuticals advanced the first formulated siRNA drug into human testing with its cancer therapy CALAA-01.

That drug comprises unmodified siRNAs against the M2 subunit of ribonucleotide reductase delivered via Arrowhead's proprietary Rondel cyclodextrin-based polymer technology. Despite publishing promising phase I data showing that the drug could knock down its target mRNA and protein inside a tumor via RNAi, Arrowhead was prompted to run a phase Ib trial amid adverse effects linked to the siRNA payload.

Although that trial was supposed to have wrapped up this summer, Arrowhead has not made public any details about CALAA-01 and its future. Instead, the company has been sharply focused on ARC-520, which was under development by Roche prior to the sale of its RNA drug assets to Arrowhead.

Earlier this year, Arrowhead President and CEO Chris Anzalone said that the firm has implemented an “aggressive development timeline” for the HBV treatment, and anticipates filing an investigational new drug application or foreign equivalent by mid-2013 (GSN 8/16/2012). A phase I trial in chronically infected HBV patients would begin in the third quarter.

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