WO2009033204A1

WO2009033204A1 - Multi-component compositions and methods for delivery of anti-cancer agents

Info

Publication number: WO2009033204A1
Application number: PCT/AU2008/001242
Authority: WO
Inventors: John Barnard Bremner; Philip Clingan; Marie Ranson; Tamantha Stutchbury; Julie Myree Locke
Original assignee: University Of Wollongong
Priority date: 2007-09-12
Filing date: 2008-08-22
Publication date: 2009-03-19

Abstract

The present invention relates to a composition for the treatment or prevention of cancer, the composition comprising 5-fluorouracil, or a pharmaceutically acceptable salt or analogue thereof; folinic acid, or a pharmaceutically acceptable salt thereof, oxaliplatin and a cyclodextrin or a pharmaceutically acceptable salt or derivative thereof.

Description

Multi-component Compositions and Methods for Delivery of Anticancer Agents

Technical Field

The present invention relates to pharmaceutical compositions for the treatment of cancer. In particular, the present invention relates to compositions comprising 5- fluorouracil, a cyclodextrin, oxaliplatin and folinic acid and their use in the treatment of cancer.

Background of the Invention

Chemotherapy plays a key role in the treatment of a range of cancers including colorectal, breast and liver cancer. One drug frequently used in chemotherapy is 5- fluorouracil (5-FU) which is known to be a particularly potent anti-cancer agent. While 5-

FU can be used independently in chemotherapeutic applications, typically the drug is used in combination with folinic acid as its calcium salt (leucovorin) (D. B. Longley, D. P.

Harkin, and P. G. Johnston, "5-Fluorourαcil: mechanisms of action and clinical strategies", Nature Reviews: Cancer, Vol.3, 330-338, 2003). The presence of folinic acid synergises the effect of 5-FU by specifically enhancing the capacity of 5-FU to inhibit thymidylate synthase.

Oxaliplatin (sold under the trade name Eloxatin®) is a platinum-based chemotherapeutic drug that is useful in the treatment of cancers that have become resistant to cisplatin. Oxaliplatin is also used in the treatment of advanced colorectal cancer, in combination with 5-FU and folinic acid. This chemotherapy regimen is sometimes referred to as "FOLFOX".

In clinical use, the administration of 5-FU, folinic acid and oxaliplatin to a patient is performed separately, meaning that the optimal synergistic effect of the combination may not be realised. According to the FOLFOX regimen, the following dose schedule is recommended for patients with stage III colon cancer:

Day 1 : Eloxatin® 85 mg/m² IV infusion in 250-500 mL D5W and leucovorin 200 mg/m²

IV infusion in D5W both given over 120 minutes at the same time in separate bags using a

Y-line, followed by 5-FU 400 mg/m² IV bolus given over 2-4 minutes, followed by 5-FU 600 mg/m² IV infusion in 500 mL D5W as a 22-hour continuous infusion. Day 2: Leucovorin 200 mg/m² IV infusion over 120 minutes, followed by 5-FU 400 mg/m² IV bolus given over 2-4 minutes, followed by 5-FU 600 mg/m² IV infusion in 500 mL D5W as a 22-hour continuous infusion.

On day 1 of this regimen, Eloxatin® and leucovorin are first administered intravenously. Shortly thereafter, two separate intravenous administrations of 5-FU are given. When administered intravenously, the solution comprising 5-FU has a pH of about 9 which results in severe and painful vein damage (phlebitis) in the patient. Consequently, a central line is often used for delivery of 5-FU, and this is a significant disadvantage of the present administration technique. As a result of their incompatibility in solution at physiological pH, 5-FU, folinic acid and oxaliplatin are administered separately in solutions having different pH values. As noted above, the separate administration may result in the optimal synergistic effect of the three compounds not being realised. Accordingly there is a need for a composition in which 5-FU, folinic acid and oxaliplatin can be co-administered at a physiologically acceptable pH at concentrations that are appropriate for clinical use.

The present inventors have found that in the presence of a cyclodextrin derivative, a homogeneous solution of 5-FU, folinic acid and oxaliplatin can be prepared at a physiological pH which minimises harmful side effects.

Summary of the Invention In a first aspect, the present invention provides a composition comprising:

(i) 5-FU, or a pharmaceutically acceptable salt or analogue thereof; (ii) folinic acid (FA), or a pharmaceutically acceptable salt thereof; (iii) oxaliplatin; and

(iv) a cyclodextrin or a pharmaceutically acceptable salt or derivative thereof. The cyclodextrin or salt or derivative thereof may be present in a concentration sufficient to prevent the 5-FU or salt or analogue thereof from precipitating out of solution.

The pH of the composition may be about 5 to about 9 or about 6.5 to about 8 or about 7.0 to about 7.8 or about 7.1 to about 7.5. The cyclodextrin or salt or derivative thereof may be present at a concentration of between about 0.1 and about 45% or between about 1 and about 45% on a w/v basis, or between about 1 and about 450mg/ml or between about 10 and about 450mg/ml or between about 1 and about 300mg/ml. The 5-FU or salt or analogue thereof may be present at a concentration of between about 1 and about 50mg/ml or between about 5 and about 50mg/ml. The folinic acid or salt thereof may be present at a concentration of between about 0.1 and about 25mg/ml or between about 0.1 and about 15mg/ml or between about 0.1 and about 12mg/ml or between about 0.1 and about 5mg/ml. The oxaliplatin may be present at a concentration of between about 0.005 and about 30mg/ml or between about 0.01 and about 20mg/ml or between about 0.1 and 15 mg/ml or between about 0.05 and about 10 mg/ml or between about 0.1 and about 10mg/ml, or between about 1 and about 8 mg/ml, or about 3 mg/ml.

The composition may be a composition for the treatment and/or prevention of cancer. The 5-FU and cyclodextrin may be present in the composition in the form of a non- covalent complex, i.e. the 5-FU may be complexed to the cyclodextrin but not covalently bound thereto.

The folinic acid or salt thereof and the cyclodextrin may be present in the composition in the form of a non-covalent complex, i.e. the folinic acid or salt thereof may be complexed to the cyclodextrin but not covalently bound thereto.

The oxaliplatin and the cyclodextrin may be present in the composition in the form of a non-covalent complex, i.e. the oxaliplatin may be complexed to the cyclodextrin but not covalently bound thereto.

The composition may comprise one or more pharmaceutically acceptable carrier, diluent and/or adjuvant. The carrier may be an aqueous carrier. The composition may be an aqueous composition.

The analogue of 5-FU may be for example 5-fluoro-2-deoxyuridine (5-FUdr).

The analogue of 5-FU may be a prodrug, for example, tegafur.

The composition may be in the form of a solution, e.g. an aqueous solution. Each of the components (i, ii, iii and iv above) may be in solution in the composition. The composition may contain no solid matter. Thus each of the components (i, ii, iii and iv above) may be completely in solution in the composition.

The composition may be a synergistic composition.

The pharmaceutically acceptable salt of folinic acid may be calcium folinate (leucovorin) or sodium folinate. The salt may be in the form of a hydrate, for example calcium folinate pentahydrate.

The folinic acid, or pharmaceutically acceptable salt thereof, may be present in - A - either of its enantiomeric forms, or as a racemic mixture. It may be the 6(S) diastereomer or the 6(R) diastereomer (wherein the glutamic acid residue of the folinic acid or folinate is in the L form), or it may be a mixture of these in any desired ratio, such as about 1 : 1 or some other ratio. It may be a mixture of these in which the 6(5) isomer is enriched. It may be between about 51 and about 100% enriched, or between about 55 and 99%, 55 and 95%, 55 and 75%, 55 and 65%, 75 and 85%, 75 and 95%, 85 and 95% or 75 and 99% enriched, e.g. about 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% enriched. It should be noted that the 6(5) form having the glutamic acid residue in the L form is pharmaceutically active. The cyclodextrin may be α-cyclodextrin, γ-cyclodextrin or a derivative or salt thereof, or may be a β-cyclodextrin or a derivative or salt thereof.

The cyclodextrin may be a cyclodextrin sulfate or a salt thereof.

In one embodiment, the cyclodextrin is a sulfated sodium salt of β-cyclodextrin, optionally a polysulfated sodium salt or a mixture of polysulfated sodium salts with differing degrees of sulfation. In another embodiment, the cyclodextrin is a hydroxypropyl cyclodextrin, for example a hydroxypropyl β-cyclodextrin. Alternatively, the cyclodextrin may be a mixture of different types of cyclodextrins, for example a mixture of hydroxyalkyl β-cyclodextrin and sulfated β-cyclodextrin.

In another embodiment there is provided a composition comprising, or consisting of:

(i) 5-FU, or a pharmaceutically acceptable salt or analogue thereof;

(ii) folinic acid, or a pharmaceutically acceptable salt thereof;

(iii) oxaliplatin;

(iv) a cyclodextrin or a pharmaceutically acceptable salt or derivative thereof; and

(v) a pharmaceutically acceptable carrier; wherein (i), (ii), (iii) and (iv) are dissolved in (v).

The cyclodextrin may be selected from the group consisting of: a hydroxyalkyl β- cyclodextrin and a sulfated β-cyclodextrin, or a combination thereof. The hydroxyalkyl β- cyclodextrin may be hydroxypropyl-β-cyclodextrin, or more specifically 2-hydroxypropyl- β-cyclodextrin. In another embodiment there is provided a composition comprising, or consisting of:

(i) 5-FU, or a pharmaceutically acceptable salt or analogue thereof in a concentration of between about 10 and about 20mg/ml; (ii) folinic acid, or a pharmaceutically acceptable salt thereof, in a concentration of between about 0.5 and about 2mg/ml;

(iii) oxaliplatin in a concentration between about 0.1 and about 20 mg/ml;

(iv) a cyclodextrin or a pharmaceutically acceptable salt or derivative thereof in a concentration of between about 20 and about 450mg/ml; and (v) a pharmaceutically acceptable carrier; wherein (i), (ii), (iii) and (iv) are dissolved in (v).

The cyclodextrin may be selected from the group consisting of: a hydroxyalkyl β- cyclodextrin and a sulfated β-cyclodextrin, or a combination thereof. The hydroxyalkyl β- cyclodextrin may be hydroxypropyl-β-cyclodextrin. In another embodiment there is provided a composition comprising, or consisting of:

(i) 5-FU, or a pharmaceutically acceptable salt or analogue thereof in a concentration of between about 1 and about 50mg/ml;

(ii) folinic acid, or a pharmaceutically acceptable salt thereof, in a concentration of between about 0.1 and about 5mg/ml;

(iii) oxaliplatin at a concentration of between about 1 and about 10 mg/ml; and

(iv) a cyclodextrin or a pharmaceutically acceptable salt or derivative thereof in a concentration of between about 10 and about 300 mg/ml;

The cyclodextrin may be selected from the group consisting of: a hydroxyalkyl β- cyclodextrin and a sulfated β-cyclodextrin, or a combination thereof. The hydroxyalkyl β- cyclodextrin may be hydroxypropyl-β-cyclodextrin.

In another embodiment there is provided a composition comprising, or consisting of: (i) 5-FU, or a pharmaceutically acceptable salt or analogue thereof in a concentration of between about 10 and about 20mg/ml;

(ii) folinic acid, or a pharmaceutically acceptable salt thereof, in a concentration of between about 0.5 and about 2mg/ml; (iii) oxaliplatin in a concentration between about 1 and about 10mg/mL;

(iv) a cyclodextrin or a pharmaceutically acceptable salt or derivative thereof in a concentration of between about 20 and about 200mg/ml; and

(v) a pharmaceutically acceptable carrier; wherein (i), (ii), (iii) and (iv) are dissolved in (v). The cyclodextrin may be selected from the group consisting of: a hydroxyalkyl β- cyclodextrin and a sulfated β-cyclodextrin, or a combination thereof. The hydroxyalkyl β- cyclodextrin may be hydroxypropyl-β-cyclodextrin.

In another embodiment there is provided a composition comprising, or consisting of: (i) 5-FU at a concentration of about 15mg/ml;

(ii) folinic acid calcium salt at a concentration of about lmg/ml; (iii) oxaliplatin in a concentration of about 3mg/ml; (iv) β-cyclodextrin sulfate salt at a concentration of about 200mg/ml; and (v) a pharmaceutically acceptable carrier; wherein (i), (ii), (iii) and (iv) are dissolved in (v).

(i) 5-FU at a concentration of about 15mg/ml; (ii) folinic acid calcium salt at a concentration of about 1 mg/ml; (iii) oxaliplatin at a concentration of about 3mg/mL;

(iv) (2-Hydroxypropyl)-β-cyclodextrin at a concentration of about 135mg/ml; and

(v) a pharmaceutically acceptable carrier; wherein (i), (ii), (iii) and (iv) are dissolved in (v). In the above embodiments, the β-cyclodextrin sulfate salt may comprise between 6 and 14 sulfate groups, the pharmaceutically acceptable carrier may be saline solution (e.g. 0.9% saline solution) and the β-cyclodextrin sulfate salt may be a sodium salt.

In a second aspect of the invention there is provided a process for making a composition, comprising combining:

(i) 5-FU, or a pharmaceutically acceptable salt or analogue thereof;

(ii) folinic acid, or a pharmaceutically acceptable salt thereof;

(iii) oxaliplatin; and

(iv) a cyclodextrin or a pharmaceutically acceptable salt or derivative thereof. The concentrations and identities of the components may be as described in the first aspect of this invention. The process may comprise combining (i) with (iv), and then adding (ii) and (iii) in any order. When adding (ii), it may be in solution, optionally in aqueous solution. Alternatively, (ii) may be added as a solid. When adding (iii), it may be in solution, optionally in aqueous solution. The process may also comprise sterilising the composition. The sterilising may comprise filtration (e.g. microfiltration), UV radiation, gamma radiation, some other form of sterilising radiation or some other form of sterilising.

The sterilising may be conducted so as not to damage or degrade any of components (i) to

(iv) above. Thus sterilising radiation (UV, gamma or other), if used, should be in a dose sufficiently high to sterilise the composition but sufficiently low to avoid damage or degradation of the components specified.

The composition may be a composition for the prevention and/or treatment of cancer.

In an embodiment, (i) and (iv) are combined in the presence of a carrier, optionally an aqueous carrier, to form a mixture, and (ii) and (iii) are then added to the mixture. The mixture may be a solution, optionally an aqueous solution. The carrier may be a solvent, optionally an aqueous solvent, (i) and (iv) may, independently, be partially soluble in the carrier, optionally sparingly soluble. Following combination of (i) and (iv) in the presence of the carrier to form a mixture, the mixture may be agitated to at least partially dissolve (i) and (iv) in the carrier. The dissolution may comprise formation of a non-covalent complex between (i) and (iv). The agitation may comprise stirring, swirling, shaking, sonicating or a combination of two or more thereof. The agitation may be conducted at elevated temperature, e.g. between about 30 and about 80°C. The composition may be agitated following addition of (ii) and (iii) and/or between the addition of (ii) and (iii). The agitation may be as described above.

In another embodiment there is provided a process for making a composition, comprising: • combining 5-FU, or a pharmaceutically acceptable salt or analogue thereof, and a cyclodextrin or a pharmaceutically acceptable salt or derivative thereof in the presence of an aqueous solvent to form a mixture;

• agitating, and optionally heating, the mixture to form a solution; and

• adding an aqueous solution of folinic acid, or of a pharmaceutically acceptable salt thereof, and further adding an aqueous solution of oxaliplatin to the solution to form the composition.

The step of adding the aqueous solutions may be conducted so as to form a composition in which the 5-FU or salt or analogue thereof, the cyclodextrin or salt or derivative thereof, the folinic acid or salt thereof and the oxaliplatin are all in solution in the composition, optionally such that no solid matter is present in the composition. The composition may be agitated, and optionally heated, following the step of adding the aqueous solution. The agitation of the composition may be sufficient to render the composition homogeneous, or to ensure that all of the components of the composition are completely in solution. In another embodiment there is provided a process for making a composition, comprising:

• combining 5-FU, or a pharmaceutically acceptable salt or analogue thereof, and a cyclodextrin or a pharmaceutically acceptable salt or derivative thereof in the presence of an aqueous solvent to form a mixture; • agitating, and optionally heating, the mixture to form a solution;

• adding an aqueous solution of folinic acid, or a pharmaceutically acceptable salt thereof, and an aqueous solution of oxaliplatin to the solution to form the composition;

• agitating, and optionally heating, the composition sufficient to ensure that all components are in solution; and

• sterilising the composition. In another embodiment there is provided a process for making a composition, comprising:

• combining 5-FU, or a pharmaceutically acceptable salt or analogue thereof, in an amount of between about 1 and about 50mg per ml of final composition, and a cyclodextrin or a pharmaceutically acceptable salt or derivative thereof in an amount of between about 20 and about 450mg per ml of final composition in the presence of an aqueous solvent to form a mixture;

• agitating, and optionally heating, the mixture to form a solution;

• adding folinic acid, or a pharmaceutically acceptable salt thereof in an amount between about 0.5 and 20mg per ml of final composition;

• adding oxaliplatin in an amount between about 0.1 and about 10 mg/ml of final composition to the solution to form the composition;

• agitating, and optionally heating, the composition sufficient to ensure that all components are in solution; and • sterilising the composition.

In a third aspect, the present invention provides a method for the treatment or prevention of cancer in a patient in need of said treatment or prevention, said method comprising administration of a therapeutically effective amount of the composition of the first aspect. In a fourth aspect, the present invention provides a method for inhibiting the proliferation of cancer cells, said method comprising exposing the cancer cells to the composition of the first aspect.

The cancer or cancer cells may be primary or secondary and may include one or more of the following: prostate cancer, colorectal cancer (which is to be understood to encompass colon cancer and bowel cancer), lung cancer (for example non-small cell lung cancer), cervical cancer, breast cancer, ovarian cancer, gastric cancer, primary hepatoma, skin cancer, pancreatic cancer, cholangiocarcinoma or a cancer of the head or neck, and cancer cells therefrom.

The skin cancer may be non-melanoma skin cancer. The patient may be a mammal, for example a human. Definitions

In the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings. As used herein, the term "oxaliplatin" includes c/5-oxalato(trans-l-l,2- diaminocyclohexane) platinum(II), its optical enatiomer czs-oxalato(trans-d-l,2- diaminocyclohexane) platinum(II), and any racemic mixture thereof.

As used herein, the term "synergistic" refers to a greater than additive effect that is produced by a combination of folinic acid, 5-FU and oxaliplatin, which exceeds the effect that would otherwise result from use of 5-FU, folinic acid and oxaliplatin alone or in combinations not including the three components.

A "therapeutically effective amount", as used herein, includes within its meaning non-toxic but sufficient amounts of 5-FU, folinic acid and oxaliplatin to provide the desired therapeutic effect. The exact amounts required may vary from subject to subject depending on factors such as the subject's general health, the subject's age and the stage and severity of the cancer.

As used herein, the terms "treating", "treatment" "preventing" and "prevention" include any and all uses which remedy cancer or symptoms thereof, prevent the establishment of cancer, or otherwise prevent, hinder, retard or reverse the progression of cancer or other undesirable symptoms in any way whatsoever. The terms "treating" and "preventing" and the like are therefore to be considered in their broadest context. For example, treatment does not necessarily imply that a patient is treated until total recovery.

As used herein the term "complex" is understood to mean a non-covalent bonding physical interaction between two chemical entities. Brief Description of the Figures

A preferred embodiment of the present invention will now be described, by way of example only, with reference to the accompanying drawings wherein:

Figure 1 shows cell proliferation (MTS assay) dose-response curves for (A) Oxaliplatin and (B) Oxaliplatin/S-FU/Cyclodextrin/Folinic acid composition against HCT- 116 colorectal cancer cells. The x-axis in both (A) and (B) is representative of Oxaliplatin concentrations. Figure 2 shows cell proliferation dose-response curves for formulations with and without oxaliplatin after 72 hours treatment. The x-axis is representative of 5-FU concentrations in equivalent FD and FX formulations to simplify comparisons between components. Values shown are mean ± SEM (n = 4) from a representative experiment and are normalized to controls (100% proliferation).

Detailed Description of the Invention

Compositions and methods of preparation

The present invention relates to compositions comprising 5-FU, or a pharmaceutically acceptable salt or analogue thereof, oxaliplatin, folinic acid, or a pharmaceutically acceptable salt thereof, and a cyclodextrin, or a pharmaceutically acceptable salt or derivative thereof, which prevents 5-FU from precipitating out of solution at a pH of about 5 to about 9. The invention also contemplates use of the compositions for the treatment of cancer.

In the present context, an analogue of 5-FU is a compound having comparable or similar pharmacological activity, particularly in a human patient, to 5-FU, and similar solubility/complexation properties, so that in a formulation according to the present invention, when made with the analogue, the analogue remains in solution. In the present specification, reference to 5-FU will be taken to include a corresponding reference to a pharmaceutically acceptable salt or analogue thereof, reference to folinic acid will be taken to include a corresponding reference to a pharmaceutically acceptable salt or derivative thereof, and reference to cyclodextrin will be taken to include a corresponding reference to a pharmaceutically acceptable salt or derivative thereof, unless the contrary is indicated or implied.

The use of the compositions of the invention provide the following advantages over current methods of cancer treatment using 5-FU, folinic acid and oxaliplatin:

(i) improving the delivery of 5-FU, folinic acid and oxaliplatin by allowing simultaneous administration of the three compounds;

(ii) minimisation of side effects that are prevalent when 5-FU is administered at higher pH values of about 9; (iii) reduction in administration time and costs (in the clinic) by enabling the combination of anticancer agents to be administered in a single dose; (iv) lower pH means that a central line usually required to prevent phlebitis may not be required, thereby simplifying administration; and

(v) possibly reduced stress on the patient.

In the compositions of the present invention, cyclodextrin and 5-FU may form a complex in solution. This complex may be a "host-guest" or "inclusion complex" or a

"non-inclusion complex". The complex may result in the otherwise insoluble 5-FU being soluble in aqueous solution at pH values of between about 5 to about 8. Alternatively the cyclodextrin and 5-FU may be associated through some other form of non-covalent interaction. Cyclodextrin and folinic acid and/or cyclodextrin and oxaliplatin may form a complex in solution. This complex may be a "host-guest" or "inclusion complex".

The 5-FU present in the compositions of the invention may be present as a pharmaceutically acceptable salt. By "pharmaceutically acceptable salt" it is meant those salts of 5-FU which are suitable for use in contact with the tissues of humans and other animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art.

For instance, suitable pharmaceutically acceptable salts of 5-FU include the sodium salt, which may be obtained from reaction with sodium hydroxide. The folinic acid present in the composition may be present as a salt of an alkali metal (e.g. sodium, potassium) or a salt of an alkaline earth metal (e.g. magnesium, calcium). In one embodiment, the folinic acid may be present as calcium folinate.

Cyclodextrins suitable for use in accordance with the present invention include natural and chemically modified cyclodextrins. The cyclodextrin may be an α- cyclodextrin, a β-cyclodextrin, a γ-cyclodextrin, or a derivative or pharmaceutically acceptable salt thereof. Mixtures of any two or more of the above cyclodextrins are also contemplated in the compositions of the invention.

Derivatives of cyclodextrins that may be used in the compositions may include cyclodextrins whereby one, or some, or all of the hydroxy groups are converted to other functional groups. As a result, the degree of substitution of the cyclodextrin may vary. In one embodiment, one or more of the hydroxy groups may be converted to OR groups. R may be a hydrocarbon group having between 1 and 20 carbon atoms, or between 1 and 15, or between 1 and 10, or between 1 and 9, or between 1 and 8, or between 1 and 7, or between 1 and 6, or between 1 and 5, or between 1 and 4 carbon atoms, for example methyl, allyl, ethyl, propyl, isopropyl, propargyl, butyl, but-2-enyl, secbutyl, pentyl, 3- methyl-pent-2-enyl, hexyl, heptyl, octyl, nonyl, decyl etc. In one embodiment, the hydrocarbon group is a branched or straight chain alkyl group. The hydrocarbon group may be optionally substituted with one or more halo groups and/or one or more hydroxy groups, such that R is a halo-substituted hydrocarbon or a hydroxy-substituted hydrocarbon, for example a hydroxyalkyl group. Examples of hydroxyalkyl groups include, but are not limited to -CH₂CH(OH)CH₃, -CH₂CH₂OH, -CH₂CH₂CH₂(OH)CH₃, - CH(OH)CH₂CH₂CH₃ and -CH(OH)CH₂CH₂CH₂CH₃. In one embodiment, the cyclodextrin is a hydroxyalkyl β-cyclodextrin, for example hydroxypropyl-β-cyclodextrin (HP-β-CD).

Derivatives of cyclodextrins also include anionic cyclodextrins such as carboxymethyl cyclodextrins, cyclodextrin sulfates, cyclodextrin sulfonates, cyclodextrin phenolates and cyclodextrin phosphates, or mixtures thereof. In one embodiment, the cyclodextrin derivative may be a sulfated α-, β-, or γ- cyclodextrin, or a salt thereof, for example a sodium salt. The sulfated cyclodextrin may be polysulfated or comprise a mixture of sulfate and hydroxy groups. In one embodiment, the sulfated cyclodextrin is a β-cyclodextrin comprising between 3 and 20, or between 3 and 18, or between 4 and 16, or between 4 and 14, or between 5 and 13, or between 5 and 12, or between 5 and 1 1, or between 6 and 10, or between 7 and 9, or between 9 and 12, or between 5 and 10 sulfate groups. The sulfate groups may be present as salts, for example sodium salts. In one embodiment, the cyclodextrin derivative is heptakis(6-O-sulfo)-β-cyclodextrin, or a salt thereof.

Further cyclodextrin derivatives which may be used include sulfoether cyclodextrins such as sulfoalkylether cyclodextrins, wherein the alkyl group comprises between 1 and 15 carbon atoms, or between 1 and 12, or between 1 and 10, or between 1 and 9, or between 1 and 8, or between 1 and 7, or between 1 and 6, or between 1 and 5, or between 1 and 4 carbon atoms, for example sulfobutyl β-cyclodextrin (Captisol^®). The alkyl group may be of the formula -(CH₂)_n-, wherein n is a number between 1 and 8, or between 1 and 7, or between 1 and 6, or between 1 and 5 or between 1 and 4. Additional cyclodextrin derivatives that may be suitable for use in the compositions of the present invention are those disclosed in "Chemical Reviews: Cyclodextrins", V. T. D'Souza and K. B. Lipkowitz, Vol.98, No.5 (American Chemical Society, 1998), which is incorporated herein by reference.

5 As noted above, the degree of substitution of the cyclodextrin may vary. Suitable cyclodextrins therefore include for example those cyclodextrins wherein one, or some, or all of the hydroxy groups are substituted. The degree of substitution may be between about 1 and 20, or between about 5 and 20, 10 and 20, 1 and 10, 1 and 5, 5 and 15, 5 and 10 or 10 and 15, e.g. about 1,2, 3,4, 5,6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. The io cyclodextrin may be a mixture of cyclodextrins of different degrees of substitution. In such a case the average degree of substitution may be between about 1 and 20, or between about 5 and 20, 10 and 20, 1 and 10, 1 and 5, 5 and 15, 5 and 10 or 10 and 15, e.g. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In one embodiment where the cyclodextrin is a hydroxypropyl-β -cyclodextrin, the average degree of substitution may be

]₅ between about 4 and about 12, or between about 5 and about 10, or between about 5 and about 9, or between about 6 and about 8, or about 7.

Generally the amount of cyclodextrin used in the compositions of the invention is an amount sufficient to ensure that the 5-FU present in the compositions is maintained in solution. The molar ratio of CD:5-FU may be between about 1:10 and about 3:1, or

20 between about 1:10 and 1:1, 1:10 and 1:2, 1:10 and 1:5, 1:5 and 3:1, 1:5 and 1:1, 1:3 and 3:1, 1:8 and 3:1, 1:8 and 2:1, 1:8 and 1:1, 1:8 and 1:2, 1:8 and 1:5, 1:3 and 2:1, 1:3 and 1:1, 1:3 and 1.1:1, 1:3 and 1.2:1,1:3 and 1.3:1, 1:3 and 1.4:1, 1:3 and 1.5:1, 1:3 and 2:1, 1:3 and 2.5:1, 1:2.5 and 3:1, 1:2 and 3:1, 1:1.5 and 3:1, 1:1.4 and 3:1, 1:1.3 and 3:1, 1:1.2 and 3:1, 1:1.1 and 3:1, 1:1 and 3:1, 1.5:1 and 3:1, 2:1 and 3:1, 2.5:1 and 3:1, 2:1 and 1:2, 1.5:1 and S 1:1.5, 1.4:1 and 1:1.4, 1,3:1 and 1:1.3, 1.2:1 and 1:1.2 or 1.1:1 and 1:1.1, e.g. about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4.5, 1:4, 1:3.5, 1:3, 1:2.5, 1:2, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 2:1, 2.5:1 or 3:1. This ratio may depend on the particular CD used.

The composition may be mildly or moderately acidic, neutral or mildly or 0 moderately basic. The pH of the composition may be between about 5.0 and about 9.0, or between about 5.0 and about 8.0, or between about 5.0 and about 7.0, or between about 5.5 and about 8.0, or between about 6.0 and about 8.0, or between about 6.5 and about 8.0, or between about 7.0 and about 8.0, or between 7.5 and about 8.0, or between about 7.0 and about 9.0 or between about 7.0 and about 9.0 or between about 7.5 and 7.8, or between about 7.5 and about 7.7, or between about 7.6 and about 7.7, or between about 6.7 and about 7.7, or between about 6.8 and about 7.6, or between about 7.0 and about 7.6, or between about 7.2 and about 7.6, or between about 7.3 and about 7.6, or between about 7.3 and 7.5. For example the pH may be about 5.0, 5.1, 5.3, 5.5, 5.7, 5.9, 6.1, 6.3, 6.5, 6.7, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

In one embodiment, the composition of the invention may be prepared by mixing solid 5-FU (15mg), sodium hydroxide solution (0.9M, 30 μL), a cyclodextrin, (e.g. β- cyclodextrin sulfated sodium salt as a mixture of 9-12 sulfates per cyclodextrin residue: between 45mg and 200mg) and water to give a total volume of 400 μL. The resulting suspension is sonicated at about 30 to 60 °C until almost all of the 5-FU has dissolved. Oxaliplatin solution (600 μL x 5 mg/ml) and folinic acid (for example, calcium folinate pentahydrate equivalent to lmg of folinic acid) are then added, and the mixture subjected to sonication at 30 to 60 °C until a homogenous solution is formed. The temperature of the sonication may be between about 30 and 50, 30 and 40, 40 and 50, or 40 and 60°C, e.g. about 30, 35, 40, 45, 50, 55 or 60°C, or may be above 60°C or below 30°C.

The homogeneous solution is cooled and allowed to stand at room temperature for about 1 hour. The solution may then be filtered under aseptic conditions and stored at room temperature in the absence of light. The pH of the solution may be between about 7.0 and about 7.5.

In another embodiment, the composition of the invention may be prepared by mixing solid 5-FU (15mg), sodium hydroxide solution (0.9M, 20 μL), a cyclodextrin, (e.g. 2-Hydroxypropyl-β-cyclodextrin, 133mg) and water to give a total volume of 400 μL. The resulting suspension is heated to about 80°C so as to dissolve the 5-FU. Oxaliplatin solution (600 μL x 5mg/ml) and folinic acid (for example, calcium folinate pentahydrate 1.2mg) are then added, and the mixture is shaken and then filtered into a sterile vial.

The solution may then be stored at room temperature in the absence of light. The pH of the solution may be between about 7.0 and about 7.5.

In a further embodiment it may be possible to remove the solvent and dry the composition for resuspension in solution at a later date. Lyophilized formulations have disadvantages with respect to the liquid formulation. The freeze-dried formulation requires extensive sonication and extra preparation by medical staff.

Pharmaceutical compositions and methods of administration

The compositions of the invention may be useful in the treatment of cancer in a patient by administering a therapeutically effective amount of composition to a subject.

Cancers which the compositions and methods of the present invention may treat include primary or secondary cancers including for example, prostate cancer, lung cancer (for example non-small cell lung cancer), cervical cancer, breast cancer, ovarian cancer, gastric cancer, upper GI cancers, primary hepatoma, pancreatic cancer, cholangiocarcinoma, colorectal cancer, pancreatic cancer, head and neck cancer, breast cancer and skin cancer, for example non-melanoma skin cancer.

In a therapeutic application, compositions may be administered to a patient already suffering from cancer, in an amount sufficient to cure, or at least partially arrest the cancer and its complications. The compositions should provide a quantity of 5-FU, folinic acid and oxaliplatin sufficient to effectively treat the cancer.

In general, suitable compositions for use in accordance with the methods of the present invention may be prepared according to methods and procedures that are known to those of ordinary skill in the art, and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant. The diluents, adjuvants and excipients must be "acceptable" in terms of being compatible with the other components of the composition, and not deleterious to the recipient thereof.

Compositions may be administered by standard routes. In general, the compositions may be administered intravenously, intraperitoneally, intraarterially, or topically. They may also be administered intramuscularly, subcutaneously or transdermally (e.g. by means of a patch). Administration may be systemic, regional or local. The particular route of administration to be used in any given circumstance will depend on a number of factors, including the type of cancer being treated, the severity and extent of the cancer, the required dosages of 5-FU, folinic acid and oxaliplatin to be delivered and any potential side-effects. Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water, BP or USP water, Ringer's solution, glucose solution, vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

The compositions of the invention may be in a form suitable for administration by injection, in the form of an ointment, cream or lotion suitable for topical administration, or in a form suitable for administration by injection intravenously, intraperitoneally or intraarterially.

For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, glucose solution, distilled water, ethanol and 1,2 propylene glycol. Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.

Methods for preparing parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed. (1975), Mack Publishing Company, Easton, Pa., hereby incorporated by reference herein.

Topical formulations comprise the composition of the invention together with one or more acceptable carriers, and optionally any other therapeutic ingredients. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treatment is required, such as liniments, lotions, creams, ointments or pastes.

Lotions according to the present invention include those suitable for application to the skin. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturiser such as glycerol, or oil such as castor oil or arachis oil.

The compositions may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

The therapeutically effective dose level for any particular patient will depend upon a variety of factors including: the disorder being treated and the severity of the disorder, the age, body weight, general health, sex and diet of the patient, the time of administration, the route of administration, the duration of the treatment, drugs used in combination or coincidental with the treatment, together with other related factors well known in medicine.

One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of 5-FU, folinic acid and oxaliplatin which would be required to treat applicable cancers.

Generally, an effective dosage of 5-FU is expected to be in the range of about 0.0001 mg to about lOOOmg per kg body weight per 24 hours; typically, about 0.001 mg to about 750mg per kg body weight per 24 hours; about 0.0 lmg to about 500mg per kg body weight per 24 hours; about O.lmg to about 500mg per kg body weight per 24 hours; about 0.1 mg to about 250mg per kg body weight per 24 hours; about l.Omg to about 250mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about l.Omg to about 200mg per kg body weight per 24 hours; about l.Omg to about lOOmg per kg body weight per 24 hours; about l .Omg to about 50mg per kg body weight per 24 hours; about l.Omg to about 25mg per kg body weight per 24 hours; about 5.0mg to about 50mg per kg body weight per 24 hours; about 5.0mg to about 20mg per kg body weight per 24 hours; about 5.0mg to about 15mg per kg body weight per 24 hours; or about 5.0 to 10.0mg/kg body weight per 24 hours.

Generally, an effective dosage of folinic acid is expected to be in the range of about O.OOOlmg to about lOOOmg per kg body weight per 24 hours; typically, about O.OOlmg to about 750mg per kg body weight per 24 hours; about 0.0 lmg to about 500mg per kg body weight per 24 hours; about O.lmg to about 500mg per kg body weight per 24 hours; about O.lmg to about 250mg per kg body weight per 24 hours; about 1.Omg to about 250mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about l.Omg to about 200mg per kg body weight per 24 hours; about l.Omg to about lOOmg per kg body weight per 24 hours; about l.Omg to about 5 Omg per kg body weight per 24 hours; about 1.Omg to about 25mg per kg body weight per 24 hours; about 5. Omg to about 5 Omg per kg body weight per 24 hours; about 5. Omg to about 20mg per kg body weight per 24 hours; about 5. Omg to about 15mg per kg body weight per 24 hours. It may be a low dose or high dose folinic acid composition.

Generally, an effective dosage of oxaliplatin is expected to be in the range of about O.OOOlmg to about lOOOmg per kg body weight per 24 hours; typically, about O.OOlmg to about 750mg per kg body weight per 24 hours; about O.Olmg to about 500mg per kg body weight per 24 hours; about 0.1 mg to about 500mg per kg body weight per 24 hours; about O.lmg to about 250mg per kg body weight per 24 hours; about 0.5mg to about 200mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about O.lmg to about 150mg per kg body weight per 24 hours; about O.lmg to about lOOmg per kg body weight per 24 hours; about 0.5mg to about 50mg per kg body weight per 24 hours; about 0.5mg to about 25mg per kg body weight per 24 hours; about 0.5mg to about 20mg per kg body weight per 24 hours; about 0.5mg to about 15mg per kg body weight per 24 hours; about lmg to about lOmg per kg body weight per 24 hours. In a composition according to the present invention the 5-FU, folinic acid and oxaliplatin may be present in pharmaceutically effective concentrations. The 5-FU may be present in a concentration of between about 1 and about 50mg/ml, or between about 1 and 40, 1 and 30, 1 and 20, 1 and 10, 1 and 5, 5 and 50, 10 and 50, 20 and 50, 5 and 30, 5 and 20 or 10 and 20mg/ml, e.g. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50mg/ml.

The folinic acid may be present in a concentration of between about 0.1 and about 25mg/ml, or between about 0.1 and 20, 0.1 and 15, 0.1 and 10, 0.1 and 5, 0.1 and 2, 0.1 and 1, 1 and 25, 5 and 25, 10 and 25, 15 and 25, 1 and 15, 1 and 10, 1 and 5, 0.5 and 5, 0.5 and 2, 5 and 20, 5 and 10 or 10 and 20mg/ml, e.g. about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25mg/ml.

The oxaliplatin may be present in a concentration of between about 0.005 and about 30 mg/mL, or between about 0.01 and 25, 0.01 and 20, 0.01 and 17, 0.01 and 15, 0.01 and 12, 0.05 and 10, 0.05 and 7.5, 0.05 and 5, 0.1 and 5, or 1 and 4mg/ml, e.g. about 0.005, 0.01, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 mg/ml.

The CD may be present in a concentration of between about 1 and about 450mg/mL, or between about 1 and about 300mg/ml, or between about 1 and 250, 1 and

200, 1 and 150, 1 and 100, 1 and 50, 1 and 20, 1 and 10, 5 and 300, 5 and 250, 5 and 200, 5 and 150, 5 and 100, 5 and 50, 5 and 20, 5 and 10, 10 and 300, 20 and 300, 30 and 300, 40 and 300, 50 and 300, 100 and 300, 200 and 300, 50 and 200, 5 and 100 or 100 and

200mg/ml, e.g. about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 25 or 300mg/ml.

Typically, in therapeutic applications, the treatment would be for the duration of the cancer.

Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the cancer being treated, the form, route and site of administration, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques.

It will also be apparent to one of ordinary skill in the art that the optimal course of treatment, such as, the number of doses of the composition given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

Those skilled in the art will appreciate that in accordance with the methods of the present invention the composition may be administered alone or in conjunction with one or more additional agents as part of a combination therapy. For example, the composition of the invention may be administered together with one or more additional compounds capable of decreasing cell proliferation and invasion and increasing apoptosis in cancer cells.

For such combination therapies, each component of the combination therapy may be administered sequentially in any order, at the same time or at different times, so as to provide the desired effect. Alternatively, the components may be formulated together in a single dosage unit as a combination product. When administered separately, it may be preferred for the components to be administered by the same route of administration, although it is not necessary for this to be so.

Combination regimens

Therapeutic advantages may be realised through combination regimens. In combination therapy the respective agents may be administered simultaneously, or sequentially in any order. Accordingly, methods of treatment according to the present invention may be applied in conjunction with conventional therapy, such as radiotherapy, chemotherapy, surgery, or other forms of medical intervention. Examples of chemotherapeutic agents include adriamycin, taxol, melphalan, cisplatin, Irinotecan, bevacizumab, cetuximab, α-interferon, vincristine, vinblastine, angioinhibins, TNP -470, pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG and the like. Other chemotherapeutic agents include alkylating agents such as nitrogen mustards including mechloethamine, melphan, chlorambucil, cyclophosphamide and ifosfamide, nitrosoureas including carmustine, lomustine, semustine and streptozocin; alkyl sulfonates including busulfan; triazines including dicarbazine; ethyenimines including thiotepa and hexamethylmelamine; folic acid analogues including methotrexate; pyrimidine analogues, cytosine arabinoside; purine analogues including 6-mercaptopurine and 6-thioguanine; antitumour antibiotics including actinomycin D; the anthracyclines including doxorubicin, bleomycin, mitomycin C and methramycin; hormones and hormone antagonists including tamoxifen and cortiosteroids and miscellaneous agents including cisplatin and brequinar, and regimens such as COMP (cyclophosphamide, vincristine, methotrexate and prednisone), etoposide, mBACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and dexamethasone), and PROMACE/MOPP (prednisone, methotrexate (w/leucovin rescue), doxorubicin, cyclophosphamide, taxol, etoposide/mechlorethamine, vincristine, prednisone and procarbazine).

The present invention will now be described with reference to specific examples, which should not be construed as in any way limiting the scope of the invention.

Examples Example 1 - Preparation of compositions To make 1 mL of composition: β-cyclodextrin sulfated sodium salt (200 mg), water

(355 μL), solid 5-FU (15 mg) and NaOH (0.9M, 30μL) were agitated or sonicated until the cyclodextrin and/or solid 5FU had dissolved. Oxaliplatin was added (600 μL, 5 mg/mL solution) and the mixture agitated. Calcium folinate pentahydrate (1.2 mg) was added to the solution and the combination shaken and then filtered into a sterile vial using aseptic techniques. The pH of this solution was 7.3 ± 0.1. The solution was stored at room temperature, protected from light.

To make 1 mL of composition: 15 mg of powdered 5-FU was mixed with a solution of (2-Hydroxypropyl)-β-cyclodextrin (380 μL, 35 %) and NaOH (20 μL, 0.9 M) and heated to 80 ⁰C for 2 min to dissolve the 5-FU. An aqueous oxaliplatin solution (600 μL, 5 mg/mL) and solid calcium folinate pentahydrate (1.2 mg) were added to the 5-FU solution and the combination shaken and then filtered into a sterile vial using aseptic techniques. The pH of this solution was 7.3 ± 0.1. The formula was stored at room temperature; protected from light.

Example 2 - Compositions

Injectable Parenteral Composition A pharmaceutical composition suitable for administration by injection may be prepared by mixing 1-5% by weight of the composition of the invention in 10% by volume propylene glycol and water. The solution is sterilised by filtration. The total amount of the composition may vary from patient to patient.

Composition for Parenteral Administration

A composition for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and 1 mL of the composition of the invention.

Similarly, a composition for intravenous infusion may comprise 250 ml of sterile Ringer's solution, and 5 mL of the composition of the invention. Injectable Parenteral Composition

A composition suitable for administration by injection may be prepared by mixing 1% by weight of the composition of the invention by volume propylene glycol and water. The solution is sterilised by filtration.

Example 3 - Cell proliferation assay A cell proliferation (MTS) assay of oxaliplatin and a composition of the invention against HCT-116 colorectal cancer cells was performed as follows. Compounds had a starting concentration as follows: oxaliplatin 38 μM, 5-FU 582 μM, folinic acid 10 μM, sulfated β-cyclodextrin 0.13%. All samples were 1 in 2 serially diluted from their starting concentration. IC_5O values are based on the concentration of oxaliplatin. Plates were seeded with 20,000 cells 24 hours prior to treatment, after 24 hours the cells were washed and the media changed immediately prior to treatment to a volume of 200 μL. Plates were then incubated for 72 hours. MTS (20 μL) was then added and the plates were developed for 3 hours prior to measurement at 490 run. The values were calculated form logarithmic sigmoidal dose response curves generated from Graphpad Prism V 4.02 software. Values were normalised to the saline controls. Representative graphs depicting normalized percentage of cell survival versus concentration are shown in Figure 1.

Example 4 - Cell Proliferation Assay

Methods

Shortly before use in the cell assays stock solutions were diluted with dH₂O and sterile filtered through 0.22 μm filters. The starting concentration of components in the sterile diluted stock solutions used for the dilution series were:

• OX - (Oxaliplatin at 0.3 mg.ml^"1)

• 5-FU/FA - (5-FU/folinic acid at 1.5 mg.ml^"1/ 0.1 mg.ml^"1)

• FD (5-FU/folinic acid/2 -hydroxypropyl-β-cyclodextrin at 1.5 mg.ml^'Vo.l mg.ml^" '/13 mg.ml^"1) • FX (5-FU/folinic acid/2-hydroxypropyl-β-cyclodextrin/oxaliplatin at 1.5 mg.ml^"

VO.1 mg.ml^"'/13 mg.ml^"1/ 0.3 mg.ml^"1)

The above solutions were diluted 1 :5 maintaining all of the components at the fixed ratios. Results are presented for 5-FU concentrations.

The cytotoxicity of the formulations and individual components was determined using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS, Promega) against the human colorectal carcinoma cell line COLO-201. Briefly, cells (1.3 xlO⁴ in a total volume of 130 μL, complete media) were seeded into 96 well microtitre plates in quadruplicate and incubated for 24 hours (37 ⁰C, 95% humidity, 5% CO₂) prior to the addition of the test compounds. Test compounds were made up fresh, on the day of testing by dilution of stock solutions in endotoxin free water and sterile filtering. Test compounds of various concentrations or water (10 μL of each) were then added in quadruplicate to give a final volume of 150 μL. Background controls containing equivalent dilutions of the test compounds, but no cells, were also prepared. Cells were incubated for a further 72 hours (37 ⁰C, 95% humidity, 5% CO₂). The MTS assay substrate (20 μL) was then added to each well and cells incubated for 2 hours to allow colour development. The absorbance at 490 nm was measured using the Spectromax^® 250 UV plate reader (Molecular Devices, USA) utilising Softmax Pro software. Data was analysed using a logarithmic sigmoidal dose response curve to determine the concentration of drug required to provoke a response halfway between the baseline and maximum responses (GraphPad Prism, Version 5.0). By constraining the baseline and maximum responses to 0 and 100%, respectively, the IC₅₀, defined as the concentration of each treatment causing 50% of maximal growth inhibition was calculated.

Results

Treatment of the COLO-201 cell line with 5-FU/FA or FD or FX or OX alone resulted in a dose-dependent inhibition of cell proliferation after 72 hours of exposure (Figure 2). The IC₅₀ values, based on the concentration of 5 -FU, for the various drug formulations are presented below in Table 1. These results were analysed by an extra sum- of-squares F test (Graphpad Prism 5) which indicated that the LOgIC₅₀ values are different for each data set (pO.0001). Except for oxaliplatin alone, all formulations demonstrated in vitro cytotoxicity equivalent to, or where oxaliplatin was present, better than 5-FU/FA. It was noted that when oxaliplatin and FU/FA were formulated into an all-in-one formulation with 2-hydroxypropyl-β-cyclodextrin (FX) cytotoxicity was not diminished. It was also noted that all inactive individual components (i.e., 2-hydroxypropyl-β- cyclodextrin or FA) were not cytotoxic at levels corresponding to those present in the formulation (data not shown).

Table 1

Claims

The claims defining the invention are as follows:

1. A composition for the treatment or prevention of cancer, said composition comprising:

(i) 5-fluorouracil (5-FU), or a pharmaceutically acceptable salt or analogue 5 thereof;

(ii) folinic acid, or a pharmaceutically acceptable salt thereof;

(iii) oxaliplatin; and

(iv) a cyclodextrin or a pharmaceutically acceptable salt or derivative thereof.

2. The composition of claim 1, wherein the pH of the composition is about 5 I₀ to about 9.

3. The composition of claim 2, wherein the pH of the composition is about 6.5 to about 7.5.

4. The composition of any one of claims 1 to 3, wherein the cyclodextrin or salt or derivative thereof is present in the composition at a concentration of between is about 10 and about 300mg/ml.

5. The composition of any one of claims 1 to 4, wherein the 5-FU or salt or analogue thereof is present in the composition at a concentration of between about 5 and about 50mg/ml.

6. The composition of any one of claims 1 to 5, wherein the folinic acid or₀ salt thereof is present in the composition at a concentration of between about 1 and about

15mg/ml.

7. The composition of any one of claims 1 to 6, wherein the oxaliplatin is present in the composition at a concentration of between about 0.05 and about 10 mg/ml.

8. The composition of any one of claims 1 to 7, wherein the molar ratio of the₅ cyclodextrin or pharmaceutically acceptable salt or derivative thereof to the 5-FU, or pharmaceutically acceptable salt or analogue thereof, is between about 1 :10 and about 2: 1.

9. The composition of any one of claims 1 to 8, additionally comprising one or more pharmaceutically acceptable carrier, diluent and/or adjuvant. 0

10. The composition of claim 9, comprising an aqueous carrier.

11. The composition of any one of claims 1 to 10, said composition being in the form of an aqueous solution.

12. The composition of any one of claims 1 to 1 1, wherein the pharmaceutically acceptable salt of folinic acid is calcium folinate or sodium folinate.

13. The composition of any one of claims 1 to 12, wherein the cyclodextrin is selected from the group consisting of: a hydroxyalkyl cyclodextrin, a sulfated

5 cyclodextrin and a sulfoalkylether cyclodextrin, or a salt thereof.

14. The composition of any one of claims 1 to 13, wherein the cyclodextrin is a β-cyclodextrin, or a salt thereof.

15. A process for making a composition for the prevention and/or treatment of cancer, said process comprising combining: io (i) 5-FU, or a pharmaceutically acceptable salt or analogue thereof;

(ii) folinic acid, or a pharmaceutically acceptable salt thereof;

(iii) oxaliplatin; and

16. The process of claim 15, comprising combining (i) with (iv), and then ]₅ adding (ii) and (iii).

17. The process of claim 15, wherein (ii) is added as an aqueous solution.

18. The process of any one of claims 15 to 17, wherein the step of combining (i) with (iv) is conducted in the presence of an aqueous solvent to form an aqueous solution.

20 19. The process of any one of claims 15 to 18, wherein the step of combining

(i) with (iv) involves agitation so as to provide a homogeneous solution of (i) and (iv).

20. The process of any one of claims 15 to 19, additionally comprising sterilising the composition.

21. A composition for the treatment or prevention of cancer when made by the 2₅ process of any one of claims 15 to 20.

22. A method for the treatment or prevention of cancer in a patient in need of said treatment or prevention, said method comprising administration of a therapeutically effective amount of a composition according to any one of claims 1 to 14 or 21.

23. The method of claim 22, wherein the cancer is selected from the group 0 consisting of prostate cancer, colorectal cancer, lung cancer, cervical cancer, breast cancer, ovarian cancer, gastric cancer, primary hepatoma, skin cancer, pancreatic cancer, cholangiocarcinoma and a cancer of the head or neck.

24. A method for inhibiting the proliferation of cancer cells, said method comprising exposing the cancer cells to a composition according to any one of claims 1 to 14 or 21.

25. Use of a composition according to any one of claims 1 to 14 or 21 for the treatment or prevention of cancer, or for inhibiting the proliferation of cancer cells.

26. Use of 5-fiuorouracil, or a pharmaceutically acceptable salt or analogue thereof, folinic acid, or a pharmaceutically acceptable salt thereof, oxaliplatin and cyclodextrin, or a pharmaceutically acceptable salt or derivative thereof in the manufacture of a medicament for the treatment or prevention of cancer.