Renta Hutabarat

Safety and Efficacy of RNAi Therapy for Transthyretin Amyloidosis

BACKGROUND Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart. A therapeutic approach mediated by RNA interference (RNAi) could reduce the production of transthyretin. METHODS We identified a potent antitransthyretin small interfering RNA, which was encapsulated in two distinct first- and second-generation formulations of lipid nanoparticles, generating ALN-TTR01 and ALN-TTR02, respectively. Each formulation was studied in a single-dose, placebo-controlled phase 1 trial to assess safety and effect on transthyretin levels. We first evaluated ALN-TTR01 (at doses of 0.01 to 1.0 mg per kilogram of body weight) in 32 patients with transthyretin amyloidosis and then evaluated ALN-TTR02 (at doses of 0.01 to 0.5 mg per kilogram) in 17 healthy volunteers. RESULTS Rapid, dose-dependent, and durable lowering of transthyretin levels was observed in the two trials. At a dose of 1.0 mg per kilogram, ALN-TTR01 suppressed transthyretin, with a mean reduction at day 7 of 38%, as compared with placebo (P=0.01); levels of mutant and nonmutant forms of transthyretin were lowered to a similar extent. For ALN-TTR02, the mean reductions in transthyretin levels at doses of 0.15 to 0.3 mg per kilogram ranged from 82.3 to 86.8%, with reductions of 56.6 to 67.1% at 28 days (P<0.001 for all comparisons). These reductions were shown to be RNAi-mediated. Mild-to-moderate infusion-related reactions occurred in 20.8% and 7.7% of participants receiving ALN-TTR01 and ALN-TTR02, respectively. CONCLUSIONS ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov numbers, NCT01148953 and NCT01559077.).

First-in-Humans Trial of an RNA Interference Therapeutic Targeting VEGF and KSP in Cancer Patients with Liver Involvement

UNLABELLED RNA interference (RNAi) is a potent and specific mechanism for regulating gene expression. Harnessing RNAi to silence genes involved in disease holds promise for the development of a new class of therapeutics. Delivery is key to realizing the potential of RNAi, and lipid nanoparticles (LNP) have proved effective in delivery of siRNAs to the liver and to tumors in animals. To examine the activity and safety of LNP-formulated siRNAs in humans, we initiated a trial of ALN-VSP, an LNP formulation of siRNAs targeting VEGF and kinesin spindle protein (KSP), in patients with cancer. Here, we show detection of drug in tumor biopsies, siRNA-mediated mRNA cleavage in the liver, pharmacodynamics suggestive of target downregulation, and antitumor activity, including complete regression of liver metastases in endometrial cancer. In addition, we show that biweekly intravenous administration of ALN-VSP was safe and well tolerated. These data provide proof-of-concept for RNAi therapeutics in humans and form the basis for further development in cancer. SIGNIFICANCE The fi ndings in this report show safety, pharmacokinetics, RNAi mechanism of action, and clinical activity with a novel fi rst-in-class LNP-formulated RNAi therapeutic in patients with cancer. The ability to harness RNAi to facilitate specifi c multitargeting, as well as increase the number of druggable targets, has important implications for future drug development in oncology.

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.

Multivalent N‑Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing

Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.

First-in-Human Evaluation of Anti–von Willebrand Factor Therapeutic Aptamer ARC1779 in Healthy Volunteers

Background— ARC1779 is a therapeutic aptamer antagonist of the A1 domain of von Willebrand Factor (vWF), the ligand for receptor glycoprotein 1b on platelets. ARC1779 is being developed as a novel antithrombotic agent for use in patients with acute coronary syndromes. Methods and Results— This was a randomized, double-blind, placebo-controlled study in 47 healthy volunteers of doses of ARC1779 from 0.05 to 1.0 mg/kg. Pharmacodynamic effects were measured by an ELISA for free vWF A1 binding sites and by a platelet function analyzer. In terms of pharmacokinetics, the concentration-time profile of ARC1779 appeared monophasic. The observed concentration and area under the curve were dose proportional. The mean apparent elimination half-life was ≈2 hours, and mean residence time was ≈3 hours. The mean apparent volumes of distribution (at steady state and during terminal phase) were approximately one half the blood volume, suggesting that ARC1779 distribution is in the central compartment. The mean clearance ranged from ≈10% to ≈21% of the glomerular filtration rate, suggesting that renal filtration may not be a major mechanism of clearance of ARC1779. Inhibition of vWF A1 binding activity was achieved with an EC90 value of 2.0 μg/mL (151 nmol/L) and of platelet function with an EC90 value of 2.6 μg/mL (196 nmol/L). ARC1779 was generally well tolerated, and no bleeding was observed. Adverse events tended to be minor and not dose related. Conclusions— This is the first-in-human evaluation of a novel aptamer antagonist of vWF. ARC1779 produced dose- and concentration-dependent inhibition of vWF activity and platelet function with duration of effect suitable for the intended clinical use in acute coronary syndromes.

An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia

Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.

Inhibition of von Willebrand factor-mediated platelet activation and thrombosis by the anti-von Willebrand factor A1-domain aptamer ARC1779

Summary.  Background: von Willebrand factor (VWF) has a role in both hemostasis and thrombosis. Platelets adhere to damaged arteries by interactions between the VWF A1‐domain and glycoprotein Ib receptors under conditions of high shear. This initial platelet binding event stimulates platelet activation, recruitment, and activation of the clotting cascade, promoting thrombus formation. Objective: To characterize the inhibitory activity of a VWF inhibitory aptamer. Methods: Using in vitro selection, aptamer stabilization, and conjugation to a 20‐kDa poly(ethylene glycol), we generated a nuclease‐resistant aptamer, ARC1779, that binds to the VWF A1‐domain with high affinity (KD ∼ 2 nm). The aptamer was assessed for inhibition of VWF‐induced platelet aggregation. In vitro inhibition of platelet adhesion was assessed on collagen‐coated slides and injured pig aortic segments. In vivo activity was assessed in a cynomolgus monkey carotid electrical injury thrombosis model. Results and Conclusion: ARC1779 inhibited botrocetin‐induced platelet aggregation (IC90 ∼ 300 nm) and shear force‐induced platelet aggregation (IC95 ∼ 400 nm). It reduced adhesion of platelets to collagen‐coated matrices and formation of platelet thrombi on denuded porcine arteries. ARC1779 also inhibited the formation of occlusive thrombi in cynomolgus monkeys. We have discovered a novel anti‐VWF aptamer that could have therapeutic use as an anti‐VWF agent in the setting of VWF‐mediated thrombosis.

Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics

In recent years, RNA interference (RNAi) therapeutics, most notably with lipid nanoparticle-based delivery systems, have advanced into human clinical trials. The results from these early clinical trials suggest that lipid nanoparticles (LNPs), and the novel ionizable lipids that comprise them, will be important materials in this emerging field of medicine. A persistent theme in the use of materials for biomedical applications has been the incorporation of biodegradability as a means to improve biocompatibility and/or to facilitate elimination. Therefore, the aim of this work was to further advance the LNP platform through the development of novel, next-generation lipids that combine the excellent potency of the most advanced lipids currently available with biodegradable functionality. As a representative example of this novel class of biodegradable lipids, the lipid evaluated in this work displays rapid elimination from plasma and tissues, substantially improved tolerability in preclinical studies, while maintaining in vivo potency on par with that of the most advanced lipids currently available.

A randomised pilot trial of the anti-von Willebrand factor aptamer ARC1779 in patients with type 2b von Willebrand disease

Desmopressin aggravates thrombocytopenia in type 2B von Willebrand disease (VWF type 2B) by release of large and hyper-adhesive von Willebrand Factor (VWF) multimers. This pilot study investigated whether the anti-VWF aptamer ARC1779 can prevent desmopressin-induced thrombocytopenia and interferes with the excessive VWF turnover in patients with VWF type 2B. Concentration effect curves of ARC1779 were established for five patients in vitro and two patients with VWF type 2B were treated by infusion of ARC1779, desmopressin, or their combination in a randomised, controlled, double-blind design. ARC1779 concentrations in the range of 1-3 microg/ml blocked free A1 domain binding sites by 90% in vitro. In vivo, desmopressin alone induced a profound (-90%) drop in platelet counts in one of the patients. ARC1779 (4-5 microg/ml) completely inhibited VWF A1 domains and prevented this desmopressin-induced platelet drop. Desmopressin alone increased VWF antigen two- to three-fold, accompanied by concordant changes in VWF Ristocetin cofactor activity (RCo) and coagulation factor VIII activity. ARC1779 substantially enhanced the desmopressin-induced maximal increase in these parameters, and improved multimer patterns. No treatment related adverse events were observed and no bleeding occurred despite marked thrombocytopenia. These data provide first proof of concept in humans and evidence that ARC1779 is a potent inhibitor of VWF. ARC1779 prevented the rapid consumption of VWF multimers together with agglutinated platelets that occurred in response to desmopressin challenge in patients with VWD type 2B.

Anti–von Willebrand factor aptamer ARC1779 for refractory thrombotic thrombocytopenic purpura

BACKGROUND: Plasma exchange is the main therapy for thrombotic thrombocytopenic purpura (TTP). No treatments other than plasma exchange have been documented to be effective nor are approved for treatment of TTP. The anti–von Willebrand factor (VWF) aptamer ARC1779 effectively inhibits VWF activity in plasma samples of TTP patients and thus shear‐dependent platelet (PLT) function as measured by the PLT function analyzer PFA‐100 (Dade Behring). It was hypothesized that ARC1779 would offer a potentially effective treatment option for a critically ill patient, refractory to standard care.