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.

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.

Concomitant Chemoradiation Therapy with Gold Nanoparticles and Platinum Drugs Co-Encapsulated in Liposomes.

A liposomal formulation of gold nanoparticles (GNPs) and carboplatin, named LipoGold, was produced with the staggered herringbone microfluidic method. The radiosensitizing potential of LipoGold and similar concentrations of non-liposomal GNPs, carboplatin and oxaliplatin was evaluated in vitro with the human colorectal cancer cell line HCT116 in a clonogenic assay. Progression of HCT116 tumor implanted subcutaneously in NU/NU mice was monitored after an irradiation of 10 Gy combined with either LipoGold, GNPs or carboplatin injected directly into the tumor by convection-enhanced delivery. Radiosensitization by GNPs alone or carboplatin alone was observed only at high concentrations of these compounds. Furthermore, low doses of carboplatin alone or a combination of carboplatin and GNPs did not engender radiosensitization. However, the same low doses of carboplatin and GNPs administered simultaneously by encapsulation in liposomal nanocarriers (LipoGold) led to radiosensitization and efficient control of cell proliferation. Our study shows that the radiosensitizing effect of a combination of carboplatin and GNPs is remarkably more efficient when both compounds are simultaneously delivered to the tumor cells using a liposomal carrier.

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.

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.

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.

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.

Development and optimization of an injectable formulation of copper diethyldithiocarbamate, an active anticancer agent.

Copper diethyldithiocarbamate (Cu(DDC)2) is the active anticancer agent generated when disulfiram (DSF) is provided in the presence of copper. To date, research directed toward repurposing DSF as an anticancer drug has focused on administration of DSF and copper in combination, efforts that have proven unsuccessful in clinical trials. This is likely due to the inability to form Cu(DDC)2 at relevant concentrations in regions of tumor growth. Little effort has been directed toward the development of Cu(DDC)2 because of the inherent aqueous insolubility of the complex. Here, we describe an injectable Cu(DDC)2 formulation prepared through a method that involves synthesis of Cu(DDC)2 inside the aqueous core of liposomes. Convection-enhanced delivery of a Cu(DDC)2 formulation prepared using 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol liposomes into a rat model of F98 glioma engendered a 25% increase in median survival time relative to vehicle-treated animals. In a murine subcutaneous MV-4-11 model, treatment resulted in a 45% reduction in tumor burden when compared to controls. Pharmacokinetic studies indicated that the Cu(DDC)2 was rapidly eliminated after intravenous administration while the liposomes remained in circulation. To test whether liposomal lipid composition could increase Cu(DDC)2 circulation lifetime, a number of different formulations were evaluated. Studies demonstrated that liposomes composed of DSPC and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-(carboxy[polyethylene glycol]-2000) (95:5) enhanced Cu(DDC)2 concentrations in the circulation as reflected by a 4.2-fold increase in plasma AUC(0-∞) relative to the DSPC/cholesterol formulation. The anticancer activity of this Cu(DDC)2 formulation was subsequently evaluated in the MV-4-11 model. At its maximum tolerated dose, this formulation exhibited comparable activity to the DSPC/cholesterol formulation. This is the first report demonstrating the therapeutic effects of an injectable Cu(DDC)2 formulation in vivo.

Optimization of liposomal topotecan for use in treating neuroblastoma.

The purpose of this work was to develop an optimized liposomal formulation of topotecan for use in the treatment of patients with neuroblastoma. Drug exposure time studies were used to determine that topotecan (Hycamtin) exhibited great cytotoxic activity against SK-N-SH, IMR-32 and LAN-1 neuroblastoma human cell lines. Sphingomyelin (SM)/cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes were prepared using extrusion methods and then loaded with topotecan by pH gradient and copper-drug complexation. In vitro studies showed that SM/Chol liposomes retained topotecan significantly better than DSPC/Chol liposomes. Decreasing the drug-to-lipid ratio engendered significant increases in drug retention. Dose-range finding studies on NRG mice indicated that an optimized SM/Chol liposomal formulation of topotecan prepared with a final drug-to-lipid ratio of 0.025 (mol: mol) was better tolerated than the previously described DSPC/Chol topotecan formulation. Pharmacokinetic studies showed that the optimized SM/Chol liposomal topotecan exhibited a 10-fold increase in plasma half-life and a 1000-fold increase in AUC0-24 h when compared with Hycamtin administered at equivalent doses (5 mg/kg). In contrast to the great extension in exposure time, SM/Chol liposomal topotecan increased the life span of mice with established LAN-1 neuroblastoma tumors only modestly in a subcutaneous and systemic model. The extension in exposure time may still not be sufficient and the formulation may require further optimization. In the future, liposomal topotecan will be assessed in combination with high-dose radiotherapy such as 131 I-metaiodobenzylguanidine, and immunotherapy treatment modalities currently used in neuroblastoma therapy.

In vitro assay for measuring real time topotecan release from liposomes: release kinetics and cellular internalization.

Topotecan is a drug that is under investigation for the treatment of neuroblastoma and has been encapsulated into liposomes to improve its therapeutic efficacy. However, liposomal formulations still need to be optimized for drug retention and new techniques to measure drug release are required to better understand this process. Here, a novel in vitro method based on fluorescence de-quenching and an automated microscopy imaging platform were developed for monitoring, in real time, the release of topotecan from a liposomal formulation. Drug release from liposomes was monitored for up to 15 h under different conditions including topotecan concentrations, fetal bovine serum amounts (0-20%), and temperatures (25 and 37 °C). A cell-based assay was used to assess liposome association with cells in culture and to quantify amounts of topotecan internalized into cells after release from liposomes. Our results show that the liposomal topotecan concentration had an influence on drug release kinetics: there was a reduction in release rate as a function of increasing concentration. Our data also show that topotecan release from the liposomal formulation was dependent on serum concentration where faster release was observed at higher serum concentrations, and on temperature where faster release was found at 37 °C. This real-time liposomal drug release assay allows for better understanding of the factors important in governing release of topotecan. The assay will be essential towards designing liposomal formulations of topotecan (and potentially of other camptothecin derivatives such as irinotecan) with optimized retention times and better therapeutic efficacy for testing in the clinic.

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

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 (45mol%) or low (<20mol%) 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) (55:45mol 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 (40mg/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 1h 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 (<20mol%) 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 (AUC0-24h of CBDCA=0.016µg·hr/mL; AUC0-24h of the DSPC/Chol CBDCA formulation=1014.0µg·hr/mL and AUC0-24h of the DSPC/DSPG/Chol CBDCA formulation=583.96µg·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.

Nanoscale Reaction Vessels Designed for Synthesis of Copper-Drug Complexes Suitable for Preclinical Development.

The development of copper-drug complexes (CDCs) is hindered due to their very poor aqueous solubility. Diethyldithiocarbamate (DDC) is the primary metabolite of disulfiram, an approved drug for alcoholism that is being repurposed for cancer. The anticancer activity of DDC is dependent on complexation with copper to form copper bis-diethyldithiocarbamate (Cu(DDC)2), a highly insoluble complex that has not been possible to develop for indications requiring parenteral administration. We have resolved this issue by synthesizing Cu(DDC)2 inside liposomes. DDC crosses the liposomal lipid bilayer, reacting with the entrapped copper; a reaction that can be observed through a colour change as the solution goes from a light blue to dark brown. This method is successfully applied to other CDCs including the anti-parasitic drug clioquinol, the natural product quercetin and the novel targeted agent CX-5461. Our method provides a simple, transformative solution enabling, for the first time, the development of CDCs as viable candidate anticancer drugs; drugs that would represent a brand new class of therapeutics for cancer patients.

Irinophore C™, a lipid nanoparticulate formulation of irinotecan, improves vascular function, increases the delivery of sequentially administered 5-FU in HT-29 tumors, and controls tumor growth in patient derived xenografts of colon cancer.

PURPOSE: A liposomal formulation of irinotecan, Irinophore C™ (IrC™) is efficacious in a panel of tumor models, normalizes tumor vasculature, and increases the accumulation of a second drug in the same tumor. We now show that Irinophore C™ is also effective against patient derived xenografts (PDX) of colon cancer, and examine the kinetics of vascular normalization in the HT-29 tumor model and assess how these changes might be used with 5-FU sequentially. MATERIALS AND METHODS: Rag2M mice bearing HT-29 tumors were treated with IrC™ (25mg/kg; Q7D×3) for up to three weeks. Groups of tumors were harvested for analysis at 7, 14 and 21days after the start of treatment. Drug and lipid levels in the tumor were evaluated using HPLC and scintillation counts, respectively. Changes in tumor morphology (H&E), vasculature (CD31), perfusion (Hoechst 33342) and apoptosis (TUNEL) were quantified using microscopy. The accumulation of a second drug ([(14)C]-5-FU, 40mg/kg) given 3h before sacrifice was determined using liquid scintillation. The efficacy of IrC™ (Q7D×3) followed by 5-FU treatment (Q7D×3) was assessed in mice bearing established HT-29 tumors. The efficacy of IrC™ was also evaluated in primary human colorectal tumors grown orthotopically in NOD-SCID mice. RESULTS: Following a single dose of IrC™ the active lactone forms of irinotecan and its metabolite SN-38 were measurable in HT-29 tumors after 7days. The treatment reduced tumor cell density and increased apoptosis. Hoechst 33342 perfusion and accumulation of [(14)C]-5-FU in the treated tumors increased significantly on days 7 and 14. This was accompanied by an increase in the number of endothelial cells relative to total nuclei in the tumor sections. Pre-treatment with IrC™ (Q7D×3) followed by 5-FU (Q7D×3) delayed the time taken for tumors to reach 1cm(3) by 9days (p<0.05). IrC™ was just as effective as free irinotecan when used on patient derived xenografts of colorectal cancer. CONCLUSIONS: Treatment with IrC™ reduces tumor cell viability and appears to normalize the vascular function of the tumor after a single treatment cycle. A concomitant increase in the accumulation of a second drug (5-FU) in the tumor was observed in tumors from IrC™ treated animals and this was correlated with changes in vascular structure consistent with normalization. The treatment effects of sequential 5-FU dosing following IrC™ are additive with no additional toxicity in contrast to previous studies where concurrent 5-FU and IrC™ treatment exacerbated 5-FU toxicity. The studies with PDX tumors also indicate that IrC™ is just as effective as free irinotecan on PDX tumors even though the delivered dose is halved.

Irinophore C™, a lipid nanoparticle formulation of irinotecan, abrogates the gastrointestinal effects of irinotecan in a rat model of clinical toxicities.

Irinotecan is a water-soluble camptothecin derivative with clinical activity against colorectal and small cell lung cancers and is currently a standard of care therapeutic in the treatment of colorectal cancer in combination with 5-fluorouracil. One of the major clinical issues limiting the use of irinotecan is gastrointestinal toxicity manifested as life-threatening diarrhea which is reported in up to 45% of treated patients. The studies summarized here tested, in a rat model of irinotecan-associated gastro-intestinal toxicity, whether a lipid nanoparticle formulation of irinotecan, Irinophore C™, mitigated early-onset or late-onset diarrhea when given at doses equivalent to unformulated irinotecan that engenders both early- and late-onset diarrhea. Specifically, rats administered intravenously on two consecutive days with unformulated irinotecan at 170 mg/kg then 160 mg/kg experienced transient early-onset diarrhea after each administration and then experienced significant late-onset diarrhea peaking 4 days after treatment. Irinophore C™ given at the identical dose and schedule did not elicit either early- or late-onset diarrhea in any animals. When Irinophore C™ was combined with 5-fluorouracil there was also no early- or late-onset diarrhea observed. Histopathological analysis of the gastro-intestinal tract confirmed that the effects associated with irinotecan treatment were absent in rats given Irinophore C™ at the identical dose. Pharmacokinetic analysis demonstrated significantly higher systemic concentrations of irinotecan in rats given the nanoparticle formulation compared to those given unformulated irinotecan. These results demonstrate that the Irinophore C™ formulation is significantly less toxic than irinotecan, used either as a single agent or in combination with 5-fluorouracil, in a rat model of irinotecan-induced gastrointestinal toxicity.

Treatment of colorectal cancer using a combination of liposomal irinotecan (Irinophore C™) and 5-fluorouracil.

PURPOSE: To investigate the use of liposomal irinotecan (Irinophore C™) plus or minus 5-fluorouracil (5-FU) for the treatment of colorectal cancer. EXPERIMENTAL DESIGN: 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. RESULTS: 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). CONCLUSIONS: 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.

Topophore C: a liposomal nanoparticle formulation of topotecan for treatment of ovarian cancer.

We have recently developed a liposomal nanoparticle (LNP) formulation of irinotecan based on loading method that involves formation of a complex between copper and the water soluble camptothecin. The loading methodology developed for irinotecan was evaluated to develop a LNP topotecan formulation (referred to herein as Topophore C) and test its activity in pre-clinical model of ovarian carcinoma. Topotecan was encapsulated into preformed liposomes containing 300 mM copper sulfate and the divalent metal ionophore A23187. Formulation optimization studies included assessments of loading efficiency, influence of temperature on drug loading and in vitro stability of the resulting formulation. In vivo assessments included drug and liposome pharmacokinetics, drug levels within plasma and the peritoneal cavity following intravenous (i.v.) administration in mice and efficacy studies on ES2 ovarian cancer model. Topotecan loading into liposomes was optimized with encapsulation efficiency of >98 % at a final drug-to-lipid (D/L) mole ratio of 0.1. Higher D/L ratios could be achieved, but the resulting formulations were less stable as judged by in vitro drug release studies. Following Topophore C administration in mice the topotecan plasma half-life and AUC were increased compared to free topotecan by 10-and 22-fold, respectively. Topophore C was 2-to 3-fold more toxic than free topotecan, however showed significantly better anti-tumor activity than free topotecan administered at doses with no observable toxic effects. Topophore C is a therapeutically interesting drug candidate and we are particularly interested in developing its use in combination with liposomal doxorubicin for treatment of platinum refractory ovarian cancer.

Lipid-based nanoformulation of irinotecan: dual mechanism of action allows for combination chemo/angiogenic therapy.

A number of studies have outlined the antiangiogenic effects of cytotoxic agents when administered frequently at low doses. These studies suggest that the effect of the cytotoxic agent is on the vasculature within the tumor and it is assumed that there is little or negligible cytotoxicity. Liposomal drug delivery systems have the ability to provide a dual mechanism of activity where tumor accumulation can deliver high local concentrations of the drug at the site of action with concomitant slow release of the drug from carriers in the blood compartment that results in antivascular effects, similar to that achieved when dosing frequently at low levels. Although this dual mechanism of activity may be linked to other lipid nanoparticle formulations of anticancer drugs, this article summarizes the evidence supporting direct (cytotoxic) and indirect (antivascular) actions of a liposomal formulation of irinotecan.

Vascular normalization in orthotopic glioblastoma following intravenous treatment with lipid-based nanoparticulate formulations of irinotecan (Irinophore C™), doxorubicin (Caelyx®) or vincristine.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Validating the use of a luciferase labeled breast cancer cell line, MDA435LCC6, as a means to monitor tumor progression and to assess the therapeutic activity of an established anticancer drug, docetaxel (Dt) alone or in combination with the ILK inhibitor, QLT0267.

A significant issue in drug efficacy studies is animal study design. Here we hypothesize that when evaluating new or existing therapeutics for the treatment of cancer, the location of disease burden will influence drug efficacy. To study this, Female NCr nude mice were inoculated with luciferase-positive human breast cancer cells (LCC6WT-luc) orthotopically (o.t.), intraperitoneally (i.p.) or intracardiacly (i.c.) to create localized, ascites or disseminated disease, respectively. Tumor development was monitored using bioluminescence imaging. Docetaxel (Dt) pharmacokinetics and distribution to sites of tumor growth were determined. Disease progression was followed in animals treated with Dt alone and in combination with QLT0267, an Integrin Linked Kinase inhibitor. Tumor related morbidity was most rapid when cells were inoculated i.c., where disease progression was observed in brain, ovaries, adrenal glands, and lungs. Dt pharmacokinetics were comparable regardless of the model used (mean plasma AUC0-24 hrs 482.6 ng/ml*hr), however, Dt levels were lowest in those tissues developing disease following i.c. cell injection. Treatment with low dose Dt (5 mg/kg) increased overall survival and reduced tumor cell growth in all three models but the activity was greatest in mice with orthotopic tumors. Higher doses of Dt (15 mg/kg) was able to prolong survival in animals bearing i.p. tumors but not i.c. tumors. Addition of QLT0267 provided no added benefit above Dt alone in the disseminated model. These studies highlight a need for more comprehensive in vivo efficacy studies designed to assess multiple disease models and multiple endpoints, focusing analysis of drug parameters on the most chemoresistant disease.