ALN-VSP , a systemically delivered RNAi or RNA interferencetherapeutic for the treatment of advanced solid tumors, demonstrates evidence for anti-tumor activity, confirming proof of mechanism in clinical trials.The investigational drug comprising two siRNAs designed to target two genes critical for the growth and development of cancer cells, includiing vascular endothelial growth factor (VEGF) and kinesin spindle protein (KSP), also known as eglin 5 (Eg5).

The drug candidate, which is formulated using Tekmira Pharmaceuticalsfirst generation lipid nanoparticle (LNP), is being developed by Alnylam Pharmaceuticals, one of the leaders in RNA interference therapy.The two companies have been partners for many years andTekmira’s lipid nanoparticle technology is the only RNAi delivery technology supporting multiple clinical candidates being advanced by Tekmira and its partners in several different disease indications.

The phase I clinical data were presented at the 47th annual meeting of the American Society of Clinical Oncology (ASCO) in a poster titled ?Phase I dose-escalation study of ALN-VSP02, a novel RNAi therapeutic for solid tumors with liver involvement,? in the Developmental Therapeutics. In this study, ALN-VSP was generally well tolerated, demonstrated evidence for anti-tumor activity, and was found to mediate RNAi activity in both hepatic and extra-hepatic tumors.

Poor survival and unmet needs
Cancer affecting the liver, known as either primary or secondary liver cancer, is associated with one of the poorest survival rates in oncology and represents a major unmet medical need affecting a large number of patients worldwide. Primary liver cancer, or hepatocellular carcinoma (HCC), is one of the most common cancers worldwide, with more than 600,000 people diagnosed each year. Secondary liver cancer, also known as metastatic liver cancer, is cancer that spreads to the liver from another part of the body due to other common cancers like colon, lung, or breast cancer. Worldwide, more than 500,000 people are diagnosed with secondary liver cancer each year.

A revolution in biology
RNAi -which seeks to specifically silence disease-related genes -represents a breakthrough in understanding how genes are turned on and off in cells, and a completely new approach to drug discovery and development. Its discovery has been heralded as ?a major scientific breakthrough that happens once every decade or so,? and represents one of the most promising and rapidly advancing frontiers in biology and drug discovery today which was awarded the 2006 Nobel Prize for Physiology or Medicine.

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RNAi is a natural process of gene silencing that occurs in organisms ranging from plants to mammals. By harnessing the natural biological process of RNAi occurring in cells, scientists are able to creation of a major new class of medicines, known as RNAi therapeutics. Small interfering RNAs (siRNAs), the molecules that mediate RNAi target the cause of diseases by potently silencing specific mRNAs, thereby preventing disease-causing proteins from being made. Lipid nano particle delivery used are thought to help get the RNA-silencing therapies where they need to go in cells.

Phase I Study Design
The trial was designed as a multi-center, open label, dose escalation study in patients with advanced solid tumors with liver involvement who have failed to respond to or have progressed after standard treatment. The primary objective was to evaluate the safety, tolerability, and pharmacokinetics of intravenous ALN-VSP. Other secondary and exploratory objectives included assessment of tumor response using Response Evaluation Criteria for Solid Tumors (RECIST), quantitation of change in tumor blood flow and vascular permeability as measured by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), and analysis of pharmacodynamic effects of ALN-VSP on tumors as measured in patients electing to proceed with voluntary pre- and post-treatment biopsies.

?We are very pleased with the Phase I results with ALN-VSP which include safety and tolerability of multiple doses of ALN-VSP, as well as evidence for anti-tumor activity in this very advanced, heavily pre-treated cancer patient population. In particular, we have seen multiple patients achieve stable disease or better, including a patient with endometrial cancer metastatic to the liver who has achieved 70% tumor regression so far and continues on study after having been on drug for over one full year,? said Jared Gollob, M.D., Senior Director of Clinical Research at Alnylam. ?It is also notable that DCE-MRI results appear to show an anti-VEGF effect with ALN-VSP in approximately 50% of evaluable patients, including those who have been previously exposed to anti-VEGF drugs. Finally, analysis of tumor biopsy samples has demonstrated siRNA delivery and proof of RNAi mechanism in both hepatic and extra-hepatic tumors.?

Trial results
Results from this Phase I study show that ALN-VSP was generally well tolerated. ALN-VSP was administered to 41 patients at doses ranging from 0.1 to 1.5 mg/kg; a total of 182 doses have been administered, including to one patient who has received 24 doses at 0.7 mg/kg over the course of more than one full year, and continues to receive treatment in the study.

Adverse events
The most common adverse events were grade 1-2 fatigue (24% of patients), nausea (17% of patients) and fever (15% of patients), with no clear dose dependence. There were also no dose-dependent changes in liver function tests. Grade 2 infusion-related reactions were observed in 15% of patients, or 3% of total doses administered; these reactions responded to slowing of the infusion of drug, and no patients discontinued therapy because of an infusion reaction. Dose-limiting toxicities included: liver failure and death in one patient with extensive hepatic metastases and prior splenectomy/partial hepatectomy at 0.7 mg/kg which was deemed possibly related to study drug; transient grade 3 thrombocytopenia in two patients at 1.25 mg/kg; and grade 3 hypokalemia in one patient at 1.5 mg/kg. Based on these safety data, the recommended dose for advancement of ALN-VSP into Phase II studies is 1.0 mg/kg.

Heavily pre-treated patients
ALN-VSP demonstrated evidence of anti-tumor activity in advanced malignancy patients. Patients participating in the study were heavily pre-treated, having received an average of 4.3 prior treatment regimens for their metastatic cancer, including both chemotherapy and anti-angiogenic drugs. Fifty percent (12 of 24) of patients evaluable for response attained stable disease (SD) or better with ALN-VSP doses greater than or equal to 0.7 mg/kg, compared to only 8% (1 of 13) at doses less than or equal to 0.4 mg/kg. Results include one major ongoing response in a patient with endometrial cancer and multiple liver metastases treated at 0.7 mg/kg. This patient, whose treatment is ongoing after over one full year, has so far had a partial response (PR) with an approximately 70% reduction in tumor burden. Sixty four percent (7 of 11) of patients achieved SD at the recommended Phase II dose of 1.0 mg/kg and 45% (5 of 11) continue to receive drug on study. In addition, DCE-MRI results were suggestive of an anti-VEGF effect. In approximately 50% of evaluable patients, the average decline in Ktrans (measure of blood flow) in liver tumors where this parameter was measured was greater than or equal to 40%, an effect that is comparable to what has been observed with other anti-VEGF drugs in solid tumors [1, 2]

Encouraging anti-tumor activity
?Both primary liver cancer and metastatic disease of the liver are associated with poor prognosis for patients, and
new therapies are clearly needed,? said Patricia LoRusso, D.O., Professor of Medicine, Director of the Phase I Clinical ? Pharmacology Team at the Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine. ?This Phase I study with ALN-VSP currently represents, to our knowledge, one of the most comprehensive clinical trials of a systemically delivered RNAi therapeutic and also one of the most extensive experiences with RNAi therapeutics in cancer. The safety data and anti-tumor activity with ALN-VSP are encouraging and I look forward to the further development of this promising agent.?

Hepatic and extra-hepatic
In this study, 29 tumor biopsies were obtained voluntarily from 15 patients across multiple dose levels (from 0.4 to 1.5 mg/kg) using a CT-guided procedure; these included hepatic (liver) tumor biopsies from 11 patients and extra-hepatic tumor biopsies from four patients. The two siRNAs targeting VEGF and KSP that comprise ALN-VSP were detected in nearly all of the biopsy samples evaluable for drug levels at siRNA concentrations ranging from 0.3 to 142 ng/g tissue.

These levels of siRNA are pharmacologically relevant since pre-clinical studies have shown that siRNA tissue levels of 1 ng/g are associated with 50% target gene silencing. [3] While there was no dose-dependence to the levels of VEGF and KSP siRNAs detected in biopsy samples, this finding was consistent with the high degree of variability in proportion of tumor, fibrotic/necrotic tissue, and normal tissue in biopsy samples, which impacts the quantitative interpretations of molecular results. Using a highly precise polymerase chain reaction (PCR)-based technique known as 5? rapid amplification of cDNA ends (5? RACE), blinded analysis of human tissue samples showed proof of RNAi-mediated target mRNA cleavage.

Specifically, three of 15 biopsy samples showed VEGF-derived mRNA fragments corresponding exactly to the predicted RNAi-mediated cleavage product based on the VEGF siRNA sequence (p<0.001) in the post-treatment biopsy sample. The samples that were positive for VEGF 5? RACE included liver tumor biopsies obtained from two patients dosed at 0.4 mg/kg and an extra-hepatic tumor biopsy from one patient dosed at 1.25 mg/kg. KSP 5? RACE assay development is in progress, with further optimization required due to low expression of KSP mRNA in tissue samples.

Pre-clinical animal studies
Pharmacokinetic data from this study showed that Cmax (peak serum concentration of drug) and area under the curve (AUC) were dose proportional with no evidence of drug accumulation. Pre-clinical animal pharmacokinetic data were predictive for the observed results in human trials participants.

?We believe the results from our ALN-VSP Phase I trial represent an important milestone in the advancement of RNAi therapeutics. Indeed, our data demonstrate for the first time both clinical activity and RNAi mechanism for an RNAi therapeutic,? said John Maraganore, Ph.D., Chief Executive Officer of Alnylam. ?Clearly, these data are not only important for the continued advancement of our ALN-VSP program, but they also significantly increase our confidence in our entire pipeline of systemically delivered RNAi therapeutics,[4] including ALN-TTR01 which is in a Phase I study for the treatment of transthyretin mediated amyloidosis, and ALN-PCS, which will soon enter clinical trials for the treatment of severe hypercholesterolemia.?

Commenting on the success of the phase I trial,Mark J. Murray M.D.,Tekmira’s President and CEO noted:”We’re excited to see Alnylam’s interim clinical data with ALN-VSP, which demonstrated RNAi activity in human tumor biopsies and confirmed that our LNP technology enables bona fide RNAi activity in man. Now, another important advancement has been made in the RNAi field as Alnylam reports that their ALN-VSP data demonstrates anti-tumor activity.”

“To date, this is the second completed human clinical trial that has reported Tekmira’s LNP technology is safe and well tolerated,” Murray added.

For more information:
Study authors:Cervantes A, Alsina M, Tabernero J, Infante JR, LoRusso P, et al.
Abstract title: Phase I dose-escalation study of ALN-VSP02, a novel RNAi therapeutic for solid tumors with liver involvement. (J Clin Oncol 29: 2011 (suppl; abstr 3025)
Saturday, June 4, 2011 from 2:00 to 6:00 p.m. CDT.
Abstract/Poster no:# 3025

[1] Cannistra SA, Matulonis U, Penson R, Wenham R, Armstrong D, et al. Bevacizumab in patients with advanced platinum-resistant ovarian cancer.ASCO Meeting Abstracts. J Clin Oncol (Meeting Abstracts) June 2006 vol. 24 no. 18 suppl. Abstract 5006.
[2] Siegel AB, Cohen EI, Ocean A, Lehrer D, Goldenberg A, Knox JJ, et al. Phase II trial evaluating the clinical and biologic effects of bevacizumab in unresectable hepatocellular carcinoma. J Clin Oncol. 2008 Jun 20;26(18):2992-8.
[3] Landesman Y, Svrzikapa N, Cognetta A 3rd, Zhang X, Bettencourt BR, et al. In vivo quantification of formulated and chemically modified small interfering RNA by heating-in-Triton quantitative reverse transcription polymerase chain reaction (HIT qRT-PCR). Silence. 2010 Aug 23;1(1):16. Full Article.
[4] Phase 1: Dose Escalation Study of ALN-VSP02. [ALN-VSP-Clinical Study Results ASCO 2011]

Clinical Trials
[A]Multi-center, Open Label, Extension Study of ALN-VSP02 in Cancer Patients Who Have Responded to ALN-VSP02 Treatment
[B]Dose Escalation Trial to Evaluate the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of Intravenous ALN-VSP02 In Patients With Advanced Solid Tumors With Liver Involvement

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