Quantification of therapeutic miRNA mimics in whole blood from nonhuman primates

Abstract

MRX34, a microRNA (miRNA)-based therapy for cancer, has recently entered clinical trials as the first clinical candidate in its class. It is a liposomal nanoparticle loaded with a synthetic mimic of the tumor suppressor miRNA miR-34a as the active pharmaceutical ingredient. To understand the pharmacokinetic properties of the drug and to rationalize an optimal dosing regimen in the clinic, a method is needed to quantitatively detect the miRNA mimic. Here, we report the development and qualification of a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay in support of pharmacokinetic and toxicokinetic assessments in the nonhuman primate. Detection and quantification were performed on total ribonucleic acid (RNA) isolated from whole blood. The qualified range of the standard curve spans 6 orders of magnitude from 2.5 × 10(-7) to 2.5 × 10(-1) ng per reverse transcription (RT) reaction, corresponding to an estimated blood concentration from 6.2 × 10(-5) to 6.2 × 10(1) ng/mL. Our results demonstrate that endogenous as well as the exogenous miR-34a can be accurately and precisely quantified. The assay was used to establish the pharmacokinetic profile of MRX34, showing a favorable residence time and exposure of the miRNA mimic in whole blood from nonhuman primates.

Figure 1

Pharmacokinetics of liposome-encapsulated miR-Rx34 in nonhuman primates. MRX34 was administered as a slow, intravenous bolus into a group of three nonhuman primates at a dose level of 1 mg/kg. Whole blood was collected at time points as indicated in the graph. Total RNA were isolated from 175 μL of whole blood per sample and eluted in 50 μL sterile, nuclease-free water. To ensure that miR-Rx34 concentrations are within the qualified range of the assay, RNA samples collected 1 min through 360 min post injection were diluted 1:10000. RNA samples collected 720 and 1440 min after administration of MRX34 were diluted 1:1000. RNA samples from untreated animals were diluted 1:10 to measure concentrations of endogenous miR-34a. For each qRT-PCR run, 2 μL of diluted RNA were used. Data are expressed as nanograms per milliter of blood.