TECHNICAL FIELD

This invention generally relates to cell biology and cancer treatment.

Disclosed herein are pharmaceutical compositions and uses thereof to induce immunogenic cell death for the treatment or prevention of cancer or other disease conditions. Further disclosed are methods for identifying compositions with enhanced ability to induce immunogenic cell death. Additional embodiments are directed to treatment methods to induce immunogenic cell death.

BACKGROUND

Immunogenic Cell Death (ICD) is a phenomenon whereby cancer cells die in a manner that activates the immune system. Activation of the immune system can in turn elicit an immune response against the cancer. Cells infected as part of an infectious disease condition can also initiate an adaptive immune response. Without being limited by theory, the phenomenon of ICD is distinct from regular apoptosis in which cell death does not generally lead to the activation of the immune system, although mechanisms involved in apoptosis may contribute to induction of ICD- related molecular markers.

In particular, during immunogenic cell death, dying tumour cells or infected cells emit signals known as damage-associated molecular patterns (DAMPs). ICD is often characterized by three distinct DAMPs, which are evidenced by the presence of one or more of the following molecular determinants (referred to in the literature as "markers") of ICD: (1 ) the pre-apoptotic expression of endoplasmic reticulin (ER) calreticulin (CRT). (2) the secretion of adenosine triphosphate (ATP), and (3) the secretion of nuclear high mobility group box 1 (HMGBi). It has been reported that the presence of these DAMPs during tumour cell death leads to the release of pro- inflammatory cytokines, activation of innate immune cells such as dendritic cells (DCs) and macrophages, and ultimately stimulation of the adaptive immune response.

A number of agents used in cancer treatment are capable of inducing immunogenic cell death as determined by one or more assays to detect one or more of the above markers. Generally, ICD inducers can be categorized as Type I or Type II inducers. It has been reported that Type I inducers induce ICD as an off-target effect, while Type II inducers typically cause primarily ER stress, which is directly related to ICD induction.

However, there is an ongoing need in the art for additional or improved compositions for inducing ICD for the treatment of cancer and other disease conditions. The present disclosure seeks to address that need or to provide useful alternatives to known approaches.

SUMMARY

According to one aspect of the disclosure, there is provided an

immunostimulatory pharmaceutical composition to treat, ameliorate and/or prevent a cancer or other condition by inducing Immunogenic Cell Death (ICD) comprising: copper and at least one agent, which is optionally an anti-cancer agent. The agent is optionally capable of complexing with copper to form a metal complex. The combination of the copper and the agent provide an increase in ICD, compared to the agent alone. The increase in the ICD response of the combination as measured by at least one of extracellular adenosine triphosphate (ATP), HMGBi release, and CRT exposure of cells in vitro may be 1 .2 to 10,000 times that of the agent alone measured under otherwise identical conditions. In alternative embodiments, the copper, the agent, or both, in certain embodiments are formulated in a liposome or other delivery vehicle known to those of skill in the art.

According to a further aspect of the disclosure, there is provided the use of copper and at least one agent, such as an anti-cancer agent, that induces ICD, in combination or sequentially, to a patient in need thereof. The combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by an assay that measures at least one of ATP secretion, HMGBi release, and CRT exposure of cells. The copper, the agent, or both, in certain embodiments, are formulated in a liposome or other delivery vehicles known to those of skill in the art.

According to another aspect of the disclosure, there is provided a method of inducing ICD comprising administering copper and at least one agent, such as an anti cancer agent, in combination or sequentially, to a patient in need thereof. The combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by measuring at least one of adenosine triphosphate (ATP) secretion, HMGB release, and CRT exposure. The copper, the agent, or both, may be formulated in a liposome or other delivery vehicle.

In yet a further aspect of the disclosure there is provided a method of producing an immunostimulatory pharmaceutical composition for inducing ICD, comprising the steps of:

(i) exposing an agent, such as an anti-cancer agent, and copper, alone and in combination, to an assay that measures at least one of ATP secretion, HMGB release, and CRT exposure in cell culture;

(ii) determining whether the agent and the copper in combination exhibit an increase in ICD response compared to results in which the ICD of the agent is measured alone; and

(iii) wherein, if the combination of the copper and the agent provide an increase in ICD, compared to the agent alone, formulating the agent, the copper, or both in one or more pharmaceutical formulations, such as a liposome or any other drug delivery vehicle, to treat, ameliorate or to prevent cancer, either sequentially or in combination, to a patient in need thereof.

In one embodiment of any of the foregoing aspects, the copper and the agent are capable of inducing the ICD response with a further, second agent.

In yet a further aspect of the disclosure, there is provided a kit comprising copper and an agent, such as an anti-cancer agent, to induce ICD, wherein a combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by measuring ATP secretion, HMGB _| release, CRT exposure or a combination thereof, and wherein the copper, the agent, or both, in non limiting embodiments, are formulated in a liposome or other drug delivery vehicle. The kit may comprise instructions to induce immunogenic cell death, e.g., dosaging or administration instructions, to a patient in need thereof. Alternatively, or in addition, the kit may comprise instructions for loading the copper and/or the agent into the delivery vehicle.

According to any of the foregoing aspects of the disclosure, in alternative embodiments, the copper and the at least one agent are capable of inducing immunogenic cell death with radiation. According to any of the foregoing aspects of the disclosure, in alternative embodiments, the increase in the ICD response in the presence of copper is measured by ATP secretion.

In a further embodiment of the any of the foregoing aspects, in alternative embodiments, the copper is incorporated in the same or a different liposome or other delivery vehicle as the agent.

According to a further embodiment of any of the above aspects, in alternative embodiments, a further, second agent is utilized.

According to any of the foregoing aspects of the disclosure, in alternative embodiments, a cytotoxic effect is realized, as measured by a cytotoxic assay.

According to any of the foregoing aspects of the disclosure, in alternative embodiments, the liposome is a pre-formed liposome. That is, the liposome is prepared in solution and subsequently the agent, such as an anti-cancer agent, is loaded into the liposome by complexation with the metal.

In alternative embodiments of any of the foregoing aspects, the agent is a therapeutic agent that can treat or prevent a disease, infection or condition. The agent may or may not complex with a metal. The agent may be an anti-cancer agent, an anti-viral agent or other therapeutic agent to treat or prevent disease, for example by inducing ICD in the presence of a metal. Moreover, in a further embodiment of any of the foregoing aspects, a nanoparticle or other suitable delivery vehicle is utilized instead of a liposome. Such alternate drug delivery vehicles are known to those of ordinary skill in the art, and may include, without limitation, a vesicle such as an exosome, a micelle, a lipid-based nanoparticle, a polymer-based nanoparticle, an emulsion or a nanotube.

BRIEF DESCRIPTION OF FIGURES FIGURE 1 A shows ATP release of CT26 murine colon cancer cells (referred to herein as“CT26 cells ^" ) treated with copper, zinc, approved chemotherapeutics, CDK inhibitors and combinations of the metal and anti-cancer agents. The approved chemotherapeutics tested included cytarabine (ARA-C), daunorubicin (DNR) and gemcitabine (GEM), and the approved CDK inhibitors included abemaciclib (ABE), palbociclib (PAL) and ribociclib (RIB). The concentration of the metal and anti- cancer agent is as indicated in the figure. The ATP release is indicated as a fold- increase in ATP release relative to an untreated control.

FIGURE 1 B shows ATP release of CT26 cells treated with copper, zinc, terpyridine derivatives or Cu-dependent cytotoxics and combinations of the metal and anti-cancer agents. The terpyridine derivatives include 2,2';6',2"-terpyridine (TER), 4 ^‘ -(4-chlorophenyl)-2, 2 ^" :6, 2"-terpyridine (CPT), 4,4\4"-tri-tert-butyl-2,2':6 ^, ,2"- terpyridine (TTBT). The Cu-dependent cytotoxics include 8-hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) and pyrithione (PYR). The concentration of the metal and anti-cancer agent is as indicated in the figure. The ATP release is indicated as a fold-increase in ATP release relative to an untreated control.

FIGURE 1 C shows ATP release of CT26 cells treated with copper, zinc and other chemical compounds, as well as combinations of the metals and chemical compounds. The chemical compounds tested include emodin (EMD),

epigallocathecin gallate (EPGG), physcion (P14Y) and PX478. The concentration of the metal and anti-cancer agent is as indicated in the figure. The ATP release is indicated as a fold-increase in ATP release relative to an untreated control.

FIGURE 2 shows the ATP and HMGBi release of CT26 cells treated with copper, zinc and an anti-cancer agent as well as combinations of the metals and the anti-cancer agent. The anti-cancer agents tested include 8-Hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) and 4"-Tri-tert-Butyl-2,2’:6\2"- terpyridine (TTBT). The concentration of the metal and anti-cancer agent is as indicated in the figure. The ATP release is indicated as a fold-increase in ATP release relative to an untreated control and HMGBi release is reported as ng/mL.

FIGURE 3 shows ATP and HMGBi release, as well as CRT exposure, of CT26 cells treated with copper and 4,4',4 ^" -Tri-tert-Butyl-2,2':6',2"-terpyridine (TTBT) alone or in combination. The concentration of the metal and anti-cancer agent is as indicated in the figure. The ATP release is indicated as a concentration in nM. HMGBi release is reported as ng/mL and CRT exposure is measured as a fold- increase relative to an untreated control. FIGURE 4A shows the HMGBi release from CT26 cells treated with various agents, with and without copper, which can complex with the metal. The agents tested include daunorubicin (DNR), PX478 and salubrinal (SAL)). HMGBi release was measured in ng/mL. The concentration of copper and the anti-cancer agents is shown in the figure.

FIGURE 4B shows the HMGB i release from CT26 cells treated with various agents, with and without copper, which cannot complex with the metal. The agents tested include cisplatin (CDDP), gemcitabine (GEM) and cytarabine (ARA-C).

HMGBi release was measured in ng/mL. The concentration of copper and the anti cancer agents is shown in the figure.

FIGURE 5 A shows the CRT exposure from CT26 cells of various agents, with and without copper, which can complex with the metal. The agents tested include daunorubicin (DNR), PX478 and salubrinal (SAL). CRT exposure was measured as a fold-increase relative to an untreated control. The concentration of copper and the anti-cancer agents is shown in the figure.

FIGURE 5B shows the CRT exposure from CT26 cells of various agents with and without copper that cannot complex with the metal. The agents tested include cisplatin (CDDP), gemcitabine (GEM) and cytarabine (ARA-C). CRT exposure was measured as a fold-increase relative to an untreated control. The concentration of copper and the anti-cancer agents is shown in the figure.

FIGURE 6A shows the ATP release from CT26 cells after treatment with daunorubicin (DNR) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.

FIGURE 6B shows the ATP release from CT26 cells after treatment with daunorubicin (DNR) and the metals manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.

FIGURE 6C shows the ATP release from CT26 cells after treatment with gemcitabine (GEM) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent. The concentration of gemcitabine and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.

FIGURE 6D shows the ATP release from CT26 cells after treatment with gemcitabine (GEM) and metals, including manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.

FIGURE 6E shows the ATP release from CT26 cells after treatment with 4’- (4-chlorophenyl)-2,2’:6',2 ^'" -terpyridine (CPT) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent. The concentration of the anti cancer agent and the metal is as indicated in the graph. Results are shown as a fold- increase in ATP release relative to an untreated control.

FIGURE 6F shows the ATP release from CT26 cells after treatment with CPT and the metals manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.

FIGURE 6G shows the ATP release from CT26 cells after treatment with 4,4',4”-tri-tert-butyl-2,2’:6 ^' ,2 ^' '-terpyridine (TTBT) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.

FIGURE 6H shows the ATP release from CT26 cells after treatment with 4,4’,4”-tri-tert-butyl-2.2 ^" :6‘,2”-terpyridine (TTBT) and the metals manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.

FIGURE 61 shows the ATP release from CT26 cells after treatment with abemaciclib (ABE) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control. FIGURE 6J shows the ATP release from CT26 cells after treatment with with abemaciclib (ABE) and the metals manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.

FIGURE 7A shows the ATP release from CT26 cells after treatment with various copper salts (copper acetate, copper chloride, copper gluconate and copper sulfate), as well as cobalt, iron, manganese, nickel and zinc. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.

FIGURE 7B shows the ATP release from CT26 cells after treatment with 4,4'.4"-tri-tert-butyl-2,2’:6\2”-terpyridine (TTBT) alone and in combination with various copper salts (copper acetate, copper chloride, copper gluconate and copper sulfate), as well as cobalt, iron, manganese, nickel and zinc. The concentration of the TTBT and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.

FIGURE 7C shows the ATP release from CT26 cells after treatment with diethyldithiocarbamate (DDC) alone and in combination with various copper salts (copper acetate, copper chloride, copper gluconate and copper sulfate), as well as cobalt, iron, manganese, nickel and zinc. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.

FIGURE 8 shows the fold-increase in reactive oxygen species (ROS) from CT26 cells after treatment with different copper salts and various metals. The copper salts examined include: copper sulfate, copper acetate, copper chloride and copper gluconate. The other metals besides copper tested for ROS were zinc, iron, manganese, cobalt and vanadium. Results are shown as a fold-increase in ROS relative to an untreated control.

FIGURE 9A shows ROS concentrations as a function of concentration (0.25- 250 mM) of copper or zinc addition to CT26 cells. Results are shown as a fold- increase in ROS relative to an untreated control.

FIGURE 9B shows the results of ROS studies with CT26 cells using copper or zinc in combination with daunorubicin (DNR), PX478 and salubrinal (SAL). Results are shown as a fold-increase in ROS relative to an untreated control. The metals were added at equimolar concentrations.

FIGURE 9C shows the results of ROS studies with CT26 cells using copper or zinc in combination with cisplatin (CDDP), gemcitabine (GEM) and cytarabine (ARA-C). Results are shown as a fold-increase in ROS relative to an untreated control. The metals were added at equimolar concentrations.

FIGURE 10A shows in vivo vaccination results with immune competent Balb/c mice that were inoculated with CT26 cells which were pre-treated with mitoxantrone (Mito), Mito and copper, cisplatin (CDDP), and CDDP and copper (Day -7). The results are reported as the number of tumour-free mice as a function of days post-challenge by inoculating the flank opposite to the “vaccination site ^" ’ with untreated CT26 cells (Day 0). The untreated control did not receive any pre-treatment or inoculation, serving as an“unvaccinated” control.

FIGURE 10B shows the percentage of tumour free Balb/c mice on Day 39 post-challenge. The mice were inoculated on Day -7 with CT26 cells which were previously treated with mitoxantrone (Mito), Mito and copper, cisplatin (CDDP), and CDDP and copper. Day 0 refers to the day when the mice were challenged with untreated CT26 cells a week after“vaccination” with pre-treated cells. The untreated control did not receive any pre-treatment or inoculation, serving as an“unvaccinated” control.

FIGURE 1 1 A shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: PX-478 alone, gemcitabine (Gem) alone, PX-478 and Gem, Cu(PX-478) alone, and Gem and Cu(PX-478) in combination.

FIGURE 1 I B shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: PX-478 alone, cisplatin (CDDP) alone, PX-478 and CDDP, Cu(PX-478) alone, and CDDP and Cu(PX-478) in combination.

FIGURE 1 1 C shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: 4,4’,4”-tri-ter-butyl- 2,2 :6 ^‘ ,2” - terpyridine (TTT) alone, cisplatin (CDDP) alone, TTT and CDDP, Cu(TTT) ₂ alone, and cisplatin and Cu(TTT) ₂ in combination. FIGURE 1 I D shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: flavopiridol (FLV) alone, mitoxantrone (Mito) alone, FLV and Mi to, Cu(FLV) alone, and Mito and Cu(FLV) in combination.

FIGURE 1 1 E shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: clioquinol (CQ) alone, gemcitabine (Gem) alone, CQ and Gem in combination, Cu(CQ)2 alone, and Gem and CU(CQ) ₂ in combination.

FIGURE 1 I F shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: 4 (4-chlorophenyl) - 2,2':6 ^' ,2”-terpyridine (4CPT) alone, cisplatin (CDDP) alone, 4CPT and CDDP, CU(4CPT) ₂ alone, and CDDP and Cu(4CPT) ₂ in combination.

FIGURE 12A shows secretion of HMGBi (ng/mL) from CT26 cells for the following: untreated, PX-478 alone, gemcitabine (Gem) alone, PX-478 and Gem, Cu(PX-478) alone, and Gem and Cu(PX-478) in combination.

FIGURE 12B shows secretion of HMGBi (ng/mL) from CT26 cells for the following: untreated, flavopiridol (FLV) alone, mitoxantrone (Mito) alone, FLV and Mito, Cu(FLV) alone, and Mito and Cu(FLV) in combination.

FIGURE 13A shows ATP release (nM) of CT26 cells for the following:

untreated, copper, PX478, gemcitabine (GEM), PX478 and GEM, and copper, PX478 and GEM in combination.

FIGURE 13B shows HMGBi release (ng/mL) of CT26 cells for the following: untreated, copper, PX478, gemcitabine (GEM), PX478 and GEM, and copper, PX478 and GEM in combination.

FIGURE 13C shows CRT exposure (fold-increase) of CT26 cell for the following: untreated, copper, PX478, gemcitabine (GEM), PX478 and GEM, and copper, PX478 and GEM in combination.

The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

DETAILED DESCRIPTION

Induction and measurement of Immunogenic Cell Death (ICD)

The compositions and methods described in the present disclosure enhance immunogenic cell death phenotypes, which is also referred to herein as“JCD". In particular, the inventors have examined the ability of various agents to induce ICD by utilizing in vitro assays to measure molecular determinants indicative of ICD and have found that copper in combination with an agent can exhibit an increased ICD response relative to the agent alone. According to one embodiment, the increase in ICD response is attributable to the copper. In another embodiment, the copper potentiates the ICD response of the agent. In certain non-limiting embodiments, after selection of suitable combinations by the foregoing assay, the agent is most advantageously loaded into delivery vehicles. This may include loading of the agent into pre-formed liposomes comprising copper, using, for example, methods described herein. However, as discussed below, other delivery vehicles can be utilized as well for delivery of the metal and/or the agents.

As noted, during ICD, cells emit signals known as damage-associated molecular patterns (DAMPs). ATP is one such DAMP that is secreted during ICD and, without being limiting, is believed to attract monocytes to the site of tumour cell death, which differentiate to dendritic cells (DC) expressing inflammatory DC markers (e.g., CD1 l ^c+ CDl l ^b+ Ly6C ^hl ). Without being limited by any particular theory, it has been reported that tumours treated with mitoxantrone are infiltrated by

CD1 E ⁺ CD 1 l ^b+ Ly6C ^hl cells within 12 h of treatment and subsequently by

macrophages. ATP secretion can be measured in vitro by exposing cells in culture to the agents being tested for a pre-determ ined period of time. ATP is subsequently measured by a reaction in which the ATP secreted is measured by luminescence.

ATP secretion is measured in vitro as follows. Prior to measuring ATP secretion, cells of interest, such as CT26 murine colon cancer cells, are seeded at an appropriate density in multi-well plates, such as 24-well plates. At 24 hours, cells are treated with the agents and the metal ion. Optionally, the metal ion and agent are incubated under conditions that are optimal for metal complexation formation prior to their addition to the cells. However, as noted, in certain embodiments, 1CD induction is not dependent on formation of a complex between the metal and the agent.

Subsequently, the cells are exposed to the tested agents (copper and/or agent) for 24 hours (or left untreated), after which the supernatant of each well is transferred to a new plate. The Promega CellTiter-Glo™ 2.0 Kit is then used, as per the manufacturer ^' s instructions, to determine the extracellular ATP level under each treatment condition. The assay measures ATP by luminescence. Luciferin is converted to oxyluciferin in the presence of ATP, Mg ²⁺ and 0 ₂ . Oxyluciferin in turn emits light that is quantified by a luminometer and is correlated with the amount of ATP present by a standard curve. The assay for use in the present disclosure is described in Promega Technical Manual, CellTiter-Glo® 2.0 Assay, Instructions for Use of Products, G9241 , G9242 and G9243 available on-line at www.promega.com and which is incorporated herein by reference.

A further DAMP is the high-mobility group box 1 , also referred to herein as "HMGBi”. Without being limited to any particular mechanism, HMGBi has been reported to be a late apoptotic marker and its release to the extracellular space facilitate dendritic cell maturation and promote tumour antigen presentation to induce CDS T cells. This molecular determinant of 1CD can be measured by the assay described below.

Similar to the assay for ATP release, cells of interest, such as CT26 murine colon cancer cells, are seeded at an appropriate density in 24-well plates. At 24 hours, cells are treated with the agents of interest. Where copper in combination with an agent or copper complexes are tested, the metal ion may be added to the agent at a predefined molar ratio. For example, in those embodiments in which complex formation is desirable, the metal complexes are formed at a pre-determined metal-to- ligand ratio prior to addition to the cells for optimal metal complexation. Cells are exposed to the copper and/or tested agents for 24 hours, after which the supernatant of each well is collected and the amount of HMGBi release is assessed using a commercial ELISA kit purchased from IBL International™. The test quantifies HMGBi by an anti-HMGB antibody that is immobilized. HMGBi binds to the antibody and a second enzyme marked antibody recognizes the immobilized antibody. Substrate reaction catalyzed by the enzyme leads to a change in colour intensity, which is quantified by known methodology.

Calreticulin (CRT) is another DAMP that is exposed on the surface of tumour cells during cell death. Without being limited by theory, Ecto-CRT is reported to act as a signal that activates phagocytosis by certain antigen presenting cells, contributing to the induction of an immune response. CRT can be measured by flow cytometric analysis of CRT cell surface expression. According to this assay, after 24 hours of treatment of the cells, such as CT26 cells, with copper and/or an agent, the copper and/or agent is removed and replaced with culture media. Cells are harvested at 0, 24, 48, and 96 hours post-treatment and stained with anti-CRT antibody followed by Alexa-488-conjugated secondary antibody. Propidium iodide (PI) is used as a counterstain to differentiate between viable and dead cells. The relative CRT mean fluorescence intensity (MF1) of Pi-negative viable cells is determined by subtracting the MFI of isotype control-stained cells from the MFI of anti-CRT stained cells, and then normalising to HBSS-treated cells. An immunofluorescence assay may also be used to visualize cell surface expression of CRT on cells treated with an agent for 4 hours and stained with the anti-CRT antibody (green), as described above. To enhance visualization, the cell membrane may be labelled with wheat germ agglutinin (red) and the nucleus with Hoechst 33342 (blue). Images may be taken using an IN Cell Analyzer 2200™ and the fluorescent signal (green) can be quantitated and analyzed at a per-well and per-cell level to analyze relative cell surface CRT expression.

As would be appreciated by those of skill in the art, the procedures and/or reaction conditions for the ATP, HMGB 1 and/or CRT assays described above can be modified to achieve optimal results relative to an untreated control.

In a further embodiment, ICD induction by copper is evidenced by each of the three assays or any combination of two of any of the three assays or one of any of the three assays. In a further embodiment, the presence of copper increases the levels of ATP secretion over that of the ICD inducer alone. In yet a further embodiment, the presence of copper enhances the level of FIMGB released with or without an agent that is an ICD inducer. According to yet further embodiments described herein, ICD induction by copper is evidenced by CRT expression. In one embodiment, the ICD response of the copper and the agent in combination as measured by extracellular ATP, HMBG 1 and/or CRT exposure is 1.2 to 10,000, 1.5 to 10,000, 2 to 10,000, 3 to 10,000, 4 to 10,000, or 5 to 10,000 times that of the agent alone measured under otherwise identical conditions. In another embodiment, the ICD response of the copper and the agent in combination as measured by extracellular ATP, HMBG 1 and/or CRT exposure is at least 1.2, 1.3, 1.4, 1.5, 1 .6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 or more times that of the agent alone measured under otherwise identical conditions. In alternative embodiments, the upper limit of the ICD response of the copper and the agent in combination as measured by extracellular ATP, HMBG 1 and/or CRT exposure may be 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 or 10,000 times relative to agent alone. Any combination of the foregoing lower and upper limits is included in select embodiments of the disclosure.

As discussed, the ICD of an agent in combination with copper is determined by measuring a phenotype of ICD (ATP secretion, CRT release and/or HMGBi), also referred to herein as a“marker” of ICD. In alternative embodiments, the cytotoxicity of a particular pharmaceutical formulation and/or treatment regime can be measured using a cytotoxicity assay. In certain non-limiting embodiments, an additive or synergistic cytotoxic effect between copper and an agent may be exhibited (non- antagonistic). Such determination may be made by the Chou Talalay method, which is known to those of ordinary skill in the art.

Without being limiting, in one embodiment, the cytotoxicity assay can be used to determine whether a cytotoxic effect of the agent of interest in combination with copper is present. Moreover, in those embodiments that additionally include radiation and/or a second agent, the composition or treatment w ill be considered to have a cytotoxic effect if any combination selected from at least two of (i) copper, (ii) an agent, (iii) a second agent if two or more are used, and (iii) radiation treatment exhibits a cytotoxic effect in vitro. By way of example, if a pharmaceutical composition comprises copper as well as a first and a second agent in combination, and if the second agent exhibits a cytotoxic effect in combination with copper, but the first agent in combination with copper does not, nor the first agent in combination with the second agent, the pharmaceutical composition will still be considered to have a cytotoxic effect as used herein. Yet in a further example, if a pharmaceutical composition comprises copper and a first and a second agent in combination, and if the first agent exhibits a cytotoxic effect in combination with the second agent, but the first or second agent in combination with copper does not, the pharmaceutical composition will still be considered to have a cytotoxic effect as used herein. In another non-limiting, illustrative example, if a treatment comprises an agent in combination with the metal and additionally radiation treatment, and if the agent and the radiation treatment exhibit a cytotoxic effect in combination, then the treatment will be considered cytotoxic, even if the metal and agent demonstrate no cytotoxic effect in combination. Additional examples will be readily envisioned by those of ordinary skill in the art.

As noted, whether a cytotoxic effect is observed can be measured using an in vitro assay. Cells are seeded in 384-well plates and treated with various

concentrations of copper and agent (0.001 nM to 10 mM) for 72 hours. Cells are then stained with Hoechst 33342 and ethidium homodimer-1 for total and non-viable (cells that have lost membrane integrity), respectively. The cells are subsequently imaged using an automated fluorescent microscopic platform such as the IN Cell Analyzer 2200. All images are processed through software such as the IN Cell Developer Toolbox which provides total and dead cell counts. The viable cell counts are used to generate dose responsive curves using curve-fitting programs such as PRISM™ (GraphPad) to determine the potency of each treatment as determined by IC ₅ o values.

For combinations involving radiation, the cells are seeded in plates and treated with the agent of interest with or without copper for 24 hours, after which the cells are irradiated at different doses (2, 4, 6, 8, 10 Gy). The irradiated cells are immediately plated in culture dishes to achieve 100-500 colonies two weeks later. The colonies are stained with aqueous malachite green or crystal violet and dried overnight prior to colony counting. The surviving fraction (SF) is calculated using the equation SF =

[no. of colonies formed after treatment / (no. of cells seeded x plating efficiency)].

The dose response curves from each treatment are then compared to assess treatment efficacy in vitro.

As noted, the metal ion of select embodiments is copper. The metal ion may also be an ion of a transition metal or a Group lllb metal. The transition metal may be from Group IB, 2B, 3B, 4B, 5B, 6B, 7B and 8B (groups 3- 12). Examples of transition metals include copper, zinc, vanadium, manganese, iron, cobalt and nickel. In another embodiment, the metal has d-orbitals. The Group lllb metal is from the boron family, which includes boron, aluminum, gallium, and indium. In one embodiment, the metal is in the 2 ⁺ oxidation state.

Agents

By the term "agent ^" , as used herein it is meant any compound or molecular species to treat, ameliorate, and/or prevent a disease or condition; a compound or molecular species that serves as a carrier for transporting the metal ion to facilitate loading into a given delivery vehicle and/or to facilitate delivery of the metal after administration; or a compound or molecular species that serves as an adjuvant. The disease or condition includes any proliferate disorder or disease and includes, but is not limited to, cancer, infectious diseases or any other condition in which 1CD is desirable.

In alternative embodiments, the agent of select embodiments used in combination with copper typically comprises at least one complexation moiety or ligand to enable complexation with the copper. However, it will be appreciated that, in certain embodiments, the agent for use in the pharmaceutical compositions described herein need not complex with the metal.

In those non-limiting embodiments in which the agent complexes with a metal, the agent may have a chemical group, often referred to as a ligand, which has atoms that allow for the coordination of the agent with the metal. The chemical group may be selected from an S-donor, O-donor, N, O-donor, a Schiff base, hydrazones, P- donor phosphine, N-donor or a combination thereof. In another embodiment, the moiety is a hard electron donor. Other moieties or ligands known to those of skill in the art suitable for complexation with a metal ion are included within the scope of certain embodiments as well. In a further embodiment, the complexation via metal coordination facilitates drug loading as described in co-owned WO 2017/100925 (Bally et al.). This could be measured by incubating an agent of interest with a liposome containing copper, or other metal ion of interest, under optimal conditions to facilitate drug uptake and measuring drug uptake after one hour. In one embodiment, the drug uptake after one hour is at least 30%, 40%, 50%, 60%, 70% or 80% as determined by measuring the drugilipid ratio relative to the theoretical drugdipid ratio that could ideally be obtained. In another embodiment, the drug uptake after one hour is between 60% and 100% as measured above. The agent tnay be selected from antineoplastic agents, for example cytotoxic agents. Such agents include, but are not limited to, nucleoside analogues, anthracyclines, anti-folates, topoisomerase I inhibitors, taxanes, vinca alkaloids, alkylating agents, platinum compounds, targeted antineoplastics, including monoclonal antibodies, tyrosine kinase inhibitors, mTOR inhibitors, CDK inhibitors, retinoids, immunomodulatory agents and histone deacetylase inhibitors, terpyridine derivatives and Cu-dependent cytotoxics.

In one embodiment, an agent suitable for use in certain embodiments is selected from PX-478, Emodin, clioquinol (CQ), pyrithione (PYR), flavopiridol (FLV), diethyldithiocarbamate (DDC), epigallocathecin gallate (EPGG), cisplatin (CDDP), gemcitabine (GEM), mitoxantrone (MITO), 4' (4-chlorophenyl) - 2.2 ^‘ :6 ^' ,2 ^" -terpyridine (4CPT), 4, 4\4”-tri-ter-butyl-2,2':6\ 2" - terpyridine (TTT), shikonin, wogonin, cytarabine (ARA-C), oxaliplatin (OXP), salubrinal (SAL), abemaciclib (ABE), palbociclib (PAL) and ribociclib (RIB), 2,2';6’,2"-terpyridine (TER), 4 ^' -(4-chlorophenyl)-2, 2’:6, 2”-terpyridine (CPT), 4,4',4"-tri-tert-butyl- 2,2 ^' :6\2 ^” -terpyridine (referred to herein as“TTBT" or“TTT"), 8-hydroxyquinoline (8HQ), physcion (PHY), diacetyl-bis(N4-methylthiosemicarbazone) (ATSM) and glyoxal-bis (N4-methylthiosemicarbazone) (GTSM). In one embodiment, the agent is not daunorubicin or doxorubicin.

In those embodiments in which the agent complexes with copper, or another metal, the agent may function as a carrier moiety, such as an ionophore. The ionophore may facilitate loading of the metal into a delivery vehicle. For example, the agent may function as an ionophore to facilitate the delivery of a metal complexed therewith across a lipid bilayer of a delivery vehicle, such as a liposome. According to such embodiments, the agent and copper may be added to the external solution of a liposome preparation. The agent may form a complex with the metal in the solution external to the liposome and the resultant copper-ionophore complex may

subsequently cross the lipid bilayer and enter the aqueous interior of the delivery vehicle.

Moreover, without being limiting, after administration of a delivery vehicle comprising the metal-carrier complex, the carrier agent may facilitate the transport of the bound copper in the complex to a disease site. Without being bound by theory, such a carrier agent may facilitate the transport of the metal across a cellular membrane. In one embodiment, the agent additionally exerts a therapeutic effect rather than simply functioning as a carrier agent.

Non-limiting examples of agents that complex with copper and that may function as a carrier for the metal include diacetyl-bis(N4-methylthiosemicarbazone) (ATSM), clioquinol (CQ) and diethyldithiocarbamate (DDC). Without being limiting, in certain embodiments, Cu-ATSM is permeable across cellular membranes and can be reduced in hypoxic tissue. A reduction in hypoxic conditions (such as at a tumour site) may allow the copper atom to dissociate from the ATSM. In some embodiments, this may allow copper to be continuously deposited in hypoxic tissues and cells.

Without being limiting, the agent for use in select embodiments may be poorly soluble in solution prior to or after complexation with the metal ion. By this it is meant that the poorly soluble agent in free form has a solubility of less than 1 mg/iriL in either water or a solution of the metal ion which complexes with the agent.

Solubility of the agent in water or in the presence of the metal ion (10 mM to 500 mM) is measured at conditions of physiological pH and temperature after 60 minutes of incubation under these conditions. Measurement of solubility of the agent is conducted as described in co-owned WO 2017/100925.

In yet further embodiments, the agent has a solubility that is greater than 1 mg/mL in either water or a metal ion solution, as determined by the assay described above (see WO 2017/100925).

Preparation of pharmaceutical formulations

The agent and metal may be formulated in any known delivery vehicle. As used herein, the term“delivery vehicle” means a particle of a size between 10 nm and 2000 nm that contains associated therewith the agent, copper or a combination thereof. The delivery vehicle typically controls the rate of release of its cargo.

In some embodiments, the agent and copper are both administered in free form or one of the agent and the metal are formulated in the delivery vehicle, while the other is in free form (such as formulated with a pharmaceutically acceptable carrier, such as a salt). Whether or not the agent and/or the metal are formulated in the delivery vehicle may depend on the mode of administration. For example, if the agent and copper are introduced directly to a disease site, such as by direct injection, then formulation in a delivery vehicle may not be necessary. Examples of suitable delivery vehicles include vesicles such as liposomes and exosomes, micelles, polymer-based nanoparticles, emulsions, or nanotubes. The agent may also be conjugated with a lipid or polymer to improve its ability to be formulated in a given drug delivery vehicle.

As discussed, in alternative embodiments, the agent may be formulated in a liposome. In alternative embodiments, the liposome is a vesicle comprising a bilayer having amphipathic lipids enclosing an internal solution. The liposome may be a large unilamellar vesicle (LUV), which can be prepared as described below using extrusion. In one embodiment, the average diameter of the liposome may be between 60 nm and 2,000 nm, 70 and 1 ,000 nm, 70 and 500 nm, 70 and 200 nm or 70 and 180 nm. The liposome may comprise lipids including phosphoglycerides and

sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, pahnitoy!oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophos- phatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid and glycosphingolipid families are also encompassed by certain embodiments. The phospholipids may comprise two acyl chains from 6 to 24 carbon atoms selected independently of one another and with varying degrees of unsaturation. Additionally, the amphipathic lipids described above may be mixed with other lipids including triacylglycerols and sterols. As would be appreciated by those of skill in the art, lipids that interfere with liposome formation in the presence of a metal should typically be avoided. Whether or not a given lipid is suitable for liposome formation in the presence of a metal ion can be determined by those of skill in the art. In one embodiment, the liposome comprises the lipids 1 ,2- distearoyl-sn-glycero-3- phosophocholine (DSPC) and cholesterol. The precise ratios of the lipids may vary as required. A non-limiting example of a suitable ratio of DSPC/Cholesterol is 55:45 mokmol.

The liposomes or other drug delivery vehicles may also comprise a hydrophilic polymer-lipid conjugate. The hydrophilic polymer may be a

polyalkylether, such as polyethylene glycol. The hydrophilic polymer-lipid conjugate is generally prepared from a lipid that has a functional group at the polar head moiety that is chemically conjugated to the hydrophilic polymer. An example of such a lipid is phosphatidylethanolamine. The inclusion of such hydrophilic polymer-lipid conjugates in a liposome can increase its circulation longevity in the bloodstream after administration. The hydrophilic polymer is biocompatible and has a solubility in water that permits the polymer to extend away from the outer surface of the liposome. The polymer is generally flexible and may provide uniform surface coverage of the liposome outer surface. In addition, it has been found herein that the inclusion of such a hydrophilic polymer-lipid conjugate can increase the amount of the transition metal encapsulated in the liposome. This can be used as a methodology to increase the amount of the agent encapsulated in the liposome. In one embodiment, the liposome may include a hydrophilic polymer, such as polyethylene glycol (PEG) at between 1 and 20 mol% or between 2 and 10 mol%. A non-limiting example of a liposomal formulation comprising PEG is DSPC/CHOL/PEG (50:45:5, mole ratio) or DSPC /PEG (95:5, mole ratio). The specific ratios of the lipids, however, may vary according to embodiments that would be apparent to those of ordinary skill in the art.

Liposomes can be prepared by any of a variety of suitable techniques known to those of skill in the art. An example of one suitable method involves cycles of freeze-thaw and subsequent extrusion of lipid preparations. According to one such method, lipids selected for inclusion in a liposome may be desiccated and dissolved in a solvent, such as an organic solvent, at a desired ratio. After removal of the solvent, the resultant lipids are hydrated in an aqueous solution. The solution in which the lipids are hydrated forms the internal solution of the liposomes. Subsequently the hydrated lipids may be subjected to cycles of freezing and thawing. The hydrated lipids are passed through an extrusion apparatus to obtain liposomes of a defined size. The size of the resulting liposomes may be determined using quasi-electric light scattering (e.g., using a NanoBrook ZetaPALS™ Potential Analyzer).

Without being limiting, the liposomes may be prepared so that they comprise an internal solution comprising the metal ion. For example, when preparing liposomes by freeze-thaw and subsequent extrusion as described above, the lipids are hydrated in a solution comprising a metal ion. Generally, the liposomes so formed will comprise the metal ion not only in the internal solution of the liposomes, but also in the external solution. Unencapsulated metal ion may be removed from the external solution of the liposome prior to loading of the agent. For example, the external copper solution may be exchanged with a solution containing substantially no copper ions by passage through a column equilibrated with a buffer. Other techniques may be employed such as centrifugation, dialysis, the addition of a chelating agent, such as EDTA (to chelate the metal) or related technologies. Typically the solution that exchanges with the metal-containing solution is a buffer, although other solutions may be used as desired. The liposomes may be subsequently concentrated to a desired lipid concentration by any suitable concentration method, such as by using tangential flow dialysis. In one embodiment, the solution external to the liposome contains substantially no metal ions that complex with the poorly soluble agent. By this it is meant that the concentration of metal ions in the external solution is less than that of the metal ion concentration in the liposome, for example less than one fifth of the concentration of metal ion in the liposome. Alternatively, or in addition, the external solution may comprise a chelating agent that chelates with the metal ions. As noted, the metal ion may be encapsulated in the liposome as a metal salt. Examples include copper sulfate, copper chloride or copper gluconate.

The pre-formed liposomes comprising the metal ion may be incubated with the agent to facilitate uptake via metal complexation. The agent may be added in any suitable form, including as a powder or as a solution. If the agent is insoluble in water, it can be added as a powder. The amount of free agent in solution can subsequently be increased by increasing the temperature. Incubation of the pre formed liposomes with the one or more agents is performed under conditions sufficient to allow the agent to move across the phospholipid bi layer of the liposome into the internal solution thereof. Such a method is referred to by those of skill in the art as "loading". Movement of the agent across the phospholipid bilayer of the pre formed liposome during loading may occur independently of any pH gradient across the bilayer. The loading may, however, be dependent on other factors. As will be appreciated, the loading conditions can be readily selected by those of skill in the art to achieve a desired rate of loading. For example, the diffusion of the agent across the bi layer may be dependent on the temperature and/or lipid composition of the liposome.

Without being bound by theory, the formation of the drug-metal complex incorporated in the pre-formed liposomes may be characterized as an inorganic synthesis reaction. In certain embodiments, the uptake of drug during the loading reaction is visualized as a colour change as many metal complexed agents have different spectral characteristics that can be detected by eye. For example, a colour change to purple, brown, green or yellow can be observed during loading with copper.

By formulating complexes through such an inorganic synthesis reaction occurring within the pre-formed liposome, a high drug-to-lipid ratio may be attained according to certain embodiments. For example, the drug-to-lipid ratio may be at least 0.3: 1 0.4: 1 , 0.5: 1 , at least 0.6: 1 , at least 0.7: 1 , or at least 0.8: 1. In further embodiments, the drug-to-lipid ratio is about 0.1 : 1 to about 0.6: 1 (molanol), about 0.15: 1 to about 0.5: 1 (molanol) or about 0.2: 1 to about 0.4: 1 (molanol). Such a high drug-to-lipid ratio may be dependent on the number of metal ions inside the liposome and/or the nature of the complex formed. Formation of a transition metal complex with a given agent may be rapid (e.g., Cu(DDC) ₂ ), occurring in minutes, or more gradual. The complexation reaction rate may be temperature dependent. The rate of metal-agent complex formation may also be dependent on the rate at which the externally added agent crosses the lipid bilayer of the liposome. As will be appreciated by those of skill in the art, these variables can be adjusted as desired to achieve a desired rate of uptake of the agent or metal-agent complexation if the latter mechanism facilitates loading.

In addition, loading of an agent may be facilitated by a carrier agent that acts as an ionophore, as described previously.

The liposome formulations may be either passively or actively loaded with the agent. However, active loading is preferred as it may result in higher trapping efficiencies and drug retention properties relative to passive loading. Nonetheless, in order to encapsulate more than one agent, a combination of passive and active entrapment methods can be utilized. For example, an agent may be passively entrapped in a liposome with a metal ion and a second agent could be loaded by the metal loading method described above.

In one embodiment, the liposome has a trapping efficiency of at least 80% with respect to at least one agent encapsulated in the liposome. In another

embodiment, the trapping efficiency is at least 90%. Such high trapping efficiencies may result from active loading. By contrast, passive loading may result in comparatively lower trapping efficiencies.

While liposomes are discussed above, other vesicles besides liposomes can be used to formulate the copper, the agent or both. For example, an exosome may serve as a delivery vehicle for one or more of the agents and/or copper. An exosome comprises a lipid bilayer enclosing an internal aqueous space and is derived from a living or dead organism, explanted tissues or organs and/or cultured cells. An exosome is typically derived from plasma membrane budding from a producer cell as described in U.S. Patent No. 10, 195,290, which is incorporated herein by reference. Exosomes may be utilized to deliver a variety of agents, such as DNA, RNA, protein and lipid. Unique protein and carbohydrate molecules on the surface of exosomes may allow them to be transported to a cellular target site in the body and be taken up efficiently by cells. The term exosome as used herein is meant to include a hybridosome®, which is a hybrid of a lipid nanoparticle and exosome, and vexosomes that transport a virus into cells via an exosome.

The delivery vehicle may include other particles that are not based on vesicle formation, although such delivery vehicles may include a surface stabilizing lipid bilayer or monolayer. An example is a micelle that is any particle or assembly that forms a core that is hydrophobic and comprises more hydrophilic regions that surround the core and that are in contact with the surrounding solvent. Micelles are generally used for the delivery of hydrophobic or water insoluble agents that are contained in the hydrophobic core. The micelle may be made up of lipids with hydrophilic heads facing outwardly and the hydrophobic tails facing inwardly to form the hydrophobic core of the particle. However, the components that make up a micelle can vary widely and include molecules such as lipoproteins, including high and low density lipoproteins, chylomicrons, fatty acids, lipids, polymers or any suitable combination. Moreover, micelles can be made up of a single molecular species or may include particles with a hydrophobic core composed of one molecular species and a hydrophilic region contacting the surrounding solvent that is composed of a different molecular species. For example, a core may be made of fatty acids surrounded by a mono or bilayer of lipids or other amphipathic molecules.

The delivery vehicle may also be a polymer-based nanoparticle or microparticle. The polymer-based nanoparticles or microparticles are typically produced from synthetic polymers, but can include natural materials as well as lipid monolayers and bi layers. Non-limiting examples of nanoparticles and microparticles comprise a core of drug surrounded by a polymeric shell or a drug dispersed throughout a polymeric matrix. The drug may be dispersed as a solid or liquid in the nanoparticle or microparticle. The agent may also be covalently bonded or associated with the particle by an electrostatic interaction. The nanoparticle or microparticle may be a polymer/lipid hybrid, such as a polymer core containing an agent surrounded by a lipid monolayer or bilayer.

Other delivery vehicles include emulsions or nanotubes. The emulsion may be an oil-in-water emulsion that contains one or more water insoluble agents in the oil phase. A nanotube is a carbon-based cylindrical tube in which an agent can be loaded. The particles may comprise a graphene sheet that forms into the shape of a cylinder.

Alternative delivery vehicles may be employed besides those specifically exemplified above.

Although the incorporation of a metal and an agent, optionally as a metal- agent complex, within a liposome or other drug delivery vehicle is described, other embodiments encompass formulations for 1CD induction in which copper is administered to a patient separately from the delivery vehicle comprising the agent. According to such embodiments, the copper is typically administered separately or together with the delivery vehicle. Typically, a copper chelate is administered by injection. Examples of copper chelates include Cu(ATSM) and Cu(GTSM). In another embodiment, the copper is formulated as a salt. For example, copper gluconate may be administered orally. The agent may be administered as a liposomal or other drug delivery formulation before, during or after administration of the free copper. In those embodiments utilizing liposomal delivery of the agent wdthout co encapsulation with copper, the agent may be loaded into the liposome using active loading methods besides metal loading, such as pH gradient loading, or passive loading methods as known to those of ordinary skill in the art. After administration, the free copper may complex with the agent or can remain uncomplexed. In a further embodiment, a liposome or other drug delivery vehicle comprising copper is administered separately or together with one or more ICD inducing agents that are in free form. Likewise, after administration, the copper may complex with the agent in free form, or remain uncomplexed.

Additional combinations of anti-cancer agents and metal ions for inducing ICD

According to certain features of select embodiments, one or more additional agents may be used to induce ICD as described herein. The combinations of two or more drugs may exhibit increased ATP secretion, HMBG _] release and/or CRT expression relative to treatment with one agent alone and/or complexed with metal. The one or more additional agent may also complex with the metal or may be present in free form. These additional agents may be selected from antineoplastic agents such as cytotoxic agents, including nucleoside analogues, anthracyclines, anti-folates, topoisomerase 1 inhibitors, taxanes, vinca alkaloids, alkylating agents, platinum compounds, targeted antineoplastics, including monoclonal antibodies, tyrosine kinase inhibitors, mTOR inhibitors, CDK inhibitors, retinoids, immunomodulatory agents, histone deacetylase inhibitors, terpyridine derivatives and Cu-dependent cytotoxics. In one embodiment, a second agent suitable for use in certain

embodiments is selected from PX-478, Emodin, clioquinol (CQ), pyrithione (PYR), flavopiridol (FLV), diethyldithiocarbamate (DDC), epigallocathecin gallate (EPGG), cisplatin (CDDP), gemcitabine (GEM), mitoxantrone (MITO), 4 ^' (4-chlorophenyl) - 2,2':6 ^' ,2"-terpyridine (4CPT), 4,4’,4’ ^' -tri-ter-butyl-2,2’:6’,2 ^” - terpyridine (TTT), shikonin, wogonin. cytarabine (ARA-C), oxaliplatin (OXP), salubrinal (SAL), abemaciclib (ABE), palbociclib (PAL) and ribociclib (RIB), 2,2';6',2"-terpyridine (TER), 4’-(4-chlorophenyl)-2, 2 ^' :6, 2 ^" -terpyridine (CPT), 4,4’,4 ^!‘ -tri-tert-butyl- 2,2’:6',2”-terpyridine (TTBT), 8-hydroxyquinoline (8HQ), physcion (PHY), diacetyl- bis(N4-methylthiosemicarbazone) (ATSM) and glyoxal-bis (N4- methylthiosemicarbazone) (GTSM). In one embodiment, the agent is not daunorubicin or doxorubicin.

In a further embodiment, one or more agents complexed with a metal may be used to treat cancer in combination with an additional therapy such as radiation.

Advantageously, the methods described in select embodiments herein can be used to load multiple agents, either simultaneously or sequentially into a liposome or other delivery vehicle. If each of the agents is incorporated into a liposome, the agents can be loaded by the complexation method described herein. Moreover, the liposomes into which the agent is loaded may themselves be prepared so that the internal solution comprises not only the metal ion but also an additional agent.

Loading of an agent in this manner is often referred to as passive loading. The subsequent loading of a second agent which complexes with the metal in the pre formed liposome (as described above) will result in incorporation of two agents in the liposome, one of which is loaded passively and the other actively via complexation. Since the passively loaded agent need not complex with a metal ion to effect loading, this approach provides greater flexibility in preparing liposome-encapsulated agent combinations for use to treat or prevent a disease of interest. Moreover, in certain non-limiting embodiments, the two or more agents may be loaded at a predetermined ratio that exhibits synergistic or additive effects as elucidated by the Chou-Talalay determination, which is a known methodology for measuring such effects.

A formulation of liposomes may also comprise two or more liposome populations, which incorporate the same or different agents, comprise different lipid formulations, or comprise liposomes of different vesicle sizes. Moreover, one or more agents may be in free form, while one or more agents may be incorporated in a liposome. For example, the copper may be in free form and the agent or agents incorporated in a liposome. In another embodiment, the copper may be incorporated in a liposome and the agent or agents may be in free form in the pharmaceutical composition. The combinations of agents selected for the pharmaceutical formulation may achieve greater therapeutic efficacy, safety, prolonged drug release or targeting after administration.

According to one embodiment, a second agent in free form is included in a treatment regime such that the drug becomes active in the presence of the metal ion. Examples of such drug combinations include co-encapsulation of metal-CQ and free DSF, the precursor of DDC. The DSF is metabolized to form DDC and DDC and is subsequently activated in the presence of a metal ion, such as copper, at the tumour site.

Although liposomal formulations incorporating copper and two or more agents are described, it is also possible to incorporate the copper and/or two or more agents into other delivery vehicles, such as those described previously.

Administration

In alternative embodiments, the pharmaceutical composition is generally administered to treat and/or prevent cancer, although other diseases, infections and conditions are contemplated in which 1CD induction by a metal provides a prophylactic (preventive), ameliorative or a therapeutic benefit. The pharmaceutical composition will be administered at any suitable dosage. In one embodiment, the pharmaceutical compositions is administered parentally, i.e., intra-arterially, intravenously, subcutaneously or intramuscularly. In other embodiments, the pharmaceutical compositions described herein may be administered topically. In still further alternative embodiments, the pharmaceutical compositions described herein may be administered orally. In yet a further embodiment, the pharmaceutical compositions are for pulmonary administration by aerosol or powder dispersion.

In yet a further embodiment, the pharmaceutical compositions are for intra- tumoural administration. If intra-tumoural injection is employed, incorporation of the metal and agent in a drug delivery vehicle is optional.

The compositions described herein may be administered to a patient. The term patient as used herein includes a human or a non-human subject.

The following examples are given for the purpose of illustration only and not by way of limitation on the scope of the invention.

EXAMPLES

Example 1 : Copper enhances ATP release

This example shows that ATP secretion, which is a marker of immunogenic cell death, is enhanced in vitro when cells are treated with copper, but not zinc. The cells were treated with various combinations of copper, zinc and metal-binding anti cancer agents of interest. The results are shown in Figure 1 A, I B and 1 C.

CT26 murine colon cancer cells were seeded at 200,000 cells/well in 24-well plates. After 24 hours, the cells were treated with copper or zinc alone and in combination with the indicated agents, which included:

(i) approved chemotherapeutics including cytarabine (ARA-C),

daunorubicin (DNR), gemcitabine (GEM) (Figure 1 A);

(ii) CDK inhibitors including abemaciclib (ABE), palbociclib (PAL) and ribociclib (RIB) (Figure 1 A);

(iii) terpyridine derivatives, including 2,2';6',2"-terpyridine (TER), 4’-(4- chlorophenyl)-2, 2':6, 2"-terpyridine (CPT), 4,4\4"-tri-tert-butyl- 2,2 ^' :6 ^? ,2"-terpyridine (TTBT) (Figure I B); (iv) Cu-dependent cytotoxics, including 8-hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) and pyrithione (PYR) (Figure 1 B); and

(v) other chemical compounds including emodin (EMD), epigallocathecin gal late (EPGG), physcion (PHY) and PX478 (Figure 1C).

Where copper or zinc complexes were tested, the metal ion was added to the ligand (agent) at the indicated molar ratio such that metal complexes between the copper or zinc and the anti-cancer agent were formed prior to addition to the cells. (However, it should be understood that the invention is not limited to the formation of any metal-agent complexes, as discussed below). The cells were exposed to the tested agents for 24 hours, after which the supernatant of each well was transferred to a new plate. The Promega CellTiter-Glo® 2.0 Kit was then used immediately, as per the manufacturer ^' s instructions, to determine the extracellular ATP level under each treatment condition. The assay for measuring extracellular ATP involves measuring luminescence as described previously.

The results of ATP secretion from CT26 cells for each anti-cancer agent in the presence and absence of copper or zinc are shown in Figures 1 A- 1 C. Copper and zinc were mixed with the anti-cancer agent at the indicated molar ratio (ligandunetal ion). The data in Figures 1 A to 1 C demonstrate that treatment of the cells with the anti cancer agents yields increased ATP release when the agents were used in combination with Cu ²⁺ , but not with Zn ²⁺ . In all cases, the increase in extracellular ATP of the combination of the agent and Cu ²⁺ was increased by a statistically significant amount relative to treatment of cells with agent alone. It is also notable that enhancements in ATP release in the presence of copper were still observed for agents that do not bind copper (GEM and ARA-C), suggesting that copper complexation may not be required for induction of ICD markers in vitro.

Example 2: Conner enhances at least two ICD markers including ATP and HMGBi release

The ability of copper to enhance two markers of ICD, namely ATP secretion and HMGBi release, was also examined. Copper or zinc were added to cells, either alone or together with 8- hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) and 4,4',4”- tri-tert-butyl-2,2':6\2”-terpyridine (TTBT) at the molar concentrations indicated, followed by measurement of extracellular ATP levels and HMGBi release.

ATP secretion was tested following the procedure set forth in Example 1. For the measurement of HMGBi release, similar to the assay for ATP release, CT26 murine colon cancer cells were seeded at an appropriate density in 24-well plates. At 24 hours, cells are treated with the agents of interest. Cells were exposed to the copper and/or tested agents for 24 hours, after which the supernatant of each well was collected and the amount of HMGBi release was assessed using a commercial ELISA kit purchased from IBL International™. The test quantifies HMGBi by an anti- HMGBi antibody that is immobilized. HMGBi binds to the antibody and a second enzyme marked antibody recognizes the immobilized antibody. A substrate reaction catalyzed by the enzyme leads to a change in colour intensity, which is quantified by known methodology as described previously.

Copper and zinc were added to cells, together with 8-hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) or 4,4’,4”-tri-tert-butyl-2,2’:6’,2”- terpyridine (TTBT) at the molar concentrations indicated, followed by measurement of the HMGBi .

As shown in Figure 2, combinations with copper and the anti-cancer agents examined showed enhancements in ATP release relative to untreated cells and the agent alone, while zinc did not display such enhancements (top panel). As further shown in Figure 2. copper in combination with the anti-cancer agents examined showed enhancements in HMGBi release relative to untreated cells and the agent alone (bottom panel). Similar to the ATP studies, the addition of zinc did not result in any enhancements in HMGBi release.

Thus, the data shown in Figure 2 reveals that copper can enhance both ATP and HMGB i release in combination with an anti-cancer agent and that the effects observed are greater than those observed with the anti-cancer agent alone.

Example 3: Conner enhances each of the three markers of ICD The ability of copper to enhance all three markers of ICD, as measured by ATP secretion, HMGB, release and CRT exposure was also examined.

Copper and 4, 4',4"-Tri-tert-Butyl-2,2':6\2”-terpyridine (TTBT) alone or in combination were added to cells at concentrations of 25 mM.

ATP secretion and HMGB _] release were determined in vitro as described previously.

To quantify CRT exposed on the cell membranes, a 96-well black-walled, clear bottom plate was coated with 50 pL/well of 25 pg/mL poly-D-lysine in water for 1 h at room temperature to facilitate cell adherence. The coating solution was aspirated and 20,000 CT26 cells/well were seeded at 100 pL/well. Cells were grown for 24 h and thereafter 100 pL of anti-cancer agents or medium was added to each well. The cells were exposed to the drugs for 24 h, at which time the cells were washed 3x with 100 pL/well of HBSS with calcium and magnesium without phenol red and fixed with 50 pL/well of 4% methanol-free formaldehyde diluted in phosphate-buffered saline (PBS) for 20 min at room temperature. Methanol-free formaldehyde was used to minimize permeabilization of cell membranes during fixation. Cells were washed 3x with HBSS and then 50 pL/well of primary antibody against CRT (diluted 1 :200 in staining buffer) was added for 1 h on ice. Cells were washed 3x with HBSS and stained with 50 pL/well of secondary antibody conjugated to DyLighf ^1* 488 (diluted 1 :500 in staining buffer) for 30 min on ice and in the dark. The plate was washed 3x with HBSS and 50 pL/well of Hoechst 33342 (diluted 1 :2000 in PBS) and CellMask™ Deep Red (diluted 1 : 1000) was added for 20 min at room temperature to stain the cell nuclei. Cells were finally washed 3x with HBSS and imaged in 100 pL/well of PBS with the IN Cell Analyzer 2200. Twenty images were acquired for each well. The flat field correction was applied during the image acquisition. Mean fluorescence intensity of CRT on the cell membranes was quantified for each individual cell by conducting a multi-target analysis using the IN Cell 2200 Workstation 3.7 software (GE Healthcare). The average of mean fluorescence intensities for all cells under the same condition was calculated and normalized to untreated cells in order to express the results as fold-increase in CRT exposure.

Primary antibody against CRT (product number PA3-900), secondary antibody conjugated to DyLight® 488 (product number 35552), Hoechst 33342 and CellMask™ Deep Red were obtained from Thermo Fisher Scientific, Waltham, MA, USA.

The results in Figure 3 show that copper in combination with the anti-cancer agent, TTBT, increased each of the three molecular markers associated with 1CD.

Example 4: Determination of HMGB release and CRT exposure with and without copper-agent complexation

The ability of anti-cancer agents to increase HMGET release or CRT exposure with and without complex formation with copper was tested. The agents examined that complex with copper include daunorubicin (DNR). PX478 and salubrinal (SAL) and those that are not capable of forming complexes include CDDP, gemcitabine (GEM) and cytarabine (ARA-C). FlMGBi release and CRT exposure was determined according to the above-described HMGB and CRT assays in CT26 cells.

FlMGBi release in the presence of copper, as shown in Figures 4A and 4B, was comparable for the agents that complex with copper (Figure 4A; DNR, PX478 and SAL) and those that do not (Figure 4B; CDDP, GEM and ARA-C).

Likewise, as shown in Figures 5A and 5B, the addition of copper to the anti cancer agents tested had no significant impact on CRT exposure levels observed, regardless of whether a copper binding agent (Figure 5A; DNR, PX478 and SAL) or a non-binding agent (Figure 5B; CDDP, GEM and ARA-C) was used.

These results suggest that complexation of an agent with copper may not be required for 1CD induction as evidenced by assays that respectively measure HMGB; release and CRT exposure.

Example 5: Enhancement of ICD as measured by at least ATP secretion is copper specific

As suggested by the data above, copper enhances ICD in vitro , as measured by the release of ATP and HMBGi while zinc does not. To determine whether the ICD effect is copper-specific, other metals besides zinc were tested to determine whether or not they could induce ICD as measured by ATP secretion. As shown below, the ICD effect as determined by ATP release suggests that ICD is induced by only copper and not by other metals. Extracellular ATP release was measured as set forth above with various anti cancer agents in combination with copper, zinc, iron, manganese, cobalt and vanadium at the indicated molar concentrations. The anti-cancer agents examined included daunorubicin (DNR; Fig. 6A and 6B), gemcitabine (GEM; Fig. 6C and 6D), 4’-(4-chIorophenyl)-2, 2':6, 2 ^" -terpyridine (CPT; Fig. 6E and 6F), 4,4’ ,4”-tr i-tert- butyl-2,2’:6’,2”-terpyridine (TTBT; Fig. 6G and 6H) and abemaciclib (ABE; Fig. 61 and 6J). The results in Figures 6A-J show that the induction of ATP in vitro occurs only with copper and an anti-cancer agent, and not with the agent combinations including other metals, namely zinc, iron, manganese, cobalt and vanadium.

Figure 7 shows similar results with various copper salts (copper acetate, copper chloride, copper gluconate and copper sulfate), as well as cobalt, iron, manganese, nickel and zinc. The anti-cancer agents examined alone and in combination with each metal and metal salt included TTBT and DDC. Each of the combinations with the copper salts tested exhibited enhanced extracellular ATP release, while the other metals tested did not exhibit such effects (Fig. 7A and 7B).

The ability of copper and various other metals to induce reactive oxygen species (ROS) was next investigated since it is known that ROS are generated at high levels by ICD-inducing chemotherapeutic agents.

To measure ROS generation by CT26 cells, 10,000 cells/well were seeded on a 96-well white-walled, clear bottom plate (Greiner Bio-One catalog number 655098) in 80 pL/well of medium. Cells were allowed to attach and grow for 24 h. Thereafter, 20 pL/well were replaced with 20 mT/well of medium supplemented with drugs or medium (untreated controls). Combinations of drugs and metals were first pre-mixed to allow complexation of the compounds for those that are able to bind copper or zinc as determined by their chemical structure. Subsequently, the combinations of drugs and metals were diluted to the appropriate concentrations in medium. Combinations of drugs and metals were prepared in the same way (pre-mixing in water and further dilution in medium) for the cytotoxicity, ATP, HMGB 1 and CRT assays described above. To detect ROS, cells were incubated with the compounds for 4 h. The amount of ROS generated was measured by the ROS-Glo™ FEO2 assay (Promega, Madison, WI, USA). Two hours before the end of the experiment, 10 pL/well of H2O2 substrate solution was added directly to each well. At the end of the experiment, 50 mT/well of ROS-Glo™ detection solution was applied to all wells, and the plate was incubated for 20 min at room temperature. The luminescence signal was measured in arbitrary units using the FLUOstar OPTIMA microplate reader (BMG Labtech, Ortenberg, Germany).

Figure 8 shows the results of the ROS studies using various metals. The results are shown as a fold-increase relative to an untreated control. As shown, the copper salts examined exhibited increases in ROS, while most of the other metals tested, including zinc, iron, manganese, and vanadium, did not exhibit such effects. Menadione (MEN) was utilized as a positive control.

Figure 9A shows fold-increase of ROS as a function of concentration (0.25- 250 mM) of copper or zinc addition to the cells. The magnitude of the fold increase of ROS increased with increasing concentrations of copper from 0 to 125 mM (left graph). However, these effects were not observed with zinc (right graph). ROS were reported as fold-increase relative to an untreated control.

Figures 9B and 9C show the results of ROS studies using copper or zinc in combination with daunorubicin (DNR), PX478, salubrinal (SAL), cisplatin (CDDP), gemcitabine (GEM) and cytarabine (ARA-C). Copper in combination with DNR, PX478, SAL, CDDP and ARA-C displayed increased ROS relative to the agent alone or the agent in combination with zinc (as measured by a fold increase in ROS relative to an untreated control).

Example 6: In vivo vaccination studies with combinations of copper and anti cancer agents

An in vivo study was conducted to investigate if the vaccination of immune competent animals with cells pretreated with copper and selected anti-cancer agents would elicit an anti-tumour immunity by preventing the development of tumours when the same animals were challenged with untreated cancer cells seven days later. Mitoxantrone (MITO), an inducer of 1CD, was selected as a positive control and cisplatin (CDDP) was used as a negative control. The cytotoxicity pattern of MITO and the presence of ICD markers (ATP, FIMGBi and CRT) on treated CT26 cells were confirmed in vitro (not shown).

Immune competent Balb/c mice (6-8 weeks old) were “vaccinated" by inoculating the left flank subcutaneously (s.c.) with 3 x 10 ⁶ CT26 cells pretreated in vitro. Briefly 2 x 10 ⁷ CT26 cells were seeded in each 175 cm ² culture flask. At 24 hours later, the cells were treated with 1 mM MITO, 25 mM CDDP, 25 mM copper sulfate (C11SO ₄ ) or combinations of the cytotoxic agent with CuS0 ₄ at the same doses. The copper concentration was selected based on studies demonstrating that a significant increase in intracellular copper levels could be achieved when cells were exposed to 25 mM CuS0 ₄ (not shown). The cells were washed with HBSS and harvested at 24 h post-treatment on Day 0 using enzyme-free cell dissociation buffer (Thermo Fisher Scientific). The cells were counted using a Cellometer Auto T4 (Nexcelom Bioscience, Lawrence, MA, USA) and re-suspended in PBS for inoculation (50 pL per mouse; n = 10 per treatment group). One week later (day 7), all mice were challenged with untreated CT26 cells by inoculating 5 x 10 ⁵ cells/50 pL s.c. into the right flank. Both flanks were monitored for palpable tumours. Tumour growth was monitored and measured with a calliper. Tumour volumes were calculated using the equation L X W ² /2 with the length (mm) being the longer axis of the tumour. Mice were considered tumour-free until the first day that tumours were defined as measurable (64 mm ) using calipers. Tumours were allowed to grow to a maximum of 1000 innT before termination. Control studies were completed to ensure that the tumour take rate of untreated CT26 cells was 100% in order to establish the validity of the vaccination assay.

The results show' that mice that were not vaccinated developed tumours by day 12 following inoculation of untreated cells (Figure 10A). Vaccination w'ith cells pretreated w ith MITO resulted in 58% of the mice being tumour-free on Day 39 post cancer challenge (Figure 10B). However, the proportion of tumour-free mice increased to 70% when mice were vaccinated with cells pretreated with MITO + copper. Vaccination with CDDP or CDDP + copper pretreated cells resulted in 10% and 0% tumour-free mice, respectively.

These data provide support that the addition of copper can enhance the anti cancer immune response of certain agents in vivo and that this augmentation effect can be sustained over time.

Example 7: Combinations of anti-cancer agents with copper exhibit increased ATP secretion

This example examines the effect of anti-cancer agents alone or in

combination with copper, further in combination with existing anti-cancer agents (clinically proven) to enhance ATP secretion, a marker of immunogenic cell death. The results are shown in Figure 1 I .

CT26 cells were treated with an anti-cancer agent of interest alone, in combination with copper, or further in combination with copper and certain clinically approved anti-cancer agents. The level of ATP secretion was determined by analyzing the supernatant at 24 hours following treatment. The first anti-cancer agents tested have copper-binding ligands and included PX-478 (25 mM), flavopiridol (FLV; I mM), clioquinol (CQ; 25mM), and analogues of terpyridine 4,4 ^' 4”-Tri-ter- Butyl-2,2':6 ^‘ ,2"-terpyridine (TTT; 25 mM) and 4 ^' -(4-chlorophenyl)-2,2':6',2”- terpyridine (CPT; 25 mM). The clinically approved anti-cancer agents include the following drugs: mitoxantrone (Mito; 1 mM), gemcitabine (Gem; 0.25 mM), and cisplatin (CDDP; 25 mM).

As shown in Figures 1 1 A-F, the combination of the first anti-cancer agent (PX-478, TTT, FLV, CQ and CPT), copper and the clinically approved agent (Mito, Gem and CDDP) yielded greater ATP release than the approved anti-cancer alone.

The use of the copper plus the first anti-cancer agent, instead of the first anti-cancer agent alone, in some cases, leads to additional enhancement in ATP release.

Example 8: HMGB release of anti-cancer agent combinations with copper

The effect of combining copper-binding agents with existing anti-cancer agents, together with copper, to increase HMGB secretion from cells was next examined and the results are presented in Figure 12.

CT26 murine colon cancer cells were seeded at 200,000 cells/well in 24-well plates. After 24 hours, cells were treated with the indicated anti-cancer agents. The release of FlMGBi was assessed for the combination of PX-478 or Cu(PX-478) and gemcitabine (Gem) and the combination of flavopiridol (FLV) or Cu(FLV) with mitoxantrone (Mito). Where copper or zinc complexes were tested, the metal ion was added to the ligand at the indicated molar ratio such that the metal complexes were formed prior to addition to the cells. Cells were exposed to the tested agents for 24 hours, after which the supernatant of each well was collected and the amount of HMGBi release was assessed using a commercial ELISA kit purchased from IBL International™. Figure 12B shows that the use of flavopidirol (FLV), either in free form or as a copper complex, leads to greater HMGBi secretion compared to mitoxantrone (Mito) alone. As seen in Figure I 2A, combining PX-478 or Cu(PX-478) with gemcitabine (Gem) led to a modest to comparable increase in HMGB, release relative to gemcitabine alone.

Example 9: Combinations of copper and two anti-cancer agents can induce at least two markers of ICD

The ability of a combination of anti-cancer agents with copper to induce all three markers of ICD was also examined. The anti-cancer agents selected for the study included PX478 and GEM. The ICD markers, including release of ATP and HMGBi and CRT exposure were examined. The ICD marker assays were carried out as described previously and the anti-cancer agents and copper were added at the concentrations indicated in Figure 13.

Combinations of copper, PX478 and GEM showed enhanced levels of ATP and HMGB 1 release relative to the agents or copper measured alone and a combination of the two agents (Figure 13A and 13B). For CRT exposure, measured as a fold increase in CRT exposure, the results show that the response for a combination of copper. PX478 and GEM was comparable to that attained with the two anti-cancer agents measured in combination or GEM alone (Figure 13C).

The foregoing description should not be construed as limiting and includes embodiments and equivalents thereof that would be known to those of ordinary skill in the art. A number of embodiments of the invention have been described.

Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.