Early Development Challenges for Drug Products Containing Nanomaterials.

The vast majority of drug product candidates in early development fail to progress to clinics. This is true for products containing nanomaterials just as for other types of pharmaceuticals. Early development pathways should therefore place high priority on experiments that help candidates fail faster and less expensively. Nanomedicines fail for many reasons, but some are more avoidable than others. Some of the points of failure are not considerations in the development of small molecules or biopharmaceuticals, and so may be unexpected, even to those with previous experience bringing drug products to the clinic. This article reviews experiments that have proven useful in providing "go/no-go" decision-making data for nanomedicines in early preclinical development. Of course, the specifics depend on the particulars of the drug product and the nanomaterial type, and not every product shares the same development pathway or the same potential points of failure. Here, we focus on challenges that differ from those in the development of traditional small molecule therapeutics, and on experiments that reveal deficiencies that can only be corrected by essentially starting over-altering the nanomedicine to an extent that all previous characterization and proof-of-concept testing must be repeated. Conducting these experiments early in the development process can save significant resources and time and allow developers to focus on derisked candidates with a greater likelihood of ultimate success.

Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain.

Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent ß-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

Choice of method for endotoxin detection depends on nanoformulation.

AIMS: Many nanoparticles interfere with traditional tests to quantify endotoxin. The aim of this study was to compare the performance of limulus amoebocyte lysate (LAL) formats on clinical-grade nanoformulations, to determine whether there were disparate results among formats and to test the applicability of an alternative bioassay (the macrophage activation test [MAT]) for resolving discrepancies, if observed. MATERIALS & METHODS: Clinical-grade nanoformulations were tested using turbidimetric, gel-clot and chromogenic LAL. Formulations that cause a discrepancy among LAL tests were also tested by the MAT. RESULTS & CONCLUSION: The gel-clot LAL method cannot be relied upon to resolve discrepancies among LAL tests for certain nanoformulations. No one LAL format was shown to be optimal for all the tested clinical-grade nanoformulations. The tested alternative bioassay (the MAT) was useful for verifying LAL findings, but only for those nanoformulations not carrying/including cytotoxic drugs.