Induced Vascular Normalization-Can One Force Tumors to Surrender to a Better Microenvironment?

Immunotherapy has changed the way many cancers are being treated. Researchers in the field of immunotherapy and tumor immunology are investigating similar questions: How can the positive benefits achieved with immunotherapies be enhanced? Can this be achieved through combinations with other agents and if so, which ones? In our view, there is an urgent need to improve immunotherapy to make further gains in the overall survival for those patients that should benefit from immunotherapy. While numerous different approaches are being considered, our team believes that drug delivery methods along with appropriately selected small-molecule drugs and drug candidates could help reach the goal of doubling the overall survival rate that is seen in some patients that are given immunotherapeutics. This review article is prepared to address how immunotherapies should be combined with a second treatment using an approach that could realize therapeutic gains 10 years from now. For context, an overview of immunotherapy and cancer angiogenesis is provided. The major targets in angiogenesis that have modulatory effects on the tumor microenvironment and immune cells are highlighted. A combination approach that, for us, has the greatest potential for success involves treatments that will normalize the tumor's blood vessel structure and alter the immune microenvironment to support the action of immunotherapeutics. So, this is reviewed as well. Our focus is to provide an insight into some strategies that will engender vascular normalization that may be better than previously described approaches. The potential for drug delivery systems to promote tumor blood vessel normalization is considered.

PEG Conjugated Zein Nanoparticles for In Vivo Use.

Zein can be utilized to form nanoscale particles for drug delivery applications. Despite the ease of synthesis, these particles often aggregate when exposed to physiologically relevant conditions (e.g., pH and salt concentrations). This instability has prevented their further development in applications requiring intravenous administration. To mitigate this colloidal instability, this research explored Zein nanoparticles (NP)s that were modified with polyethylene glycol (PEG) either through functionalized PEG pre- or post-NP formation. The results suggest that the pre-functionalization of the Zein using N-hydroxysuccinimide ester terminated PEG is the method of choice for synthesizing Zein NPs with conjugated PEG (Zein:PEG-Zein NPs). Zein:PEG-Zein NPs formed using this method displayed excellent stability in physiologically relevant conditions over 72 h and were stable at 4 °C for at least 3 months. When the NPs were cultured with cells for 72 h, no cytotoxicity or early signs of apoptosis were identified. Cellular uptake of the Zein:PEG-Zein NPs did not seem to be impacted by the amount of PEG incorporated in the NP but were concentration-, time-, and temperature-dependent. The lowest percent, stable Zein:PEG-Zein NP formulation (80% unmodified Zein and 20% PEG-modified Zein) induced no observable toxicity over 14 days in CD-1 mice dosed at 70 mg/kg via the tail vein. However, repeat dose pharmacokinetic (PK) studies demonstrated that following the first dose, the second dose caused health issues that required euthanasia shortly after administration. For those animals that survived, there was faster plasma elimination of the Zein:PEG-Zein NPs. Despite this, the Zein:PEG-Zein NPs represent a significantly improved formulation approach, one that displays a long circulation half-life and is suitable for single-use administration. Repeat dose applications will require additional methods to silence the immune response that is generated when using these NPs intravenously.

DMPC/Chol liposomal copper CX5461 is therapeutically superior to a DSPC/Chol formulation.

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.

Monolignol export by diffusion down a polymerization-induced concentration gradient.

Lignin, the second most abundant biopolymer, is a promising renewable energy source and chemical feedstock. A key element of lignin biosynthesis is unknown: how do lignin precursors (monolignols) get from inside the cell out to the cell wall where they are polymerized? Modeling indicates that monolignols can passively diffuse through lipid bilayers, but this has not been tested experimentally. We demonstrate significant monolignol diffusion occurs when laccases, which consume monolignols, are present on one side of the membrane. We hypothesize that lignin polymerization could deplete monomers in the wall, creating a concentration gradient driving monolignol diffusion. We developed a two-photon microscopy approach to visualize lignifying Arabidopsis thaliana root cells. Laccase mutants with reduced ability to form lignin polymer in the wall accumulated monolignols inside cells. In contrast, active transport inhibitors did not decrease lignin in the wall and scant intracellular phenolics were observed. Synthetic liposomes were engineered to encapsulate laccases, and monolignols crossed these pure lipid bilayers to form polymer within. A sink-driven diffusion mechanism explains why it has been difficult to identify genes encoding monolignol transporters and why the export of varied phenylpropanoids occurs without specificity. It also highlights an important role for cell wall oxidative enzymes in monolignol export.

Modular Lipid Nanoparticle Platform Technology for siRNA and Lipophilic Prodrug Delivery.

Successfully employing small interfering RNA (siRNA) therapeutics requires the use of nanotechnology for efficient intracellular delivery. Lipid nanoparticles (LNPs) have enabled the approval of various nucleic acid therapeutics. A major advantage of LNPs is the interchangeability of its building blocks and RNA payload, which allow it to be a highly modular system. In addition, drug derivatization approaches can be used to synthesize lipophilic small molecule prodrugs that stably incorporate in LNPs. This provides ample opportunities to develop combination therapies by co-encapsulating multiple therapeutic agents in a single formulation. Here, it is described how the modular LNP platform is applied for combined gene silencing and chemotherapy to induce additive anticancer effects. It is shown that various lipophilic taxane prodrug derivatives and siRNA against the androgen receptor, a prostate cancer driver, can be efficiently and stably co-encapsulated in LNPs without compromising physicochemical properties or gene-silencing ability. Moreover, it is demonstrated that the combination therapy induces additive therapeutic effects in vitro. Using a double-radiolabeling approach, the pharmacokinetic properties and biodistribution of LNPs and prodrugs following systemic administration in tumor-bearing mice are quantitatively determined. These results indicate that co-encapsulating siRNA and lipophilic prodrugs into LNPs is an attractive and straightforward plug-and-play approach for combination therapy development.

Development and characterization of a novel flavopiridol formulation for treatment of acute myeloid leukemia.

For more than 30 years, treatment of acute myeloid leukemia (AML) has remained largely unchanged and reliant on chemotherapeutic drug combinations, specifically cytarabine and daunorubicin (the 7 + 3 regimen). One broad spectrum drug, flavopiridol (also known as Alvocidib) has shown significant activity against AML through the inhibition of cyclin-dependent kinases. Flavopiridol is a semisynthetic flavonoid and our research team recently described methods to formulate another flavonoid, quercetin, through the ability of flavonoids to bind divalent metals. This method relies on use of copper-containing liposomes to enhance the apparent solubility of flavopiridol and to create formulations suitable for intravenous (i.v.) use. Similar to quercetin, flavopiridol is defined as an aqueous-insoluble compound (< 1 mg/mL in water) and this research sought to evaluate whether the copper-binding capabilities of flavopiridol could be used to prepare an injectable formulation that would exhibit enhanced exposure and improved efficacy. Flavopiridol powder was added directly to preformed copper-containing liposomes (DSPC:Chol or DSPC:DSPE-PEG2000) and the resulting formulations were characterized. Pharmacokinetic and efficacy studies were then conducted. The liposomal flavopiridol formulations were well-tolerated in mice following i.v. administration at a dose of 5 mg/kg with no apparent acute or chronic toxicities. In vivo pharmacokinetics of the optimized DSPC/DSPE-PEG2000 liposomal flavopiridol formulation demonstrated a 30-fold increase in AUC (0.804 µg-hr/mL versus 26.92 µg-hr/mL) compared to the free flavopiridol formulation. The resultant liposomal formulation also demonstrated significant therapeutic activity in MV4-11 and MOLM-13 subcutaneous AML models. Additional studies will be required to define whether formulation changes can be made to enhance flavopiridol retention in the selected composition. The results suggest that further increases in flavopiridol retention will result in improved therapeutic activity.

Loss of Parkinson's susceptibility gene LRRK2 promotes carcinogen-induced lung tumorigenesis.

Pathological links between neurodegenerative disease and cancer are emerging. LRRK2 overactivity contributes to Parkinson's disease, whereas our previous analyses of public cancer patient data revealed that decreased LRRK2 expression is associated with lung adenocarcinoma (LUAD). The clinical and functional relevance of LRRK2 repression in LUAD is unknown. Here, we investigated associations between LRRK2 expression and clinicopathological variables in LUAD patient data and asked whether LRRK2 knockout promotes murine lung tumorigenesis. In patients, reduced LRRK2 was significantly associated with ongoing smoking and worse survival, as well as signatures of less differentiated LUAD, altered surfactant metabolism and immunosuppression. We identified shared transcriptional signals between LRRK2-low LUAD and postnatal alveolarization in mice, suggesting aberrant activation of a developmental program of alveolar growth and differentiation in these tumors. In a carcinogen-induced murine lung cancer model, multiplex IHC confirmed that LRRK2 was expressed in alveolar type II (AT2) cells, a main LUAD cell-of-origin, while its loss perturbed AT2 cell morphology. LRRK2 knockout in this model significantly increased tumor initiation and size, demonstrating that loss of LRRK2, a key Parkinson's gene, promotes lung tumorigenesis.

TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma.

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.

Characterization of a liposomal copper(II)-quercetin formulation suitable for parenteral use.

Quercetin (3,3',4',5,7-pentahydroxyflavone) is a naturally derived flavonoid that is commonly found in fruits and vegetables. There is mounting evidence to suggest that quercetin has potential anticancer effects and appears to interact synergistically when used in combination with approved chemotherapeutic agents such as irinotecan and cisplatin. Unfortunately, quercetin has shown limited clinical utility, partly due to low bioavailability related to its poor aqueous solutions (< 10 µg/mL). In this study, liposomal formulations of quercetin were developed by exploiting quercetin's ability to bind copper. Quercetin powder was added directly to pre-formed copper-containing liposomes (2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 M ratio)). As a function of time and temperature, the formation of copper-quercetin was measured. Using this methodology, a final quercetin-to-lipid (mol:mol) ratio of 0.2 was achievable and solutions containing quercetin at concentrations of > 5 mg/mL were attained, representing at least a > 100-fold increase in apparent solubility. The resulting formulation was suitable for intravenous dosing with no overt toxicities when administered at doses of 50 mg/kg in mice. Pharmacokinetic studies demonstrated that the copper-quercetin formulations had an AUC0-24H of 8382.1 µg h/mL when administered to mice at 50 mg/kg. These studies suggested that quercetin (not copper-quercetin) dissociates from the liposomes after administration. The resulting formulation is suitable for further development and also serves as a proof-of-concept for formulating other flavonoids and flavonoid-like compounds. Given that quercetin exhibits an IC50 of >10 µM when tested against cancer cell lines, we believe that the utility of this novel quercetin formulation for cancer indications will ultimately be as a component of a combination product.

Liposomal OTS964, a TOPK inhibitor: a simple method to estimate OTS964 association with liposomes that relies on enhanced OTS964 fluorescence when bound to albumin.

OTS964 is an inhibitor of T-lymphokine-activated killer cell-originated protein kinase (TOPK), a protein kinase important for mitosis and highly expressed in ovarian and lung cancers. This compound demonstrated potent anti-proliferative activity in a panel of cell lines positive for TOPK; however, when administered to mouse xenograft models, adverse hematopoietic toxicities were observed. To overcome this problem, OTS964 was encapsulated into liposomes and a liposomal formulation of OTS964 is now considered a lead candidate for clinical development. To support clinical development of this formulation, it is critically important to define assays that can easily distinguish between free and liposomal OTS964. Here, we develop a new assay to determine liposomal OTS964 encapsulation (percentage of drug associated with the liposomes) and OTS964 that is dissociated from the liposomes (percentage of drug released from liposomes) by monitoring the enhanced OTS964 fluorescence after its binding to albumin. The optical properties of OTS964 were investigated and three absorbance peaks were identified (235 nm, 291 nm, and 352 nm). Fluorescence was observed at 350 nm (excitation) and 470 nm (emission). Interestingly, the fluorescence of OTS964 increased 18-fold in the presence of serum proteins and more specifically albumin. This phenomenon was used to discriminate between the amounts of drug associated with the liposomes or released from the liposomes. Controls consisting of liposomal OTS964 permeabilized with saponins or octyl glucopyranoside served to confirm that drug release could be monitored by albumin-associated increases in fluorescence. The OTS964 liposomal formulation proved to be very stable with less than 10% release after 4 days in phosphate-buffered saline at 37 °C. The quantity of drug associated with the liposomal surface but not inside the liposomes could also be estimated using this approach. These studies present a novel approach to characterize liposomal release of OTS964, in real time and in a non-invasive manner while acquiring additional information about the spatial distribution of liposomal drug.

Recent Treatment Advances and the Role of Nanotechnology, Combination Products, and Immunotherapy in Changing the Therapeutic Landscape of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common acute leukemia that is becoming more prevalent particularly in the older (65 years of age or older) population. For decades, "7 + 3" remission induction therapy with cytarabine and an anthracycline, followed by consolidation therapy, has been the standard of care treatment for AML. This stagnancy in AML treatment has resulted in less than ideal treatment outcomes for AML patients, especially for elderly patients and those with unfavourable profiles. Over the past two years, six new therapeutic agents have received regulatory approval, suggesting that a number of obstacles to treating AML have been addressed and the treatment landscape for AML is finally changing. This review outlines the challenges and obstacles in treating AML and highlights the advances in AML treatment made in recent years, including Vyxeos®, midostaurin, gemtuzumab ozogamicin, and venetoclax, with particular emphasis on combination treatment strategies. We also discuss the potential utility of new combination products such as one that we call "EnFlaM", which comprises an encapsulated nanoformulation of flavopiridol and mitoxantrone. Finally, we provide a review on the immunotherapeutic landscape of AML, discussing yet another angle through which novel treatments can be designed to further improve treatment outcomes for AML patients.

What Drives Innovation: The Canadian Touch on Liposomal Therapeutics.

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.

Liposomal formulations of carboplatin injected by convection-enhanced delivery increases the median survival time of F98 glioma bearing rats.

BACKGROUND: Effectiveness of chemotherapy for treating glioblastoma (GBM) brain tumors is hampered by the blood-brain barrier which limits the entry into the brain of most drugs from the blood. To bypass this barrier, convection-enhanced delivery (CED) was proposed to directly inject drugs in tumor. However, the benefit of CED may be hampered when drugs diffuse outside the tumor to then induce neurotoxicity. Encapsulation of drugs into liposome aims at increasing tumor cells specificity and reduces neurotoxicity. However, the most appropriate liposomal formulation to inject drugs into brain tumor by CED still remains to be determined. In this study, four liposomal carboplatin formulations were prepared and tested in vitro on F98 glioma cells and in Fischer rats carrying F98 tumor implanted in the brain. Impact of pegylation on liposomal surface and relevance of positive or negative charge were assessed. RESULTS: The cationic non-pegylated (L1) and pegylated (L2) liposomes greatly improved the toxicity of carboplatin in vitro compared to free carboplatin, whereas only a modest improvement and even a reduction of efficiency were measured with the anionic non-pegylated (L3) and the pegylated (L4) liposomes. Conversely, only the L4 liposome significantly increased the median survival time of Fisher rats implanted with the F98 tumor, compared to free carboplatin. Neurotoxicity assays performed with the empty L4' liposome showed that the lipid components of L4 were not toxic. These results suggest that the positive charge on liposomes L1 and L2, which is known to promote binding to cell membrane, facilitates carboplatin accumulation in cancer cells explaining their higher efficacy in vitro. Conversely, negatively charged and pegylated liposome (L4) seems to diffuse over a larger distance in the tumor, and consequently significantly increased the median survival time of the animals. CONCLUSIONS: Selection of the best liposomal formulation based on in vitro studies or animal model can result in contradictory conclusions. The negatively charged and pegylated liposome (L4) which was the less efficient formulation in vitro showed the best therapeutic effect in animal model of GBM. These results support that relevant animal model of GBM must be considered to determine the optimal physicochemical properties of liposomal formulations.

Liposomal Formulations to Modulate the Tumour Microenvironment and Antitumour Immune Response.

Tumours are complex systems of genetically diverse malignant cells that proliferate in the presence of a heterogeneous microenvironment consisting of host derived microvasculature, stromal, and immune cells. The components of the tumour microenvironment (TME) communicate with each other and with cancer cells, to regulate cellular processes that can inhibit, as well as enhance, tumour growth. Therapeutic strategies have been developed to modulate the TME and cancer-associated immune response. However, modulating compounds are often insoluble (aqueous solubility of less than 1 mg/mL) and have suboptimal pharmacokinetics that prevent therapeutically relevant drug concentrations from reaching the appropriate sites within the tumour. Nanomedicines and, in particular, liposomal formulations of relevant drug candidates, define clinically meaningful drug delivery systems that have the potential to ensure that the right drug candidate is delivered to the right area within tumours at the right time. Following encapsulation in liposomes, drug candidates often display extended plasma half-lives, higher plasma concentrations and may accumulate directly in the tumour tissue. Liposomes can normalise the tumour blood vessel structure and enhance the immunogenicity of tumour cell death; relatively unrecognised impacts associated with using liposomal formulations. This review describes liposomal formulations that affect components of the TME. A focus is placed on formulations which are approved for use in the clinic. The concept of tumour immunogenicity, and how liposomes may enhance radiation and chemotherapy-induced immunogenic cell death (ICD), is discussed. Liposomes are currently an indispensable tool in the treatment of cancer, and their contribution to cancer therapy may gain even further importance by incorporating modulators of the TME and the cancer-associated immune response.

Histone Deacetylase Inhibitors Synergize with Catalytic Inhibitors of EZH2 to Exhibit Antitumor Activity in Small Cell Carcinoma of the Ovary, Hypercalcemic Type.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but extremely lethal malignancy that mainly impacts young women. SCCOHT is characterized by a diploid genome with loss of SMARCA4 and lack of SMARCA2 expression, two mutually exclusive ATPases of the SWI/SNF chromatin-remodeling complex. We and others have identified the histone methyltransferase EZH2 as a promising therapeutic target for SCCOHT, suggesting that SCCOHT cells depend on the alternation of epigenetic pathways for survival. In this study, we found that SCCOHT cells were more sensitive to pan-HDAC inhibitors compared with other ovarian cancer lines or immortalized cell lines tested. Pan-HDAC inhibitors, such as quisinostat, reversed the expression of a group of proteins that were deregulated in SCCOHT cells due to SMARCA4 loss, leading to growth arrest, apoptosis, and differentiation in vitro and suppressed tumor growth of xenografted tumors of SCCOHT cells. Moreover, combined treatment of HDAC inhibitors and EZH2 inhibitors at sublethal doses synergistically induced histone H3K27 acetylation and target gene expression, leading to rapid induction of apoptosis and growth suppression of SCCOHT cells and xenografted tumors. Therefore, our preclinical study highlighted the therapeutic potential of combined treatment of HDAC inhibitors with EZH2 catalytic inhibitors to treat SCCOHT.

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

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.

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.

Development of a copper-clioquinol formulation suitable for intravenous use.

Clioquinol (CQ) is an FDA-approved topical antifungal agent known to kill cancer cells. This facilitated the initiation of clinical trials in patients with refractory hematologic malignancies. These repurposing efforts were not successful; this was likely due to low intracellular levels of the drug owing to poor absorption and rapid metabolism upon oral administration. CQ forms a sparingly soluble copper complex (Cu(CQ)2) that exhibits enhanced anticancer activity in some cell lines. We have utilized a novel method to synthesize Cu(CQ)2 inside liposomes, an approach that maintains the complex suspended in solution and in a format suitable for intravenous administration. The complex was prepared inside 100-nm liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine/cholesterol (55:45). The therapeutic activity of the resultant formulation was evaluated in two subcutaneous tumor models (glioblastoma and ovarian cancers) but was not active. We also assessed the ability of the Cu(CQ)2 formulation to increase copper delivery to cancer cells in vitro and its potential to be used in combination with disulfiram (DSF). The results suggested that addition of Cu(CQ)2 enhanced cellular copper levels and the activity of DSF in vitro; however, this combination did not result in a statistically significant reduction in tumor growth in vivo. These studies demonstrate that a Cu(CQ)2 formulation suitable for intravenous use can be prepared, but this formulation used alone or in combination with DSF was not efficacious. The methods described are suitable for development formulations of other analogues of 8-hydroxyquinoline which could prove to be more potent.

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.

A Perspective - can copper complexes be developed as a novel class of therapeutics?

Although copper-ligand complexes appear to be promising as a new class of therapeutics, other than the family of copper(ii) coordination compounds referred to as casiopeínas these compounds have yet to reach the clinic for human use. The pharmaceutical challenges associated with developing copper-based therapeutics will be presented in this article along with a discussion of the potential for high-throughput chemistry, computer-aided drug design, and nanotechnology to address the development of this important class of drug candidates.