Arrowhead Research this week published preclinical data demonstrating that its lead hepatitis B drug candidate, dubbed ARC-520, could silence the hepatitis B virus in rodents for up to one month after a single dose.
The data, which were published in Molecular Therapy, also showed that the delivery technology used in ARC-520 could safely and effectively deliver siRNAs targeting coagulation factor VII in non-human primates.
Arrowhead had previously discussed the findings at the 2012 Oligonucleotide Therapeutics Society meeting (GSN11/8/2012).
“These data suggest that ARC-520 could be a powerful therapy for chronic HBV infection,” Arrowhead President and CEO Chris Anzalone said in a statement. “More broadly, this paper reports on a delivery system capable of extremely efficient gene silencing that can be used for a variety of disease targets.”
Arrowhead remains on track to initiate a phase I trial of ARC-520 in healthy volunteers in the third quarter, with a second phase I in chronic HBV patients beginning before year-end.
ARC-520 is based on a delivery technology known as dynamic polyconjugates that were originally developed by Mirus Bio, but further refined by Roche following its 2008 acquisition of Mirus (GSN 7/24/2008). Following its highly publicized departure from the RNA therapeutics field, Roche sold off the Mirus assets to Arrowhead late last year (GSN 11/27/2011).
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.
Roche researchers, however, discovered that the DPCs and their siRNA payloads did not need to be joined together as long as they were both targeted to the same cell type, which enabled them to be administered separately — a process referred to as co-injection. They also tested different types of endosomolytic polymers with the DPC molecules, ultimately selecting a peptide with improved pharmacokinetics.
In the data published in Molecular Therapy, the investigators evaluated doses of factor VII-targeted siRNAs at 2 mg/kg, with DPC doses ranging from 1 mg/kg to 16 mg/kg. “Increasing the [DPC] dose … is expected to increase the efficiency of endosomal release of [siRNAs], enabling greater target gene knockdown,” they noted.
They reported that the co-injection strategy resulted in target gene knockdown in the livers of mice of about 80 percent at that 2 mg/kg DOC dose, with no effect seen in control animals. “Still greater levels of inhibition … were achieved at higher doses … reaching 99 percent at 8 mg/kg,” the team added.
The mice showed no sign of elevated liver enzymes or blood urea nitrogen, a marker of kidney toxicity, even at the highest treatment levels.
In repeat-dosing experiments, mice were treated four times over the course of five weeks with 6 mg/kg DPC doses and 5 mg/kg doses of factor VII siRNAs. As expected at this dose level, factor VII was reduced by 99 percent through the course of the study. Meanwhile, “no elevations of liver enzymes or of kidney markers blood urea nitrogen and creatinine were observed in evaluations after each dose, indicating there is no cumulative toxicity,” the researchers wrote.
Next, cynomolgus monkeys were given intravenous co-injections of 2 mg/kg of factor VII siRNAs, and either 1 mg/kg or 3 mg/kg doses of DPCs.
Factor VII plasma levels were lowered by at least 80 percent for roughly one month in the primates, but were unchanged in those receiving 10 mg/kg of DPCs and control siRNAs. All safety markers remained unchanged relative to pre-treatment levels at all DPC dose levels, suggesting “remarkably efficient target gene knockdown in mice and non-human primates without toxicity.”
The investigators then looked to see if the DPC approach could be used to treat chronic HBV infection.
According to Arrowhead COO Bruce Given, the researchers scoured GenBank for highly conserved sequences of the HBV genome, eventually winnowing down the number of target candidates to four, which were tested in two mouse models — a transient transfection model in which infection is artificially established and the virus expressed in about 5 percent to 20 percent of hepatocytes, and a transgenic mouse that expresses the virus in 100 percent of its liver cells.
“The transient model allows us to look at the circulating viral load and allows us to look at the antigens that are thought to be central to maintaining chronic infection in humans,” Given noted. “The transgenic model allows us to very specifically look at what's going on with respect to viral RNA and DNA production … and production of virions.”
In the first model, the researchers administered DPCs at a 6 mg/kg dose, chosen because it was shown to be saturating for endosomal release in the mouse studies looking at factor VII, along with doses of the four cholesterol-conjugated siRNAs ranging from 0.25 mg/kg to 6 mg/kg.
At the highest dose of all siRNAs tested, viral DNA in serum was reduced more than 90 percent for one month. The two most effective siRNAs, when injected together along with 6 mg/kg doses of the DPCs, reduced levels of measurable HBV RNA by 87 percent at a 1 mg/kg dose and 92 percent at a 6 mg/kg dose.
These two siRNAs, which cover around 99.6 percent of the HBV genome and are therefore expected to decrease the probability of viral resistance in a patient, were then tested in the transgenic model. A single co-injection of DPCs at the 6 mg/kg level and the two siRNAs at 3 mg/kg doses each resulted in a greater than 80 percent knockdown of viral RNA after one month.
A “profound” reduction in viral proteins was also observed, the study’s authors wrote — a finding that may be key to ARC-520’s potential to cure HBV.
“Researchers have for some time postulated that reduction of viral antigens may allow awakening of an immune response that leads to a functional cure,” they noted in Molecular Therapy.
‘We have very effective ways of knocking down viremia … by knocking out the viral polymerase,” Given explained. “However, these do nothing to reduce the circulating proteins in the blood which are thought to play an important role in maintaining chronicity in those humans that go on to develop chronic HBV.”
Any viral protein knockdown that occurs with existing treatments is “indirect,” he said, adding that less than 10 percent of patients receiving current treatments such as interferon experience a reduction in circulating antigens. Notably, all patients who go on to develop antibodies to hepatitis B surface antigen — and therefore are considered to be functionally cured — experience drops in antigen levels on par with those achieved with ARC-520 prior to seroconversion, he said.
“RNAi suppression of [hepatitis B surface antigen] production as shown in our study specifically would allow the expansion of such [antigen]-specific cytotoxic T lymphocytes and facilitate … seroconversion,” the study’s authors wrote.