Benny J. Evison

Development of parameters for the fabrication of nanosized vectors is pivotal for its successful administration in therapeutic applications. In this study, homogeneously distributed chitosan nanoparticles (CNPs) with diameters as small as 62 nm and a polydispersity index (PDI) of 0.15 were synthesized and purified using a simple, robust method that was highly reproducible. Nanoparticles were synthesized using modified ionic gelation of the chitosan polymer with sodium tripolyphosphate. Using this method, larger aggregates were mechanically isolated from single particles in the nanoparticle population by selective efficient centrifugation. The presence of disaggregated monodisperse nanoparticles was confirmed using atomic force microscopy. Factors such as anions, pH, and concentration were found to affect the size and stability of nanoparticles directly. The smallest nanoparticle population was ∼62 nm in hydrodynamic size, with a low PDI of 0.15, indicating high particle homogeneity. CNPs were highly stable and retained their monodisperse morphology in serum-supplemented media in cell culture conditions for up to 72 hours, before slowly degrading over 6 days. Cell viability assays demonstrated that cells remained viable following a 72-hour exposure to 1 mg/mL CNPs, suggesting that the nanoparticles are well tolerated and highly suited for biomedical applications. Cellular uptake studies using fluorescein isothiocyanate-labeled CNPs showed that cancer cells readily accumulate the nanoparticles 30 minutes posttreatment and that nanoparticles persisted within cells for up to 24 hours posttreatment. As a proof of principle for use in anticancer therapeutic applications, a [(14)C]-radiolabeled form of the anticancer agent doxorubicin was efficiently encapsulated within the CNP, confirming the feasibility of using this system as a drug delivery vector.

Mitoxantrone is a synthetic anthracenedione originally developed to improve the therapeutic profile of the anthracyclines and is commonly applied in the treatment of breast and prostate cancers, lymphomas, and leukemias. A comprehensive overview of the drug's molecular, biochemical, and cellular pharmacology is presented here, beginning with the cardiotoxic nature of its predecessor doxorubicin and how these properties shaped the pharmacology of mitoxantrone itself. Although mitoxantrone is firmly established as a DNA topoisomerase II poison within mammalian cells, it is now clear that the drug interacts with a much broader range of biological macromolecules both covalently and noncovalently. Here, we consider each of these interactions in the context of their wider biological relevance to cancer therapy and highlight how they may be exploited to further enhance the therapeutic value of mitoxantrone. In doing so, it is now clear that mitoxantrone is more than just another topoisomerase II poison.

Proprotein convertase (PC) subtilisin kexin type 9 (PCSK9) inhibits the clearance of low density lipoprotein (LDL) cholesterol from plasma by directly interacting with the LDL receptor (LDLR). As the interaction promotes elevated plasma LDL cholesterol levels and a predisposition to cardiovascular disease (CVD), it has attracted much interest as a therapeutic target. While anti-PCSK9 monoclonal antibodies have been successful in the treatment of hypercholesteremia by decreasing CVD risk, their high cost and a requirement for injection have prohibited widespread use. The advent of an orally bioavailable small molecule inhibitor of the PCSK9-LDLR interaction is an attractive alternative, however efforts have been tempered as the binding interface is unfavourable for binding by small organic molecules. Despite its challenging nature, we report herein the discovery of compound 3f as a small molecule inhibitor of PCSK9. The kinase inhibitor nilotinib emerged from a computational screen that was applied to identify compounds that may bind to a cryptic groove within PCSK9 and proximal to the LDLR-binding interface. A subsequent in vitro PCSK9-LDLR binding assay established that nilotinib was a bona fide but modest inhibitor of the interaction (IC50 = 9.8 µM). Through multiple rounds of medicinal chemistry, 3f emerged as a lead-like molecule by demonstrating disruption of the PCSK9-LDLR interaction at nanomolar levels in vitro (IC50 = 537 nM) with no inhibitory activity (IC50 > 10 µM) against a small panel of kinases. Compound 3f restored LDL uptake by liver cells at sub-micromolar levels and demonstrated excellent bioavailability when delivered subcutaneously in mice. Most significantly, compound 3f lowered total cholesterol levels in the plasma of wild-type mice, thereby providing proof-of-concept that the notion of a small molecule inhibitor against PCSK9 is therapeutically viable.

Mitoxantrone is an anti-cancer agent used in the treatment of breast and prostate cancers. It is classified as a topoisomerase II poison, however can also be activated by formaldehyde to generate drug-DNA adducts. Despite identification of this novel form of mitoxantrone-DNA interaction, excessively high, biologically irrelevant drug concentrations are necessary to generate adducts. A search for mitoxantrone analogues that could potentially undergo this reaction with DNA more efficiently identified Pixantrone as an ideal candidate. An in vitro crosslinking assay demonstrated that Pixantrone is efficiently activated by formaldehyde to generate covalent drug-DNA adducts capable of stabilizing double-stranded DNA in denaturing conditions. Pixantrone-DNA adduct formation is both concentration and time dependent and the reaction exhibits an absolute requirement for formaldehyde. In a direct comparison with mitoxantrone-DNA adduct formation, Pixantrone exhibited a 10- to 100-fold greater propensity to generate adducts at equimolar formaldehyde and drug concentrations. Pixantrone-DNA adducts are thermally and temporally labile, yet they exhibit a greater thermal midpoint temperature and an extended half-life at 37 degrees C when compared to mitoxantrone-DNA adducts. Unlike mitoxantrone, this enhanced stability, coupled with a greater propensity to form covalent drug-DNA adducts, may endow formaldehyde-activated Pixantrone with the attributes required for Pixantrone-DNA adducts to be biologically active.

DNA methylation is an epigenetic modification of the mammalian genome that occurs predominantly at cytosine residues of the CpG dinucleotide. Following formaldehyde activation, pixantrone alkylates DNA and particularly favours the CpG motif. Aberrations in CpG methylation patterns are a feature of most cancer types, a characteristic that may determine their susceptibility to specific drug treatments. Given their common target, DNA methylation may modulate the DNA damage induced by formaldehyde-activated pixantrone. In vitro transcription, mass spectrometry and oligonucleotide band shift assays were utilized to establish that pixantrone-DNA adduct formation was consistently enhanced 2-5-fold at discrete methylated CpG doublets. The methylation-mediated enhancement was exquisitely sensitive to the position of the methyl substituent since methylation at neighboring cytosine residues failed to confer an increase in pixantrone-DNA alkylation. Covalent modification of DNA by formaldehyde-activated doxorubicin, but not cisplatin, was augmented by neighbouring CpG methylation, indicating that modulation of binding by CpG methylation is not a general feature of all alkylators. HCT116 colon cancer cells vastly deficient in CpG methylation were 12- and 10-fold more resistant to pixantrone and doxorubicin relative to the wild-type line, suggesting that these drugs may selectively recognize the aberrant CpG methylation profiles characteristic of most tumour types.