CX5461, a compound initially identified as an RNA polymerase inhibitor and more recently as a G-quadruplex binder, binds copper to form a complex. Our previous publication showed that the ...complexation reaction can be leveraged to formulate copper-CX5461 inside liposomes, improving the apparent solubility of CX5461 by over 500-fold and reducing the elimination of CX5461 from the plasma compartment following intravenous administration. In mouse models of acute myeloid leukemia, the resulting formulation was more effective than the free drug solution of CX5461 (pH 3.5) currently used in clinical trials. However, the gains observed with the liposomal formulation were minimal, despite significant increases in circulation half-life. Since the formulation technology used relied on liposomes and the fate of most compounds associated with liposomes is dependent on liposomal lipid composition, the studies described here were designed to evaluate how simple changes in lipid composition could affect therapeutic activity. The previously reported formulation method was simplified to ensure an easy scale-up process. In the modified method, pre-measured solid CX5461 was added to copper-containing liposomes prior to an incubation at 60 °C, which enabled copper-CX5461 complexation inside DSPC/Chol or DMPC/Chol liposomes. Efficacy was determined in BRCA-normal (BxPC3) and BRCA-deficient (Capan-1) models of pancreatic cancer. Both liposomal formulations enhanced the circulation lifetime of CX5461 compared to the free drug solution (pH 3.5). Unlike most compounds that are loaded using a transmembrane pH-gradient, the dissociation of CX5461 from liposomes prepared using the copper complexation method were comparable for DSPC/Chol and DMPC/Chol liposomes, in vitro and in vivo. Nonetheless, copper CX5461 prepared using DMPC/Chol liposomes exhibited superior efficacy. The reason for the improved activity of DMPC/Chol copper-CX5461 was not readily explained by the release data and may be due to the fact that DMPC/Chol liposomes are less stable following localization in the tumor. The results indicate that the therapeutic effects of copper-CX5461 will be dependent on liposomal lipid composition and that liposomal CX5461 should exhibit superior benefits when used to treat BRCA-deficient cancers.
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•A CX5461 formulation has been prepared using metal chemistry and liposomes•Liposomal formulations of CuCX5461 exhibit reduced plasma elimination rates•DMPC/Chol liposomal CX5461 is more active than DSPC/Chol liposomal CX5461•Dissociation of the metal-CX5461 complex may be rate limiting for drug release•The DMPC/Chol formulation was most active likely due to intracellular processing
Liposomes are considered one of the most successful drug delivery systems (DDS) given their established utility and success in the clinic. In the past 40⁻50 years, Canadian scientists have made ...ground-breaking discoveries, many of which were successfully translated to the clinic, leading to the formation of biotech companies, the creation of research tools, such as the Lipex Extruder and the NanoAssemblr™, as well as contributing significantly to the development of pharmaceutical products, such as Abelcet
, MyoCet
, Marqibo
, Vyxeos
, and Onpattro™, which are making positive impacts on patients' health. This review highlights the Canadian contribution to the development of these and other important liposomal technologies that have touched patients. In this review, we try to address the question of what drives innovation: Is it the individual, the teams, the funding, and/or an entrepreneurial spirit that leads to success? From this perspective, it is possible to define how innovation will translate to meaningful commercial ventures and products with impact in the future. We begin with a brief history followed by descriptions of drug delivery technologies influenced by Canadian researchers. We will discuss recent advances in liposomal technologies, including the Metaplex technology from the author's lab. The latter exemplifies how a nanotechnology platform can be designed based on multidisciplinary groups with expertise in coordination chemistry, nanomedicines, disease, and business to create new therapeutics that can effect better outcomes in patient populations. We conclude that the team is central to the effort; arguing if the team is entrepreneurial and well positioned, the funds needed will be found, but likely not solely in Canada.
Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative ...genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.
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.
5-Fluorouracil (5-FU) is a small, very membrane permeable drug that is poorly retained within the aqueous compartment of liposomal nanoparticles (LNP). To address this problem a novel method relying ...on formation of a ternary complex comprising copper, low molecular weight polyethylenimine (PEI) and 5-FU has been developed. More specifically, in the presence of entrapped copper and PEI, externally added 5-FU can be efficiently encapsulated (>95%) in DSPC/Chol (1,2-Distearoyl-sn-Glycero-3-Phosphocholine/cholesterol; 55:45mol%) liposomes (130–170nm) to achieve drug-to-lipid ratios of 0.1 (mol:mol). Drug release studies completed using this LNP formulation of 5-FU demonstrated significant improvements in drug retention in vitro and in vivo. Plasma concentrations of 5-FU were 7- to 23-fold higher when the drug was administered intravenously to mice as the LNP 5-FU formulation compared to free 5-FU. Further, the therapeutic effects of the LNP 5-FU formulation, as determined in a HT-29 subcutaneous colorectal cancer model where treatment was given QDx5, was greater than that which could be achieved with free 5-FU when compared at equivalent doses. This is the first time an active loading method has been described for 5-FU. The use of ternary metal complexation strategy to encapsulate therapeutic agents may define a unique platform for preparation of LNP drug formulations.
Ternary metal complexation drives the active uptake of 5-Fluorouracil into liposomes. Drug release properties are tuned by modifying the ligand providing a new platform technology to prepare LNP drug formulations. Display omitted
The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found ...in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line's sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.
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.
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•The 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.
Chemotherapy for glioblastoma (GBM) patients is compromised in part by poor perfusion in the tumor. The present study evaluates how treatment with liposomal formulation of irinotecan (Irinophore C™), ...and other liposomal anticancer drugs, influence the tumor vasculature of GBM models grown either orthotopically or subcutaneously.
Liposomal vincristine (2 mg/kg), doxorubicin (Caelyx®; 15 mg/kg) and irinotecan (Irinophore C™; 25 mg/kg) were injected intravenously (i.v.; once weekly for 3 weeks) in Rag2M mice bearing U251MG tumors. Tumor blood vessel function was assessed using the marker Hoechst 33342 and by magnetic resonance imaging-measured changes in vascular permeability/flow (Ktrans). Changes in CD31 staining density, basement membrane integrity, pericyte coverage, blood vessel diameter were also assessed.
The three liposomal drugs inhibited tumor growth significantly compared to untreated control (p < 0.05-0.001). The effects on the tumor vasculature were determined 7 days following the last drug dose. There was a 2-3 fold increase in the delivery of Hoechst 33342 observed in subcutaneous tumors (p < 0.001). In contrast there was a 5-10 fold lower level of Hoechst 33342 delivery in the orthotopic model (p < 0.01), with the greatest effect observed following treatment with Irinophore C. Following treatment with Irinophore C, there was a significant reduction in Ktrans in the orthotopic tumors (p < 0.05).
The results are consistent with a partial restoration of the blood-brain barrier following treatment. Further, treatment with the selected liposomal drugs gave rise to blood vessels that were morphologically more mature and a vascular network that was more evenly distributed. Taken together the results suggest that treatment can lead to normalization of GBM blood vessel the structure and function. An in vitro assay designed to assess the effects of extended drug exposure on endothelial cells showed that selective cytotoxic activity against proliferating endothelial cells could explain the effects of liposomal formulations on the angiogenic tumor vasculature.
Summary
Insensitivity to platinum, either through inherent or acquired resistance, is a major clinical problem in the treatment of many solid tumors. Here, we explored the therapeutic potential of ...diethyldithiocarbamate (DDC), pyrithione (Pyr), plumbagin (Plum), 8-hydroxyquinoline (8-HQ), clioquinol (CQ) copper complexes in a panel of cancer cell lines that differ in their sensitivity to platins (cisplatin/carboplatin) using a high-content imaging system. Our data suggest that the copper complexes were effective against both platinum sensitive (IC
50
~ 1 μM platinum) and insensitive (IC
50
> 5 μM platinum) cell lines. Furthermore, copper complexes of DDC, Pyr and 8-HQ had greater therapeutic activity compared to the copper-free ligands in all cell lines; whereas the copper-dependent activities of Plum and CQ were cell-line specific. Four of the copper complexes (Cu(DDC)
2
, Cu(Pyr)
2
, Cu(Plum)
2
and Cu(8-HQ)
2
) showed IC
50
values less than that of cisplatin in all tested cell lines. The complex copper DDC (Cu(DDC)
2
) was selected for in vivo evaluation due to its low nano-molar range activity in vitro and the availability of an injectable liposomal formulation. Liposomal (Cu(DDC)
2
) was tested in a fast-growing platinum-resistant A2780-CP ovarian xenograft model and was found to achieve a statistically significant reduction (50%;
p
< 0.05) in tumour size. This work supports the potential use of copper-based therapeutics to treat cancers that are insensitive to platinum drugs.
To investigate the use of liposomal irinotecan (Irinophore C™) plus or minus 5-fluorouracil (5-FU) for the treatment of colorectal cancer.
The effect of irinotecan (IRI) and/or 5-FU exposure times on ...cytotoxicity was assessed in vitro against HT-29 or LS174T human colon carcinoma cells. The pharmacokinetics and biodistribution of Irinophore C™ (IrC™) and 5-FU, administered alone or in combination, were compared in vivo. A subcutaneous model of HT-29 human colorectal cancer in Rag2-M mice was utilized to assess the efficacy of IrC™ alone, and in combination with 5-FU.
The cytotoxicity of IRI and 5-FU were strongly dependent on exposure time. Synergistic interactions were observed following prolonged exposure to IRI/5-FU combinations. Pharmacokinetics/biodistribution studies demonstrated that the 5-FU elimination rate was decreased significantly when 5-FU was co-administered intravenously with IrC™, versus alone. Significant decreases in 5-FU elimination were also observed in plasma, with an associated increase of 5-FU in some tissues when 5-FU was given by intraperitoneal injection and IrC™ was given intravenously. The elimination of IrC™ was not significantly different when administered alone or in combination with 5-FU. Therapeutic studies demonstrated that single agent IrC™ was significantly more effective than the combination of IRI/5-FU; surprisingly, IrC™/5-FU combinations were no more effective than IrC™ alone. The administration of combinations of 5-FU (16 mg/kg) and IrC™ (60 mg IRI/kg) showed increased toxicity when compared to IrC™ alone. Treatment with IrC™ alone (60 mg IRI/kg) delayed the time required for a 5-fold increase in initial tumor volume to day 49, compared to day 23 for controls. When IrC™ (40 mg IRI/kg) was used in combination with 5-FU (16 mg/kg), the time to increase tumor volume 5-fold was 43 days, which was comparable to that achieved when using IrC™ alone (40 mg IRI/kg).
Single agent IrC™ was well tolerated and has significant therapeutic potential. IrC™ may be a suitable replacement for IRI treatment, but its use with free 5-FU is complicated by IrC™-engendered changes in 5-FU pharmacokinetics/biodistribution which are associated with increased toxicity when using the combination.
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