Ada W. Y. Leung

Induction of Cytotoxicity in Pyridine Analogues of the Anti-metastatic Ru(III) Complex NAMI-A by Ferrocene Functionalization

A series of novel ferrocene (Fc) functionalized Ru(III) complexes was synthesized and characterized. These compounds are derivatives of the anti-metastatic Ru(III) complex imidazolium [trans-RuCl4(1H-imidazole) (DMSO-S)] (NAMI-A) and are derived from its pyridine analogue (NAMI-Pyr), with direct coupling of Fc to pyridine at the 4 or 3 positions, or at the 4 position via a two-carbon linker, which is either unsaturated (vinyl) or saturated (ethyl). Electron paramagnetic resonance (EPR) and UV-vis spectroscopic studies of the ligand exchange processes of the compounds in phosphate buffered saline (PBS) report similar solution behavior to NAMI-Pyr. However, the complex with Fc substitution at the 3 position of the coordinated pyridine shows greater solution stability, through resistance to the formation of oligomeric species. Further EPR studies of the complexes with human serum albumin (hsA) indicate that the Fc groups enhance noncoordinate interactions with the protein and help to inhibit the formation of protein-coordinated species, suggesting the potential for enhanced bioavailability. Cyclic voltammetry measurements demonstrate that the Fc groups modestly reduce the reduction potential of the Ru(III) center as compared to NAMI-Pyr, while the reduction potentials of the Fc moieties of the four compounds vary by 217 mV, with the longer linkers giving significantly lower values of E1/2. EPR spectra of the compounds with 2-carbon linkers show the formation of a high-spin Fe(III) species (S = 5/2) in PBS with a distinctive signal at g = 4.3, demonstrating oxidation of the Fe(II) ferrocene center and likely reflecting degradation products. Density functional theory calculations and paramagnetic (1)H NMR describe delocalization of spin density onto the ligands and indicate that the vinyl linker could be a potential pathway for electron transfer between the Ru and Fe centers. In the case of the ethyl linker, electron transfer is suggested to occur via an indirect mechanism enabled by the greater flexibility of the ligand. In vitro assays with the SW480 cell line reveal cytotoxicity induced by the ruthenium ferrocenylpyridine complexes that is at least an order of magnitude higher than the unfunctionalized complex, NAMI-Pyr. Furthermore, migration studies with LNCaP cells reveal that Fc functionalization does not reduce the ability of the compounds to inhibit cell motility. Overall, these studies demonstrate that NAMI-A-type compounds can be functionalized with redox-active ligands to produce both cytotoxic and anti-metastatic activity.

A simple passive equilibration method for loading carboplatin into pre-formed liposomes incubated with ethanol as a temperature dependent permeability enhancer

ABSTRACT A passive equilibration method which relies on addition of candidate drugs to pre‐formed liposomes is described as an alternative method for preparing liposome encapsulated drugs. The method is simple, rapid and applicable to liposomes prepared with high (45 mol%) or low (< 20 mol%) levels of cholesterol. Passive equilibration is performed in 4‐steps: (i) formation of liposomes, (ii) addition of the candidate drug to the liposomes in combination with a permeability enhancing agent, (iii) incubation at a temperature that facilitates diffusion of the added compound across the lipid bilayer, and (iv) quenching the enhanced membrane permeability by reduction in temperature and/or removal of the permeabilization enhancer. The method is fully exemplified here using ethanol as the permeabilization enhancer and carboplatin (CBDCA) as the drug candidate. It is demonstrated that ethanol can be added to liposomes prepared with 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC) and Cholesterol (Chol) (55:45 mol ratio) in amounts up to 30% (v/v) with no change in liposome size, even when incubated at temperatures > 60 °C. Super‐saturated solutions of CBDCA (40 mg/mL) can be prepared at 70 °C and these are stable in the presence of ethanol even when the temperature is reduced to < 30 °C. maximum CBDCA encapsulation is achieved within 1 h after the CBDCA solution is added to pre‐formed DSPC/Chol liposomes in the presence of 30% (v/v) ethanol at 60 °C. When the pre‐formed liposomes are mixed with ethanol (30% v/v) at or below 40 °C, the encapsulation efficiency is reduced by an order of magnitude. The method was also applied to liposomes prepared from other compositions include a cholesterol free formulations (containing 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[carboxy(polyethylene glycol)‐2000] (DSPE‐PEG2000)) and a low Chol (< 20 mol%) formulations prepared with the distearoyl‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol) DSPG)). The cytotoxic activity of CBDCA was unaffected when prepared in this manner and two of the resultant formulations exhibited good stability in vitro and in vivo. The cytotoxic activity of CBDCA was unaffected when prepared in this manner and the resultant formulations exhibited good stability in vitro and in vivo. Pharmacokinetics studies in CD‐1 mice indicated that the resulting formulations increased the circulation half life of the associated CBDCA significantly (AUC0–24 h of CBDCA = 0.016 &mgr;g·hr/mL; AUC0–24h of the DSPC/Chol CBDCA formulation = 1014.0 &mgr;g·hr/mL and AUC0–24h of the DSPC/DSPG/Chol CBDCA formulation = 583.96 &mgr;g·hr/mL). Preliminary efficacy studies in Rag‐2M mice with established subcutaneous H1975 and U‐251 tumors suggest that the therapeutic activity of CBDCA is improved when administered in liposomal formulations. The encapsulation method described here has not been disclosed previously and will have broad applications to drugs that would normally be encapsulated during liposome manufacturing.

Synthetic lethality in lung cancer and translation to clinical therapies

Lung cancer is a heterogeneous disease consisting of multiple histological subtypes each driven by unique genetic alterations. Despite the development of targeted therapies that inhibit the oncogenic mutations driving a subset of lung cancer cases, there is a paucity of effective treatments for the majority of lung cancer patients and new strategies are urgently needed. In recent years, the concept of synthetic lethality has been established as an effective approach for discovering novel cancer-specific targets as well as a method to improve the efficacy of existing drugs which provide partial but insufficient benefits for patients. In this review, we discuss the concept of synthetic lethality, the various types of synthetic lethal interactions in the context of oncology and the approaches used to identify these interactions, including recent advances that have transformed the ability to discover novel synthetic lethal combinations on a global scale. Lastly, we describe the specific synthetic lethal interactions identified in lung cancer to date and explore the pharmacological challenges and considerations in translating these discoveries to the clinic.

Sulfonation, an underexploited area: from skeletal development to infectious diseases and cancer

Sulfonation is one of the most abundant cellular reactions modifying a wide range of xenobiotics as well as endogenous molecules which regulate important biological processes including blood clotting, formation of connective tissues, and functionality of secreted proteins, hormones, and signaling molecules. Sulfonation is ubiquitous in all tissues and widespread in nature (plants, animals, and microorganisms). Although sulfoconjugates were discovered over a century ago when, in 1875, Baumann isolated phenyl sulfate in the urine of a patient given phenol as an antiseptic, the significance of sulfonation and its roles in human diseases have been underappreciated until recent years. Here, we provide a current overview of the significance of sulfonation reactions in a variety of biological functions and medical conditions (with emphasis on cancer). We also discuss research areas that warrant further attention if we are to fully understand how deficiencies in sulfonation could impact human health which, in turn, could help define treatments to effect improvements in health.

Combined Use of Gene Expression Modeling and siRNA Screening Identifies Genes and Pathways Which Enhance the Activity of Cisplatin When Added at No Effect Levels to Non-Small Cell Lung Cancer Cells In Vitro

Platinum-based combination chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC). While cisplatin is effective, its use is not curative and resistance often emerges. As a consequence of microenvironmental heterogeneity, many tumour cells are exposed to sub-lethal doses of cisplatin. Further, genomic heterogeneity and unique tumor cell sub-populations with reduced sensitivities to cisplatin play a role in its effectiveness within a site of tumor growth. Being exposed to sub-lethal doses will induce changes in gene expression that contribute to the tumour cell’s ability to survive and eventually contribute to the selective pressures leading to cisplatin resistance. Such changes in gene expression, therefore, may contribute to cytoprotective mechanisms. Here, we report on studies designed to uncover how tumour cells respond to sub-lethal doses of cisplatin. A microarray study revealed changes in gene expressions that occurred when A549 cells were exposed to a no-observed-effect level (NOEL) of cisplatin (e.g. the IC10). These data were integrated with results from a genome-wide siRNA screen looking for novel therapeutic targets that when inhibited transformed a NOEL of cisplatin into one that induced significant increases in lethality. Pathway analyses were performed to identify pathways that could be targeted to enhance cisplatin activity. We found that over 100 genes were differentially expressed when A549 cells were exposed to a NOEL of cisplatin. Pathways associated with apoptosis and DNA repair were activated. The siRNA screen revealed the importance of the hedgehog, cell cycle regulation, and insulin action pathways in A549 cell survival and response to cisplatin treatment. Results from both datasets suggest that RRM2B, CABYR, ALDH3A1, and FHL2 could be further explored as cisplatin-enhancing gene targets. Finally, pathways involved in repairing double-strand DNA breaks and INO80 chromatin remodeling were enriched in both datasets, warranting further research into combinations of cisplatin and therapeutics targeting these pathways.

In Vivo Validation of PAPSS1 (3′-phosphoadenosine 5′-phosphosulfate synthase 1) as a Cisplatin-sensitizing Therapeutic Target

Purpose: Our previous screening efforts found that inhibition of PAPSS1 increases the potency of DNA-damaging agents in non–small cell lung cancer (NSCLC) cell lines. Here, we explored the clinical relevance of PAPSS1 and further investigated it as a therapeutic target in preclinical model systems. Experimental Design: PAPSS1 expression and cisplatin IC50 values were assessed in 52 lung adenocarcinoma cell lines. Effects of PAPSS1 inhibition on A549 cisplatin sensitivity under hypoxic and starvation conditions, in 3D spheroids, as well as in zebrafish and mouse xenografts, were evaluated. Finally, the association between PAPSS1 expression levels and survival in patients treated with standard chemotherapy was assessed. Results: Our results show a positive correlation between low PAPSS1 expression and increased cisplatin sensitivity in lung adenocarcinoma. In vitro, the potentiation effect was greatest when A549 cells were serum-starved under hypoxic conditions. When treated with low-dose cisplatin, PAPSS1-deficient A549 spheroids showed a 58% reduction in size compared with control cells. In vivo, PAPSS1 suppression and low-dose cisplatin treatment inhibited proliferation of lung tumor cells in zebrafish xenografts and significantly delayed development of subcutaneous tumors in mice. Clinical data suggest that NSCLC and ovarian cancer patients with low PAPSS1 expression survive longer following platinum-based chemotherapy. Conclusions: These results suggest that PAPSS1 inhibition enhances cisplatin activity in multiple preclinical model systems and that low PAPSS1 expression may serve as a biomarker for platin sensitivity in cancer patients. Developing strategies to target PAPSS1 activity in conjunction with platinum-based chemotherapy may offer an approach to improving treatment outcomes. Clin Cancer Res; 23(21); 6555–66. ©2017 AACR.

Copper-CX-5461: A novel liposomal formulation for a small molecule rRNA synthesis inhibitor

ABSTRACT CX‐5461 is currently in Phase I/II clinical trials for advanced hematologic malignancies and triple negative or BRCA‐deficient breast cancer. The compound is currently administered to patients intravenously (i.v.) at low pH (3.5) due to solubility challenges. Reliance of low pH to enhance solubility of CX‐5461 can adversely impact pharmacokinetics, biodistribution and therapeutic potential. We have addressed this solubility issue through a formulation method that relies on the interactions between CX‐5461 and copper. Copper binds CX‐5461 through the nitrogens of the pyrazine ring. Here, we describe synthesizing this copper‐complexed CX‐5461 (Cu(CX‐5461)) within liposomes. CX‐5461 was added to copper‐containing liposomes and incubated at 60 °C for 30 min. The pharmacokinetics of CX‐5461 was assessed in mice following a single i.v. injection at 30 mg/kg. Efficacy studies were completed in multiple subcutaneous mouse xenografts as well as in a bone marrow engraftment model of acute myeloid leukemia (AML). The novel Cu(CX‐5461) formulation was stable at pH 7.4 and exhibited increased plasma circulation longevity, increasing the total exposure to CX5461 by an order of magnitude. Cu(CX‐5461) was more active than CX‐5461 in AML models in vivo. In HCT116‐B46 and Capan‐1 solid tumour models that are BRCA‐deficient, the Cu(CX‐5461) formulation engendered activity that was comparable to that of the low pH CX‐5461 formulation. We have generated the first Cu(CX‐5461) formulation suitable for i.v. administration that is more efficacious than the existing low‐pH formulation in pre‐clinical models of AML. The Cu(CX‐5461) formulation may serve as an alternative formulation for CX‐5461 in BRCA‐deficient cancers. Graphical abstract Figure. No caption available. HighlightsThe phase I/II targeted agent CX‐5461 is poorly soluble at physiological pH.Metal‐binding property of CX‐5461 can be exploited for formulation strategies.The first Cu(CX‐5461) nanoformulation extends CX‐5461 circulation lifetime.Cu(CX‐5461) is equally or more efficacious than the low pH CX‐5461 formulation.

Transition Metal Ions Promote the Bioavailability of Hydrophobic Therapeutics: Cu and Zn Interactions with RNA Polymerase I Inhibitor CX5461

Low aqueous solubility is a major barrier to the clinical application of otherwise promising drug candidates. We demonstrate that this issue can be resolved in medicinal molecules containing potential ligating groups, through the addition of labile transition-metal ions. Incubation of the chemotherapeutic CX5461 with Cu2+ or Zn2+ enables solubilization at neutral pH but does not affect intrinsic cytotoxicity. Spectroscopic and computational studies demonstrate that this arises from coordination to the pyrazine functionality of CX5461 and may involve bidentate coordination at physiological pH.