In addition to focusing on further advancing the analytical methodologies and manufacturing capabilities (as discussed below) for these components, Calando made substantial effort in the early days to identify an initial cancer

gene target suitable for eventual clinical application and to optimize an siRNA to downregulate that target. Calando selected the M2 subunit of ribonucleotide reductase (RRM2), an established anticancer target which catalyzes a STA-4783 rate-limiting step in the production of 2′-deoxyribonucleoside 5′-triphosphates which are necessary for DNA replication. Just as TfR overexpression Inhibitors,research,lifescience,medical across a variety of cancer types opened the possibility of RONDEL-based nanoparticles to achieve uptake in many different classes of tumors, the demonstrated sensitivity of many cell types to RRM2 inhibition maintained the Inhibitors,research,lifescience,medical potential generality of anti-RRM2 siRNA-containing nanoparticles to treat multiple types of cancers. A combination of in silico and in vitro screening of many siRNA candidates led to identification of a lead sequence (named “C05C”; also referred to as “siRRM2B + 5”) which was shown to be a potent downregulator of RRM2 in cancer cells of various types and species and induced a concomitant antiproliferative Inhibitors,research,lifescience,medical effect in those cells

[24]. Having defined the four components of Calando’s putative lead candidate formulation (CAL101, AD-PEG5000,

AD-PEG5000-Tf, and C05C), named “CALAA-01,” campaigns to scale up the manufacturing of each component were made while simultaneously expanding and improving Inhibitors,research,lifescience,medical the analytical methods employed to characterize each of them. The manufacturers of the lots of components used for IND-enabling toxicology studies and initial clinical material are listed in Table 6. Improvements Inhibitors,research,lifescience,medical in scale of up to three orders of magnitude were achieved for these molecules, and, as is customary for such projects, several challenges were identified and overcome during development. For example, in the case of CAL101, a previously unidentified impurity created in the initial β-CD functionalization step was observed that could be carried only through subsequent steps; methodologies for quantifying and removing this species were developed and employed. Ultimately, sufficient quantities of all components were obtained that satisfied all acceptance criteria and were employed for subsequent testing. Table 6 Manufacturers of primary toxicology and initial clinical lots of CALAA-01 components. Preclinical safety and efficacy testing of CALAA-01 were performed across a number of species and tumor types, respectively. A dose-range-finding study in non human primates [25] provided an early glimpse of the safety profile at each of three different dose levels (3, 9, and 27mg/kg with respect to C05C).