WO2006084248A2

WO2006084248A2 - Compositions comprising a platinum complex, lipid, and surfactant

Info

Publication number: WO2006084248A2
Application number: PCT/US2006/004152
Authority: WO
Inventors: Alemseged Truneh; Krzysztof Dziewiszek; Gui Lui
Original assignee: Antigenics, Inc.
Priority date: 2005-02-04
Filing date: 2006-02-06
Publication date: 2006-08-10
Also published as: WO2006084248A3

Abstract

Disclosed are compositions comprising a lipid, a surfactant, and a platinum complex having the formula II: Pt R K9/ \ K7 (II) wherein R6 and R7 each independently represent a leaving group; wherein R8 and R9 are each amines substituted by a group selected from a substituted or unsubstituted alkyl, -aryl, -arylalkyl, heteroalkyl, heterocyclyl, heteroaryl, heteroarylalkyl, -alkenyl, -cycloalkyl, or -cycloalkenyl groups having between 4 and 20 carbon atoms; or R8 and R9 are linked together as represented by formula (Ha): (Ha) and wherein said lipid is capable of forming a liposome. The compositions of the invention are more stable (e.g., resistant to solids formation) than are conventional liposomal platinum compositions which do not contain a surfactant. Also disclosed are methods for making the compositions and the use of the compositions for treating or preventing cancer.

Description

COMPOSITIONS COMPRISING A PLATINUM COMPLEX, LIPID, AND SURFACTANT

This application claims benefit of U.S. Provisional Application No. 60/650,327 filed February 4, 2005, which is incorporated herein by reference in its entirety.

1. FIELD OF THE INVENTION

The invention relates to compositions comprising a platinum complex, a lipid, and a surfactant, methods for making said compositions, and methods for using these compositions to treat or prevent cancer.

2. BACKGROUND OF THE INVENTION

Platinum coordination complexes were first identified as cytotoxic agents in 1965. c/s-Di ϊuninedichloroplatinum(II) (cisplatin) is a clinically significant anticancer agent useful for the treatment of a broad spectrum of neoplastic diseases in humans (see Loehrer et al., Ann. Int. Med. 100:704-713 (1984)). cw-Diamine(l,l-cyclobutane- dicarboxylato)platinum(II) (carboplatin), is a second-generation platinum analog and is the only platinum drug other than cisplatin to enjoy widespread use in the clinic. Carboplatin is effective when used in place of cisplatin in established chemotherapeutic drug regimens and although less emetic, nephrotoxic, neurotoxic, and ototoxic than cisplatin, carboplatin has undesirable myelosuppressive properties that cisplatin does not (see Go et al., J. Clin. Oncol. J/7: 409-22 (1999)). Oxaliplatin is a recently developed third-generation cisplatin analog with a 1,2-diaminocyclohexane (DACH) carrier ligand which has displayed clinical activity in a variety of tumor types and is not cross-resistant with cisplatin and carboplatin. Misset et al., Crit Rev. Oncol. Hematol. 35: 75-93 (2000) report that oxaliplatin acts synergistically with 5- fluorouracil (5-FU) in both 5-fluorouracil resistant and chemotherapy-naive disease and is currently being evaluated as a single-agent and in combination regimens against breast, lung and prostate cancer and non-Hodgkin' s lymphoma.

Other platinum analogs that have shown recent promise in clinical trials include NDDP (cz,s-bis-neodecanoato-trørø'-R,R- 1,2-diaminocyclohexane platinum(II) (see U.S. Patent No. 5,178,876 to Khohkar). U.S. Patent No. 4,256,652 to Kidani et al. describes platinum complexes comprising resolved stereoisomers of 1,2-diaminocyclohexane (DACH), including cis-OACB, trans-R,R-OACR and trans-S,S-OACJl. The trans-DkCΑ ligated platinum complexes were typically more efficacious as anti-tumor agents than the analogous czs-DACH complexes.

Liposomes are lipid vesicles, which may form spontaneously upon addition of an aqueous solution to a dry lipid film or powder, and can be used as drug carriers for both hydrophobic and hydrophilic drugs, the drugs being entrapped in the hydrophobic or hydrophilic portions of the liposome, respectively. Mayhew et ah, Liposomes, Ed., Marc J. Ostro, Marcel Dekker, Inc., New York, N.Y. (1983). Multilamellar liposomes are a class of multilayer lipid vesicles (MLVs) that are particularly suited for carrying hydrophobic drugs. When administered intravenously to animals and humans, MLVs concentrate in the liver, spleen and other organs rich in reticuloendothelial (RES) cells {see Kasi et ah, Int. J. Nucl. Med. Biol. 11:35-37 (1984); Lopez-Berestein et al, Cancer Drug Deliv. 1:199-205 (1984); and Lopez-Berestein et al, Cancer Res. 44:375-378 (1984)).

Liposomes have been utilized for the in vitro delivery of anticancer agents (see Mayhew et al, Liposomes, Ed. Ostro, Marcel Dekker, Inc., New York, N.Y.(1983), immunomodulators, and anti-fungal agents (see Mehta et al. , Immunology 5Jj 517-527 (1984)). In vivo drug delivery using liposomes has been reported in both animals and humans (see Lopez-Berestein et ah, Clin. Exp. Metastasis 2:127-137(1984); Lopez-Berestein et ah, J. Inf. Dis. 147:937-945 (1983); and Lopez-Berestein et ah, J. Inf. Dis. 151:704-710 (1985)). U.S. Patent No. 5,902,604 to Zou et al. describes a method for preparing a submicron liposome suspension comprising the anti-tumor agent annamycin and the surfactant Tween 20, which is reported to shorten the reconstitution step and improve the physical stability of the liposome.

Liposomal cis-platin formulations have been plagued by very poor encapsulation efficiency and by the relatively low stability of the resulting liposomal formulations (see Freise et ah, Arch. Int. Pharmacodynamie Therapie 258:180-192 (1982)). L-NDDP is a liposomal formulation of the complex bis-neodecanoato-cz,s'-l,2- diaminocyclohexane platinum(II) and is currently showing promise in clinical trials for pancreatic cancer, metastatic colorectal cancer and malignant mesothelioma. In some cases, DACH platinum complexes require an "activation" processes ranging from in vivo activation, addition of pH adjusting excipients to heating or mechanical agitation prior to administration. In certain cases, these processes could last for several hours. In the case of L-NDDP, however, prolonged activation processes increase the likelihood of degradation and/or precipitation of insoluble platinum complexes such as DACH-Pt (H)Cl₂.

There is a need, therefore, for lipid or liposomal formulations of NDDP that, when activated, are stable and the final compositions have a viscosity useful for injection into a subject.

The recitation of any reference in Section 2 of this application is not an admission that the reference is prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention relates to compositions comprising a lipid, a platinum anti-tumor agent, and a surfactant (the "compositions of the invention").

One aspect of the invention relates to a liposomal composition comprising a lipid, a surfactant, and a platinum (II) complex having the formula (II):

Pt R₉ R₇

(H) wherein R₆ and R₇ each independently represent a leaving group;

R₈ and R₉ are each amines substituted by a group selected from a substituted or unsubstituted alkyl, -aryl, -arylalkyl, heteroalkyl, heterocyclyl, heteroaryl, heteroarylalkyl, -alkenyl, -cycloalkyl, or -cycloalkenyl groups having between 4 and 20 carbon atoms; or R₈ and R₉ are linked together as represented by formula (Ha):

(Ha) wherein,

A is absent or represents a ring containing 4 to 10 ring atoms selected from C, N, O, and S (A, when present, may be saturated, unsaturated, or aromatic); B represents, independently for each occurrence, one or more occurrences of a group selected from H, halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate, — (CH₂)_p>alkyl, — (CH₂)_p>alkenyl, — (CH₂)_P'alkynyl, — (CH₂)p.aryl, — (CH₂Varalkyl, — O(CH₂)_P-R', — (CH₂)_P>SR\ and — (CH₂)_P>NR'₂, wherein R' represents, independently for each occurrence, H or substituted or unsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to 8-membered ring; and p' represents an integer from 0 to 4,

V and V, independently, represent a C(H)_m wherein m represents 0 or 1; W is absent or represents (Z(H)_n)_p wherein Z represents C₅ N, O, or S; and n and p independently represent 0, 1, 2, or 3; and wherein said lipid is capable of forming a liposome. In embodiments of the invention wherein R₈ and Rg nitrogens are linked together as represented by formula (Ha), the platinum complex has the following structural formula (lib):

(lib) wherein A, B, W, V, V, R₆ and R₇ are as described above.

In one embodiment, A, W, V, and V together represent a cyclohexane ring.

In another embodiment, A, B, W, V, and V together with R₈ and R₉ represent a cycloalkyl-l,2-diamino having between 3 and 7 carbon atoms or an alkyl- vicinal- diamino having between 2 and 12 carbon atoms.

In another embodiment, A, B, W, V, and V together with R₈ and R₉, represent trans-R,R- 1 ,2-diaminocyclohexane, trans-S,S- 1 ,2-diaminocyclohexane, cis- 1 ,2- diaminocyclohexane, ethylene diamine, or l,l-bis(aminomethyl)cyclohexane.

In another embodiment, A, B, W, V, and V together with R₈ and R₉, represent trans-R,R-l,2- diaminocyclohexane.

In another embodiment, A, B, W, V, and V together with R₈ and R₉, represent trans-S,S- 1 ,2-diaminocyclohexane. In one embodiment, R₆ and R₇ are each independently -neohexanoato, - neoheptanoato, -neononanoato, -neodecanoato, -neooctanoato, -neopentanoato, -2- ethylhexanoato, -2-ethylbutyrato, -2-propylpropanoato, -2-methyl-2-ethylheptanoato, -2,2- diethylhexanoato, -2,2-dimethyl-4-ethylhexanoato, -2,2-diethyl-4-methylpentanoato, -2,2- dimethyloctanoato, -2-methyl-2-ethylheptanoato, -2,2-diethylhexanoato, -2,3,5- trimethylheptanoato,-2,2-diethyl-4-methylpentanoato or -2,2,4,4-tetramethylpentanoato, or - 2,2-dimethyloctanoato.

In one embodiment, R₆ and R₇ are each -neodecanoato. In one embodiment, R₆ and R₇ are each independently a hydrophobic - alkylcarboxylate, -OH, -OH₂, -Cl, halo, -NO₃, or -HSO₄, with the proviso that R₆ and R₇ are not simultaneously both an -alkylcarboxylate.

In one embodiment, R₆ and R₇ are each independently -OH, -OH₂, -halo, - NO₃, or -HSO₄.

In certain embodiments, the invention relates to a composition comprising a lipid, a polysorbate surfactant, an isotonic agent and a platinum complex having the formula:

Pt

R₄ R₂ wherein R₁ and R₂ are both neodecanoato; R₃ and R₄ are linked to form a 1,2-diaminocyclohexane; and wherein said lipid is capable of forming a liposome. In one embodiment of this composition, the polysorbate surfactant is polysorbate 20.

In another embodiment of this composition, the lipid comprises a mixture of dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline.

In another embodiment of this composition, the isotonic agent is sodium chloride.

In another embodiment of this composition, the pH of the composition is from about 2 to about 6.

In a specific embodiment of this composition, the polysorbate surfactant is polysorbate 20, the lipid comprises a mixture of dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline, the isotonic agent is sodium chloride, and the pH of the composition is from about 2 to about 6, e.g., about 2.4.

Another aspect of the invention relates to a liposomal composition comprising a lipid, a surfactant and a platinum complex, said platinum complex being formed by a first composition comprising a lipid, a surfactant, and a platinum (II) complex of the formula (II) as described, supra, wherein the activation comprises heating, adding a chloride source, acidifying, or a combination thereof.

A specific embodiment of the invention relates to a composition comprising a lipid, a surfactant, and a platinum complex of formula (I):

(D wherein R₁ and R₂ are each independently an -alkyl-carboxylato bearing a hydrophobic radical function having 5 to 24 carbon atoms, -OH, -OH₂, -halo, -NO₃, or - HSO₄; or R₁ and R₂ are linked together to form a -dicarboxylato bearing a hydrophobic radical function;

R₃ and R₄ are each amines of the formula:

H

— N

R₅

wherein each R₅ is independently -H, -alkyl, -aryl, -arylalkyl, -heteroalkyl, - heterocyclyl, -heteroaryl, -heteroarylalkyl, -alkenyl, -cycloalkyl, or -cycloalkenyl having between 1 and 20 carbon atoms; or R₃ and R₄ are linked to form a cycloalkyl- 1,2-diamino having between 3 and 7 carbon atoms or an alkyl-vicinal-diamino having between 2 and 12 carbon atoms; and wherein said lipid is capable of forming a liposome. It has been observed that certain platinum (II) species are water-insoluble as well as lipid insoluble, thereby severely limiting their pharmaceutical efficacy. These complexes are substituted diaminoplatinum (II) dihalo compounds typically represented by formula III. (See e.g., Jain et al., J. Inorg. Biochem., 33, 1-9 (1988), Brown et al., J: Med. Chem., 25 952-956 (1982), Al-Baker et al., J. Coord. Chem., 31, 109-116 (1994), and Fanizzi et al., Inorg. Chim. Acta, 137,45-51 (1987). In certain embodiments, these compounds can be formed from lipid or water soluble precursor chemical compounds having a leaving group instead of a halo group. The present invention contemplates methods for delaying or preventing the precipitation of such substituted diamino platinum (II) dihalo compounds, without having to use pharmaceutically unacceptable solvents, so as to increase their bioactivity and make them amenable for pharmaceutical uses.

Accordingly, one aspect of the present invention relates to a liposomal composition comprising a water insoluble substituted diamino platinum (II) X₂ complex represented by formula III in a solubilized form:

(III) wherein R₈ and R₉ are as defined above;

X represents a halo; wherein the water insoluble substituted diamino platinum (II) X₂.is formed by activating a composition comprising a lipid, a surfactant, a platinum (II) complex represented by the formula (II):

Pt R₉ R₇

(H) wherein R₆, R₇, R₈, and R₉ are as defined above with a proviso that R₆ and R₇ are not halo and wherein the activation comprises adding a halide ion source, acid, or heating the composition or any combination thereof. In some embodiments R₆ and R₇ are each independently an -alkyl-carboxylato bearing a hydrophobic radical function having 5 to 24 carbon atoms, -OH, -OH₂, -NO₃, or -HSO₄; or R₁ and R₂ are linked together to form a - dicarboxylato bearing a hydrophobic radical function. The liposomal composition is more stable (e.g., resistant to solids formation) than lipid-containing platinum compositions made without a surfactant.

A skilled artisan would understand that the extent of activation, and thereby the amount of the solubilized form of the water insoluble substituted diamino platinum (II) X₂ actually administered to a subject, can be controlled by varying the amount of halide ion source or acid added, or by adjusting the temperature and time of heating. For example, in certain embodiments, exposing the liposomal formulation containing the platinum complex of the formula (I) or (II) to higher temperature can provide higher levels of activated species than would exposure of the liposomal formulation to lower temperatures.

As used herein including the claims, the term "halo" includes fluoro, chloro, bromo, and iodo. Preferred halo substituents are chloro, bromo and iodo.

In certain embodiments, the composition of the invention further comprises a liquid diluent (the "liquid composition of the invention").

In certain other embodiments, the liquid composition of the invention comprising a liquid can be lyopbilized. The resulting lyophilate can then be reconstituted by adding a pharmaceutically suitable diluent or excipient. In certain embodiments, the composition or its reconstituted product can be "activated" in vitro prior to administration.

The invention also relates to methods for making the compositions of the invention.

Accordingly, in one embodiment, the present invention relates to a method for the activation of a liquid composition comprising lipid, surfactant, an aqueous diluent, and a substituted diamino platinum (II) complex prodrug represented by formula II

II wherein R₆, R₇, R₈, and Rg are as defined above; said method comprising adding an amount of surfactant and activating the liposomal composition by heating, adding a chloride source, acidifying, or a combination thereof so as to form a second composition with enhanced biological activity and free from precipitated platinum complexes. Preferably, the amount of surfactant added is sufficient to delay the onset of precipitation by at least one hour. In certain embodiments, the activation is rapid, e.g., within 60, 50, 40, 30, 20, 15, 10, or even 5 minutes,and the activated composition is a pharmaceutically stable composition (e.g., resistant to solids formation) suitable for administration within a 24 hour window from activation.

The preparation of liquid compositions containing solubilized platinum (II) complexes of the formula (I) or (II) that do not contain surfactant has often proved problematic because these complexes precipitate out of solution before compositions could be administered to patients, hi many instances, conditions sufficient to activate the composition have often resulted in rapid precipitation of insoluble platinum species. The rapid precipitation thwarts efforts to prepare a liquid composition that is suitable for parenteral administration. Surprisingly, addition of a surfactant into the liquid compositions containing these water-insoluble platinum (II) complexes, delays the onset of precipitation of water-insoluble species thereby making these compositions suitable and stable for pharmaceutical delivery into a subject. In specific embodiments the onset of precipitation is delayed for at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, or at least 18 hours after activation of the liquid composition. In some embodiments, the onset of precipitation has been delayed for periods as long as 24 hours after activation of the liquid composition. Accordingly, the present invention also contemplates a method for delaying the precipitation of water-insoluble platinum(II) species.

Upon activation, the pH of the compositions containing a platinum complex of formula (I) or (II) increases. If left uncontrolled, the pH of the composition could rise to a point where precipitation of water-insoluble platinum (II) species occurs. To delay or prevent such precipitation, in certain embodiments, the present invention also relates to adding a buffer such as lactic acid (See, for example, U.S. Patent No. 6,476,068). The buffer can be introduced before, during, or after the activation step. The buffer can stabilize both the unactivated composition and activated composition.

In one embodiment, the invention relates to a second composition prepared by activating a first composition, said first composition comprising a lipid, a surfactant, an aqueous diluent, and a platinum complex having the formula (I) or (II), wherein:

R₁-R₄ or R₆, R₇, R₈ and R₉ are as defined above; said lipid is capable of forming a liposome; and the activating is heating, adding a chloride source, acidifying, or a combination thereof.

In another embodiment, the invention relates to a method for making a composition comprising:

(a) mixing a lipid, a surfactant, and a platinum complex of formula (I) or (II) with a first diluent to form a liquid composition; wherein R₁-R₄ or R₆, R₇, R₈ and R₉ are as defined above and said lipid is capable of forming a liposome; and

(b) lyophilizing the liquid composition to form a lyophilate.

In another embodiment, the invention relates to a method for making a composition comprising: (a) mixing a lipid and a platinum complex of formula (I) or (II) with a first diluent to form a liquid composition lacking a surfactant; wherein R₁-R₄ or R₆, R₇, R₈ and R₉ are as defined above; and said lipid is capable of forming a liposome;

(b) lyophilizing the liquid composition to form a lyophilate; (c) contacting the lyophilate with a second diluent comprising water to form a reconstituted composition; and

(d) contacting the reconstituted composition with a surfactant. In another embodiment, the invention relates to a method for making a composition comprising: (a) mixing a lipid and a platinum compound of formula (I) with a first diluent to form liquid composition lacking a surfactant; wherein R₁-R₄ or R₆, R₇, R₈ and R₉ are as defined above and said lipid is capable of forming a liposome;

(b) lyophilizing the liquid composition to form a lyophilate; and

(c) contacting the lyopbilate with a second diluent comprising water and a surfactant to form a reconstituted composition.

In a particular embodiment, the invention relates to a method for making a composition. The method includes:

(a) mixing a lipid and a platinum complex with a first diluent to form a liquid composition lacking a surfactant, the platinum compound having the formula:

^R\ /^R1

Pt ^R4 ^R2 wherein R₁ and R₂ are both neodecanoato;

R₃ and R₄ are linked to form a 1,2-diaminocyclohexane;

(b) lyophilizing the liquid composition to form a lyophilate;

(c) contacting the lyophilate with a second diluent comprising water, an isotonic agent and a polysorbate surfactant to form a reconstituted composition; and

(d) heating the reconstituted composition at a temperature of from about 30°C to about 45°C, e.g., about 37⁰C.

In one embodiment of the method, the first diluent comprises t-butanol and water. In another embodiment of the method, the polysorbate surfactant is . - polysorbate 20. In another embodiment of the method, the lipid comprises a mixture of dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline.

In another embodiment of the method, the isotonic agent is sodium chloride.

In another embodiment of the method, the pH of the reconstituted composition is from about 2 to about 6, e.g. , about 2.4.

In another embodiment of the method, step (d) comprises heating the reconstituted composition for about 0.25 to about 3 hours, e.g., about 2 hours.

In a specific embodiment of the method: the first diluent comprises t-butanol and water, the polysorbate surfactant is polysorbate 20, the lipid comprises a mixture of dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline, the isotonic agent is sodium chloride, the pH of the reconstituted composition is from about 2 to about 6, and step (d) comprises heating the reconstituted composition for about 0.25 to about 3 hours.

In a particular embodiment of this method, the composition formed has an administration window, i.e., a time period wherein at least 50, 60, 70 or 80% of the NDDP in the reconstituted composition of (c) has been consumed (converted to an activated species, e.g., DACH-Pt-Cl₂), but no precipitated platinum species are observed, that is from 2 to 10 hours, and more preferably from 3 to 8 hours, or 4 to 6 hours.

In another particular embodiment, the invention relates to a method for making a composition. The method includes:

(a) mixing a lipid, a polysorbate surfactant and a platinum complex with a first diluent to form a liquid composition, the platinum compound having the formula:

Pt

R₄ R₂ wherein R₁ and R₂ are both neodecanoato; R₃ and R₄ are linked to form a 1,2-diaminocyclohexane;

(b) lyophilizing the liquid composition to form a lyophilate; (c) contacting the lyophilate_with a second diluent comprising water and an isotonic agent to form a reconstituted composition; and (d) heating the reconstituted composition at a temperature of from about 30°C to about 45°C, e.g., about 37⁰C.

In one embodiment of the method, the first diluent comprises t-butanol and water. In another embodiment of the method, the polysorbate surfactant is polysorbate 20.

In another embodiment of the method, the lipid comprises a mixture of dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline.

In another embodiment of the method, the isotonic agent is sodium chloride. In another embodiment of the method, the pH of the reconstituted composition is from about 2 to about 6, e.g., about 2.4.

Another aspect of the invention relates to a method for reducing the viscosity of a liposomal composition comprising one or more lipids capable of forming a liposome, one or more platinum complexes (e.g, the platinum complexes of the formula I or II), an aqueous diluent or an aqueous diluent containing a carbohydrate-based isotonic agent to achieve ^pharmaceutically acceptable viscosity suitable for parenteral administration. The method comprises adding a surfactant to the liposomal composition. In some embodiments, the surfactant is added to a pre-lyophilate mixture comprising the lipid and platinum complex. In other embodiments, the surfactant is added to the pre-liposomal powder consisting essentially of the lipid and a bioactive agent. Methods for forming pre-lyophilate and pre-liposomal mixtures are described in U.S. Patent No. 5,902,604.

In preparing certain liquid compositions of the invention, the addition of an aqueous diluent containing a carbohydrate-based isotonic agent (e.g., dextrose) delays the onset of precipitation of insoluble species for certain platinum complexes of the formula I or II. However, in some embodiments, the high viscosity of the resulting liquid composition renders it unsuitable for parenteral injection. Addition of a surfactant to the liquid composition reduces the viscosity of the liquid composition to a viscosity that is suitable for parenteral injection. For parenteral administrations of these liposomal compositions, the viscosities of these compositions need to be within pharmaceutically acceptable ranges. Discussion of appropriate ranges for viscosities are presented in Claassen, V, Neglected Factors in Pharmacology and Neuroscience Research, Techniques in the Behavioural and Neural Sciences: VoI 12, Huston J P (ed), Amsterdam: Elsevier (1994). Moreover, in certain embodiments, the addition of the surfactant to the liquid composition further delays the onset of precipitation of insoluble species. Advantageously, this delay extends the time period in which the liquid composition may be safely administered to a subject. In specific embodiments of the invention, the onset of precipitation is delayed for at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, or at least 18 hours over the time period in which precipitation is observed in liquid compositions prepared in the absence of a surfactant.

Accordingly, one embodiment of the invention relates to a method for preparing a second liquid composition comprising one or more lipids capable of forming a liposome, an aqueous diluent, and a bioactive agent (e.g., a water-insoluble platinum (II) complex); wherein the second liquid composition is suitable for parenteral administration, the method comprising adding a surfactant to a first liquid composition comprising the lipid and the bioactive agent (e.g., water-insoluble platinum (II) complex) in an aqueous diluent to give the second liquid composition. In a specific embodiment, the aqueous diluent is saline. Another embodiment of the invention relates to a method for preparing a second liquid composition comprising one or more lipids capable of forming a liposome, an aqueous diluent, and a bioactive agent (e.g., water-insoluble platinum (II) complex); wherein the second liquid composition is suitable for parenteral administration, the method comprising: (a) adding an aqueous diluent comprising a carbohydrate-based isotonic agent to a first liquid composition comprising the one or more lipids and the bioactive agent (e.g., water-insoluble platinum (II) complex); and

(b) adding a surfactant to the liquid composition of (a) to give the second liquid composition.

Non-limiting examples of a bioactive agent include an antifungal agent, an antibiotic, an adjuvant, a vaccine, a contrast agent, a diagnostic agent, a drug targeting agent, an anticancer agent, and a genetic fragment.

The invention still further relates to methods for treating or preventing cancer. In one embodiment, the invention relates to a method for treating or preventing cancer comprising administering to a patient in need thereof an effective amount of the compositions of the invention, hi certain embodiments, the composition of the invention may be administered conjointly with other therapeutic agents, or may be administered in a treatment regimen involving other therapeutic agents. The details of the invention are set forth in the accompanying figures, description and examples below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, illustrative methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise.

4. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the activation of liposomal NDDP compositions (shown as the disappearance of NDDP) when reconstituted in an acidified saline solution. Figure 2 shows the survival rate in mice challenged with L1210 tumor cells

(10⁶) by intraperitoneal (i.p.) route on day 0 and treated with liposomal NDDP compositions, oxaliplatin or diluent (control) on days 1, 4 and 8 by i.v. route.

Figure 3 shows the cumulative tumor burden (volume) in mice challenged with HT29 tumor cells (10⁶) by subcutaneous route on day 0 and treated with a liposomal NDDP composition, oxaliplatin or diluent (control) on days 1, 8 and 15 by intravenous (i.v.) route. The * denotes a statistically significant difference from the diluent group. • 5. DETAILED DESCRIPTION OF THE INVENTION

5.1. THE COMPOSITIONS OF THE INVENTION

The present invention relates to compositions and methods for formulating water-insoluble bioactive agents in manner suitable for pharmaceutical administration into a subject.

Another embodiment of the present invention relates to compositions and methods for formulating these water-insoluble drugs into pharmaceutically acceptable liposomal formulations. In certain embodiments, the bioactive agents are water insoluble platinum (II) complexes. In particular, the present invention relates to ways of formulating these water-insoluble platinum (II) complexes so that these complexes do not precipitate out of the pharmaceutical compositions. In certain other embodiments, these complexes are products of activating lipid soluble platinum (II) complexes (i.e., through heating, adding a chloride source, acidifying or a combination thereof), which function as precursors of the activated products. These precursors themselves have lower bioactivity compared to the activated products. The present invention provides methods for activation of these precursors to achieve rapid activation (without precipitation of water-insoluble species), to produce compositions containing stable concentration (e.g., resistant to solids formation) of solubilized water-insoluble activated platinum (II) species. The methods of the invention allow activation of the platinum (II) complexes while delaying the precipitation of these water-insoluble activated platinum (II) species, so that the activated platinum complexes can be safely administered within a convenient administration window.

In certain embodiments, the pharmaceutical compositions of the present invention are liposomal. For parenteral administrations of these liposomal compositions, the viscosities of these compositions need to be within pharmaceutically acceptable ranges. Discussion of appropriate ranges for viscosities are presented in Claassen, V, Neglected Factors in Pharmacology and Neuroscience Research, Techniques in the Behavioural and Neural Sciences: VoI 12, Huston J P (ed), Amsterdam: Elsevier (1994). The present invention provides methods to optimize the viscosities of liposomal formulations of bioactive agents (e.g, platinum (II) complexes of the formula I or II) by introducing an effective amount of surfactant to the formulations either before, during or after the formation of liposomes. An additional benefit of adding a surfactant, is the reduction of the mean diameters of the resulting liposomes. In the case of water-insoluble platinum (II) complexes, the addition of the surfactant also prevents or delays the precipitation of insoluble species from the pharmaceutical compositions.

The present invention also contemplates use of the pharmaceutical compositions of the present invention for treatment of cancer. In certain embodiments, these compositions may also be part of a combination therapy with other chemotherapeutic agents. The combination therapies offer advantages in terms of additive or synergistic efficacy between the combined drugs, reduced toxicities (see, e.g., U.S. Patent No. 6,066,668), or the ability to treat drug resistant varieties of cancers.

Definitions As used herein, the term "leaving group," in the context of a platinum (II) complex of the invention, refers to a chemical moiety which dissociates from the platinum complex upon heating, exposure to a chloride source, exposure to an acid, or a combination thereof. Non- limiting examples of leaving groups include -alkyl-carboxylatos bearing a hydrophobic radical function having 5 to 24 carbon atoms, -OH, -OH₂, -halo, -NO₃, or -HSO₄. The term "alkyl" refers to straight or branched chain saturated aliphatic groups, cycloalkyl(alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 20 or fewer carbon atoms in its backbone (e.g., C₁-C₂₀ for straight chains, C₃-C₃₀ for branched chains), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

The "alkyl" (or "lower alkyl") can be "unsubstituted alkyl" or "substituted alkyl," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyϊ may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), — CF₃, — CN and the like. Exemplary substituted alkyls are described below. It will also be understood by a skilled artisan that one or more heteroatoms maybe substituted for the carbon(s) on an alkyl chain so as to form a heteroalkyl group. Non-limiting examples of heteroalkyl groups are 2- methoxyethyl, 2-ethoxyethyl, 2-methylthiomethyl and 2-ethylthioethyl.

Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. The term "alkenyl" refers to straight or branched chain non-cyclic hydrocarbon which contains at least one carbon-carbon double bond. As used herein, the term "lower alkenyl" refers to alkenyl groups having from one to ten carbon atoms and preferably, from one to six carbon atoms.

The term "alkynyl" refers to an unsaturated , straight or branched chain hydrocarbon group containing at least one triple bond. As used herein, the term "lower alkynyl" refers to alkynyl groups having from one to ten carbon atoms and preferably, from one to six carbon atoms.

The term "cycloalkyl", as used herein, unless otherwise indicated, refers to a non- aromatic, monocyclic or bicyclic saturated ring consisting of carbon and hydrogen atoms. Preferred cycloalkyls have from three to eight ring carbon atoms, including, but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The cycloalkyl groups may be optionally substituted one or more times, with substituents such as halogen, azide, alkyl (i.e., arylalkyl) alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, — CF₃, — CN, or the like., preferably halogen, lower alkyl. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl- substituted alkyls, — CF₃, — CN, and the like.

The term "cycloalkenyl", as used herein, unless otherwise indicated, refers to a non- aromatic, monocyclic or bicyclic ring consisting of carbon and hydrogen atoms, having at least one carbon-carbon double bond.

The term "heterocyclyl", as used herein, unless otherwise indicated, refers to a non- aromatic, single or fused ring of carbon atoms and at least one heteroatom. hi specific aspects of the invention, the heterocyclic rings have up to four heteroatoms in each ring, each of which are independently selected from O, N and S, and which rings, may be unsubstituted or substituted independently by, for example, up to three substituents. Each heterocyclic ring suitably has from 4 to 7, preferably 5 or 6, ring atoms. A fused heterocyclic ring system may include carbocyclic rings and need include only one heterocyclic ring which may be partially saturated or saturated. The heterocyclyl includes mono, bicyclic and tricyclic heteroaromatic ring systems comprising up to four, preferably 1 or 2, heteroatoms each selected from O, N and S. Each ring may have from 4 to 7, preferably 5 or 6, ring atoms. A bicyclic or tricyclic ring system may include a carbocyclic ring. Carbocyclic ring includes cycloalkyl, cycloalkenyl or aryl ring. Examples of heterocyclyl groups include pyrrolidine, pyrrolidinone, piperidine, piperidinone, piperazine, morpholine, imidazolidine, pyrazolidine, hydantoin, oxetane, tetrahydrofuran, tetrahydropyran, pyrrole, indole, pyrazole, indazole, trizole, benzotrizole, imidazole, benzoimdazole, thiophene, benzothiophene, thiozole, benzothiozole, furan, benzofuran, oxazole, benzoxazole, isoxazole, tetrazole, pyridine, pyrimidine, trizine, quinoline, isoquinoline, quinazoline, indoline, indolinone, benzotetrahydrofuran, tetrahydroquinoline, tetrahydroisoquinoline and methylenedioxyphenyl.

The term "aryl" refers to aromatic groups which have at least one ring having a conjugated pi electron system, and includes carbocyclic aryl and heterocyclic aryl (also referred to as "heteroaryl"). In specific embodiments, the aryl group is a 5-, 6-, and 7- membered single-ring aromatic groups. In certain embodiments the aryl group may include from zero to four heteroatoms. Non-limiting examples of aryl rings include benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl (i.e., arylalkyl) alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, — CF₃, — CN, or the like, m certain embodiments the aryl group can be a polycyclic ring system having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls. As discussed above, the present invention relates to compositions comprising a lipid, a platinum anti-tumor agent, and a surfactant. The compositions of the present invention are less viscous and more stable (e.g., resistant to solids formation) than lipid- containing platinum compositions made without a surfactant. In one embodiment, the invention relates to a composition comprising a lipid, a surfactant, and a platinum complex having the formula (I):

RS _{N /}R,

Pt R₄ R₂

(I) wherein R₁ and R₂ are each independently an -alkyl-carboxylato bearing a hydrophobic radical function having 5 to 24 carbon atoms, -OH, -OH₂, -halo, -NO₃, or - HSO₄; or R₁ and R₂ are linked together to form a -dicarboxylato bearing a hydrophobic radical function;

R₃ and R₄ are each amines of the formula:

H

— \

R₅

wherein each R₅ is independently -H, -alkyl, -aryl, -aryllkyl, -alkenyl,

-cycloalkyl, or -cycloalkenyl having between 1 and 20 carbon atoms; or R₃ and R₄ are linked to form a cycloalkyl- 1,2-diamino having between 3 and 7 carbon atoms or an alkyl-vicinal- diamino having between 2 and 12 carbon atoms; and wherein said lipid is capable of forming a liposome. In one embodiment, R₁ and R₂ are each independently -OH, -OH₂, -halo,

-NO₃, -HSO₄; or an -alkyl-carboxylato containing from 5 to 24 carbon atoms.

In another embodiment, R₁ and R₂ are each independently an -alkyl- carboxylato bearing a hydrophobic radical function, -OH, -OH₂, -halo, -NO₃, or -HSO₄; or an -alkyl-carboxylato containing from 5 to 24 carbon atoms, with the proviso that R₁ and R₂ are not both simultaneously -alkyl-carboxylato containing from 5 to 24 carbon atoms.

In another embodiment, R₁ and R₂ are each independently -OH, -OH₂, -halo, - NO₃, or -HSO₄.

A non-limiting example of an -alkyl-carboxylato containing from 5 to 24 carbon atoms is represented by formula Ia:

(Ia) wherein -R₆, -R₇, and -R₈ are each independently hydrogen, or straight-chain or branch-chain hydrocarbon moieties having from 1 to 13 carbon atoms. Non-limiting examples of -alkyl carboxylato groups containing from 5 to 24 carbon atoms include -neohexanoato, -neoheptanoato, -neononanoato, -neodecanoato, -neooctanoato, -neopentanoato. Since the precursors of the aforementioned alkyl carboxylato groups are often sold as alkyl carboxylic acids comprising a mixture of structural isomers, the term "alkyl carboxylato" as used herein refers to both single isomers, or a mixture of all possible structural isomers. In certain embodiments, R₁ and R₂ are each independently -pentanoato, - hexanoato, -heptanoato, -octanoato, -nonooato, -undecanoato, -dodecanoato, -tridecanoato, - tetradecanoato, -pentadecanoato, -hexadecanoato, -heptadecanoato, -octanoato, - nonadecanoato, — 2-ethylhexanoato, -2-ethylbutyrato, -2-propylpropanoato, -2-methyl-2- ethylheptanoato, -2,2-diethylhexanoato, -2,2-dimethyl-4-ethylhexanoato, -2,2-diethyl-4- methylpentanoato, -2,2-dimethyloctanoato, -2-methyl-2-ethylheptanoato, -2,2- diethylhexanoato, 2,2,5-trimethylheptanoato, -2,2-diethyl-4-methylpentanoato or -2,2,4,4- tetraniethylpentanoato. In certain preferred embodiments, R₁ and R₂, independently, represent, a neodecanoato group.

In certain embodiments, R₃ and R₄ are linked together to form an alkyl- vicinal- diamino having between 2 and 12 carbon atoms, or a cycloalkyl-l,2-diamino having between 3 and 7 carbon atoms in the cyclic portion.

In one embodiment, R₃ and R₄ are linked together to form an alkyl- vicinal- diamino. A non-limiting example of an alkyl- vicinal-diamino is ethylene diamine or 1,1-bis- (aminomethyl)cyclohexane. In one embodiment, R₃ and R₄ are linked together to form a cycloalkyl-1 ,2- diamino. hi another embodiment, the cylcoalkyl-l,2-diamino is trans-D,L-l,2- diaminocyclohexane, trans-R,R- 1 ,2-diaminocyclohexane, trans-S,S- 1 ,2-diaminocyclohexane, cis-l,2-diaminocyclohexane, or l,l-bis(aminomethyl)cyclohexane.

In certain preferred embodiments, R₃ and R₄ together represent trans-R,R-l,2- diaminocyclohexane. In another embodiment, R₁ and R₂ are each -neodecanoato, and R₃ and R₄ together represent trans-R,R-l,2-diaminocyclohexane.

In another embodiment, the platinum complex of formula (I) or (II) is cis-bis- neoheptanoato (trans-R,R-S,S-l,2-diaminocylcohexane) platinum (II), cis-bis- neoheptanoato(trans-R,R-S,S-l,2-diaminocylcohexane) platinum (II), cis-bis-neohexanoato 1 , 1 -bis-(aminomethyl)cy clohexaneplatinum (II), cis-bis-neodecanoato 1 , 1 -bis- (aminomethyl)cyclohexane platinum (II), cis-bis-neoheptanoato(trans-R,R-l,2- diaminocyclohexane) platinum (II), cis-bis-neoheptanoato[l,l-bis- (aminomethyl)cyclohexane] platinum (II), cis-bis~2,2-dimethyloctanoato(trans-R,R-l,2- diaminocyclohexane) platinum (II), cis-bis-neononanoato (trans-R,R-l,2- diaminocyclohexane) platinum (II), cis-bis-neoheptanoato(ethylenediamine) platinum (II), cis-bis-neopentanoato[l,l-bis-(aminomethyl)cyclohexane] platinum (II), cis-bis- neononanoato [1,1 -bis-(aminomethyl)cyclohexane] platinum (II), cis-bis-2-methyl-2- ethylheptanoato(trans-R,R-l,2-diaminocyclohexane) platinum (II), cis-bis-2,2- diethylhexanoato(trans-R,R- 1 ,2- diaminocyclohexane) platinum (II), cis-bis-2,2-dimethyl-4- ethylhexanoato(trans-R,R-l,2- diaminocyclohexane) platinum (II), cis-bis-2,2-diethyl-4- methylpentanoato(trans-R,R-l,2- diaminocyclohexane) platinum (II), cis-bis-2,2,5- trimethylheptanoato (trans-R,R-l,2- diaminocyclohexane) platinum (II), cis-bis-2,2- dimethyloctanoato(trans-S,S-l,2- diaminocyclohexane) platinum (II), cis-bis-2,2,4,4- tetrarnethylpentanoato(trans-R,R-l,2- diaminocyclohexane) platinum (II), cis-bis-2-methyl-2- ethylheptanoato(trans-S,S-l,2- diaminocyclohexane) platinum (II), cis-bis-2,2- diethylhexanoato (trans-S,S-l,2- diaminocyclohexane) platinum (II), cis-bis-2,2,5- trimethylheptanoato (trans-S,S-l,2- diaminocyclohexane) platinum (II), cis-bis-2,2-diethyl-4- methylpentanoato(trans-S,S-l,2- diaminocyclohexane) platinum (II), cis-bis-2,2,4,4- tetramethylpentanoato(trans-S,S-l,2- diaminocyclohexane) platinum (II), cis-bis- neodecanoato-trans-RjR-l^-diaminocyclohexane platinum (II), or cis-bis-neodecanoato (cis- 1,2-diaminocyclohexane platinum (II).

Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. AU such isomers, as well as mixtures thereof, are intended to be included in this invention. If₅ for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts may be formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. In certain embodiments of the invention wherein the platinum complex of the formula

I or II specify substitution of a chemical radical, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

In the embodiment described above, it will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

The amount of the platinum complex of formula (I) or (II) present in the composition of the invention can vary. In one embodiment, the amount of platinum present in the composition of the invention is from about 0.001 % to about 0.25%; in another embodiment from about 0.005 % to about 0.1 %; in another embodiment, about 0.03 % to about 0.08 %; and in another embodiment, about 0.06 % by weight based on the total weight of the composition of the invention. As used herein, such weight percentages are described based on the formula weight of platinum metal.

As used herein, the term "about" when used in connection with concentrations, temperatures, time, and the like means within standard experimental error. The compositions of the invention also comprise a lipid that is capable of forming a liposome (i.e., the lipid forms a liposome under appropriate conditions, e.g., in the presence of an aqueous diluent).

In certain embodiments, any lipid useful for making a conventional liposome can be used including zwitterionic lipids, anionic lipids, nonionic lipids, and/or cationic lipids. Non-limiting examples of lipids useful in the present invention include, but are not limited to, dimyristoyl phosphatidyl choline (DMPC), egg phosphatidyl choline, dilauryloyl phosphatidyl choline, dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline, 1- myristoyl-2-palmitoyl phosphatidyl choline, l-palmitoyl-2-myristoyl phosphatidyl choline, 1- palmitoyl-2-stearoyl phosphatidyl choline, l-stearoyl-2-palmitoyl phosphatidyl choline, dioleoyl phosphatidyl choline, dimyristoyl phosphatidyl glycerol (DMPG), dilauryloyl phosphatidyl glycerol, dioleoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol, distearoyl phosphatidyl glycerol, l-myristoyl-2-palmitoyl phosphatidyl glycerol, l-palmitoyl- 2-myristoyl phosphatidyl glycerol, 1 -palmitoyl-2-stearoyl phosphatidyl glycerol, 1-stearoyl- 2-palmitoyl phosphatidyl glycerol, dioleoyl phosphatidyl glycerol, dimyristoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dimyristoyl phosphatidyl serine (DMPS), dilauryloyl phosphatidyl serine, dipalmitoyl phosphatidyl serine, distearoyl phosphatidyl serine, l-myristoyl-2-palmitoyl phosphatidyl serine, l-palmitoyl-2-myristoyl phosphatidyl serine, 1 -palmitoyl-2-stearoyl phosphatidyl serine, l-stearoyl-2-palmitoyl phosphatidyl serine, dioleoyl phosphatidyl serine, brain sphingomyelin, dipalmitoyl sphingomyelin, distearoyl sphingomyelin, dimyristoyl phosphatidic acid, dilauryloyl phosphatidic acid, dioleoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, l-myristoyl-2-palmitoyl phosphatidic acid, l-palmitoyl-2-myristoyl phosphatidic acid, 1 -palmitoyl-2-stearoyl phosphatidic acid, l-stearoyl-2-palmitoyl phosphatidic acid, or dioleoyl phosphatidic acid.

Lipids are available from, e.g., Lipoid, GmbH, Ludwigshafen, Germany, NOF Corporation, Tokyo, Japan; Avanti Polar Lipids, Inc. Alabaster, Alabama, USA; Chemi S. p. a., Patrica, Italy.

In one embodiment, the lipid is a phosphatidyl glycerol. In another embodiment, the lipid is a phosphatidyl choline.

In a preferred embodiment, the lipid is dimyristoyl phosphatidyl glycerol or dimyristoyl phosphatidyl choline.

In another embodiment, the lipid comprises dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline. When the lipid comprises dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline, the ratio of the lipids can vary. In one embodiment, the molar ratio of dimyristoyl phosphatidyl glycerol to dimyristoyl phosphatidyl choline is from about 1:20 to about 20:1; in another embodiment, from about 2:10 to about 10:2; and in another embodiment, about 3:7. In another embodiment, the lipid consists essentially of dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline.

The amount of lipid used in the composition of the invention can vary. In one embodiment, the lipid is present in the composition of the invention in an amount from about 0.1 parts by weight to about 1000 parts by weight based on the amount of platinum. In another embodiment, the lipid is present in the composition of the invention in an amount from about 1 part by weight to about 100 by parts weight based on the amount of platinum. In another embodiment, the lipid is present in the composition of the invention in an amount from about 25 parts by weight to about 75 parts by weight based on the amount of platinum. In another embodiment, the lipid is present in the composition of the invention in an amount of about 50 parts by weight based on the amount of platinum, hi certain embodiments, the composition has 46 parts of lipids per 1 part of platinum, hi specific embodiments, the composition has 32 parts of DMPC and 14 parts of DMPG. As used herein, such weight ratios are described based on the formula weight of platinum metal.

The compositions of the present invention further comprise a surfactant. Surfactants (surface active agents) are compounds that facilitate the dissolution, dispersion, and/or homogeneous suspension of the compositions of the invention in the diluent. Without being limited by theory, it is believed that the surfactant prevents or delays the formation of solids, e.g., the formation of solid platinum complexes from the platinum compositions, particularly those platinum compositions comprising a chloride source (e.g. , saline or hydrochloric acid). It is also believed that the surfactant reduces the viscosity of the platinum composition, particularly those platinum compositions comprising a carbohydrate-based isotonic agent as described below.

Any pharmaceutically acceptable surfactant can be used. For example, the surfactant can be non-ionic, cationic, anionic, zwitterionic, or any combination thereof. Non-limiting examples of useful non-ionic surfactants include poloxamers such as poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407; and poly(oxyethyl)-sorbitan monooleates ("polysorbates") such as polysorbate 20, polysorbate 60, polysorbate 80; or any combination thereof. Poloxamers are available from BASF, Mount Olive NJ, under the tradename POLOXAMER^®. Polysorbates are available from Uniqema, New Castle, DE under the tradename TWEEN^® Pharma.

Non-limiting examples of cationic surfactants useful in the present invention include trialkylammonium halides such as dodecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide.

Non-limiting examples of an anionic surfactants useful in the present invention include include tetralkyl ammonium sulfates such as alkyl sulfates such as sodium dodecyl sulfate.

Non-limiting examples of zwitterionic surfactants useful in the present invention is 3-dimethyldodecylarnine propane sulfonate.

In one embodiment, the non-ionic surfactant is poloxamer 237, poloxamer 407, or a combination thereof.

In one embodiment, the non-ionic surfactant is poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or a combination thereof. In another embodiment, the non-ionic surfactant is poloxamer 188.

In another embodiment, the non-ionic surfactant is poloxamer 338.

In another embodiment, the non-ionic surfactant is poloxamer 237.

In another embodiment, the non-ionic surfactant is poloxamer 407.

In one embodiment, the non-ionic surfactant is polysorbate 20, polysorbate 80, or a combination thereof.

In another embodiment, the non-ionic surfactant is polysorbate 20.

In another embodiment, the non-ionic surfactant is polysorbate 80.

The amount of surfactant used in the composition of the invention can vary and will depend, in part, on the amount of platinum complex, the amount and type of lipid, and the composition of the aqueous diluent. In one embodiment, the surfactant is present in the composition of the invention in an amount from about 0.1 parts by weight to about 100 parts by weight based on the amount of platinum. In another embodiment, the surfactant is present in the composition of the invention in an amount from about 1 part by weight to about 10 parts weight based on the amount of platinum. In another embodiment, the surfactant is present in the composition of the invention in an amount from about 3 parts by weight to about 7 parts by weight based on the amount of platinum, hi another embodiment, the surfactant is present in the composition of the invention in an amount of about 5 parts by weight based on the amount of platinum. In another embodiment, the surfactant is present in the composition of the invention in an amount from about 1 parts by weight to about 3 parts by weight based on the amount of platinum. In another embodiment, the surfactant is present in the composition of the invention in an amount of about 2.5 parts by weight based on the amount of platinum. As used herein, such weight ratios are described based on the formula weight of platinum metal. The compositions of the present invention can further comprise one or more of an isotonic agent; an anticancer agent other than the platinum complex of formula (I) or (II); or a therapeutic agent other than the platinum complex of formula (I) or (II) such as, e.g., an antiemetic agent, a hematopoietic colony stimulating factor, an anti-depressant, analgesic agent or an immunotherapeutic agent such as vaccine or immunostimulant. One of ordinary skill in the art will recognize that certain substances can be characterized as one or more of the foregoing. For instance, a vaccine can be both an anticancer agent and an immunotherapeutic agent. The composition and methods of the invention also contemplate use of such dual functional therapeutic agents.

Non-limiting examples of useful isotonic agents include carbohydrate-based isotonic agents such as dextrose, sucrose, lactose or mannitol; glycerol, 1,2-propane-diol; inorganic isotonic agents such as saline; or any combination thereof.

In one embodiment, the isotonic agent is carbohydrate-based isotonic agent. In another embodiment, the carbohydrate-based isotonic agent is dextrose, sucrose, lactose, mannitol, inositol, saccharose, maltitol or any combination thereof. As used herein including the claims, the term "carbohydrate-based isotonic agents" refer to water-soluble mono- or oligosaccharides useful for adjusting the osmotic pressure of aqueous mixtures into which the compositions of the invention are added.

In another embodiment, the isotonic agent is glycerol or 1,2-propane-diol.

In one embodiment, the isotonic agent is an inorganic isotonic agent. In another embodiment, the inorganic isotonic agent is saline.

In another embodiment, the isotonic agent comprises a carbohydrate-based isotonic agent and an inorganic isotonic agent.

In another embodiment, the isotonic agent comprises saline and dextrose.

In another embodiment, the isotonic agent comprises saline and sucrose. In another embodiment, the isotonic agent comprises saline and lactose.

In another embodiment, the isotonic agent comprises saline and mannitol.

In another embodiment, the isotonic agent comprises saline and glycerol or 1,2-propane-diol. Compositions of the invention that comprise an isotonic agent typically further comprise an aqueous diluent {i.e., a diluent comprising water) as described below. The amount of carbohydrate-based isotonic agent, when used, is from about 0.001% to about 20% by weight based on the total weight of the aqueous diluent. In another embodiment, the amount of carbohydrate-based isotonic agent, when used, is from about 0.1% to about 10% by weight based on the total weight of the aqueous diluent, hi another embodiment, the amount of carbohydrate-based isotonic agent, when used, is from about 1% to about 6% by weight based on the total weight of the aqueous diluent.

The amount of glycerol or 1,2-propane-diol, when used, is from about 0.001% to about 20% by weight based on the total weight of the aqueous diluent. In another embodiment, the amount of glycerol or 1,2-propane-diol, when used, is from about 0.1% to about 10% by weight based on the total weight of the aqueous diluent. In another embodiment, the amount of glycerol or 1,2-propane-diol, when used, is from about 1% to aboυt 6% by weight based on the total weight of the aqueous diluent. The amount of inorganic isotonic agent, when used, is from about 0.001 % to about 2% by weight based on the total weight of the aqueous diluent. In another embodiment, the amount of inorganic isotonic agent, when used, is from about 0.1% to about 1.5% by weight based on the total weight of the aqueous diluent. In another embodiment, the amount of inorganic isotonic agent, when used, is from about 0.75% to about 1% by weight based on the total weight of the aqueous diluent. In another embodiment, the amount of inorganic isotonic agent, when used, is about 0.9% by weight based on the total weight of the aqueous diluent.

In certain embodiments, the isotonic agent may also promote activation of the platinum complex of formula (I) or (II). For instance, in embodiments wherein R₆ and R₇ are -alkyl carboxylato groups in platinum complex of the formula (I), or wherein R₃ and R₄ are — -alkyl carboxylato groups in the platinum complex of the formula (II), use of an isotonic agent that supplies a source of chloride ion is desirable. In a preferred embodiment, the isotonic agent is sodium chloride.

Non-limiting examples and amounts of anti-cancer agents other than the platinum complex of formula (I) or (II) useful in the compositions of the present invention are described in Section 5.5.5.

Non-limiting examples and amounts of therapeutic agents other than the platinum complex of formula (I) or (II) useful in the compositions of the present invention are described in Section 5.5.6. When the composition of the invention is a liposomal composition, the liposomes typically have a mean diameter of about 1000 nm (micrometer). In certain embodiments the liposomes typically have a mean diameter of less than about 1000 nm (micrometer). In one embodiment, the mean diameter of the liposomes is from about 25 nm to about 250 nm; in another embodiment, from about 50 nm to about 100 nm; and in another embodiment, from about 100 nm to about 500 nm. In another embodiment, at least about 99% of the liposomes have a mean diameter of less than about 100 nm, based on the total number of liposomes. In another embodiment, at least about 99% of the liposomes have a mean diameter of less than about 50 nm, based on the total weight of liposomes; in another embodiment, at least about 99% of the liposomes have a mean diameter of less than about 20 nm, based on the total weight of liposomes. The mean diameter of the liposomes can be measured using a COULTER COUNTER^® MULTISIZER II^® (Beckman Coulter, Fullerton, CA) or by light scattering using a PD2000DLS (Precision Detectors, Franklin, Massachusetts). Typically, a mixture a liposomal composition of the present invention (~1 wt. %) in 0.9% saline or water is used to measure liposome mean diameters.

The invention also relates to a lyophilate, which is the direct result of lyophilizing a liquid composition of the invention. It will be understood that the liquid composition can be a solution or a suspension. The lyophilate is useful for making the liposomal formulations of the present invention and is useful as a component of a pharmaceutical composition as described, e.g., in Section 5.3 below. The lyophilate can be made by the methods described in Section 5.2 below. The type and amount of lipid, surfactant, diluent, and platinum complex of formula (I) or (II) include those described above for the compositions of the invention.

The lyophilate can be a free-flowing solid or a residue such as an oil. In one embodiment, the lyophilate is a free-flowing powder.

As noted above, the composition of the invention can also comprises a liquid diluent. Accordingly, in one embodiment, the invention relates to a composition comprising a lipid, a surfactant, a platinum complex of formula (I) or (II) and a liquid diluent.

It will be understood, that when the composition comprises a liquid diluent, the resultant liquid composition can be a solution or a suspension.

In one embodiment, the liquid composition is a solution, hi another embodiment, the liquid composition is a suspension.

In one embodiment, the liquid composition does not comprise liposomes. In another embodiment, the liquid composition comprises liposomes, e.g., as a liposomal suspension. In another embodiment, the liquid composition comprising liposomes is formed from a lyophilate, e.g., by contacting a lyophilate with an aqueous diluent.

In another embodiment, the liquid composition is a liposomal suspension, wherein a platinum complex of formula (I) or (II) is entrapped in said liposomes. The type and amount of lipids and surfactants useful for making the liquid composition include those described above.

Liquid diluents useful in the present invention are those that form a solution or suspension with the lipid, the platinum complex of formula (I) or (II), and the surfactant. The liquid diluent can comprise an organic solvent, an inorganic solvent, or a combination thereof. In one embodiment, the solvent is an organic solvent, hi another embodiment, the solvent is an inorganic solvent.

A non-limiting example of a useful inorganic solvent is water. Non-limiting examples of useful organic solvents include tert-butanol, dimethyl sulfoxide, and supercritical CO₂. It will be understood that the liquid diluent can contain more than one organic solvent. In certain embodiments, the solvent can contain a high-boiling organic solvent or an oil that will form a solution or homogeneous suspension with the liquid composition. A liquid composition containing a high-boiling organic solvent or oil may not freeze, but the resultant lyophilate can be reconstituted into a liquid composition useful for administration to a patient.

If the liquid composition is used to make a lyophilate, the liquid diluent may also be capable of being lyophilized (see Section 5.2). Non-limiting examples of solvents which can be used in a lyophilization method include ethanol, propanol, isopropanol, tert- butanol, dimethylformamide, dimethylsulfoxide, water or any combination thereof. In another embodiment, the liquid diluent comprises tert-butanol. In another embodiment, the liquid diluent comprises tert-butanol and water. In another embodiment, the liquid diluent consists essentially of tert-butanol and water. In certain embodiments the percentage ratio of the amount of non-aqueous solvent, e.g., tert-butanol, in an aqueous co-solvent mixture can be about 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% by weight of the mixture.

The amount of the platinum complex of formula (I) or (II) present in the liquid composition can vary. In one embodiment, the amount of platinum present in the liquid composition is from about 0.001 % to about 0.25%; in another embodiment from about 0.005 % to about 0.1 %; and in another embodiment, about 0.03 % to about 0.08 % based on the total weight of the liquid composition.

The amount of liquid diluent present in the liquid compositions can vary. Typically, the liquid diluent is present in an amount sufficient to form a solution or a well- dispersed suspension (e.g., a homogeneous suspension) of the lipid, the complex of formula (I), and the surfactant.

In certain embodiments, the liquid diluent is an aqueous diluent. The amount of aqueous diluent can vary and will depend, in part, on the type and amount of other components of the composition of the invention. In one embodiment, the aqueous diluent is present in the composition of the invention in an amount from about 100 parts by weight to about 10,000 parts by weight based on the amount of platinum, hi another embodiment, the aqueous diluent is present in the composition of the invention in an amount from about 500 parts by weight to about 5,000 parts by weight based on the amount of platinum. In another embodiment, the aqueous diluent is present in the composition of the invention in an amount from about 1,000 parts by weight to about 5,000 parts by weight based on the amount of platinum. In another embodiment, the aqueous diluent is present in the composition of the invention in an amount of about 1,700 parts by weight based on the amount of platinum.

An aqueous diluent, when used, can further comprise one or more of a surfactant, an isotonic agent, a pH-adjusting agent, an anti-cancer agent other than the platinum complex, a therapeutic agent other than the platinum complex, or any combination thereof. The types and amounts of these components are. discussed below.

The pH of the aqueous diluent, when used, can vary. In one embodiment, the pH of the aqueous diluent is from about 1 to about 10. In another embodiment, the pH of the aqueous diluent is from about 2 to about 7. In another embodiment, the pH of the aqueous diluent is from about 2 to about 6. In another embodiment, the pH of the aqueous diluent is from about 3 to about 6. In another embodiment, the pH of the aqueous diluent is about 4. In another embodiment, the pH of the aqueous diluent is about 5. The pH of the aqueous diluent can be adjusted using a pH adjusting agent.

Non-limiting examples of useful pH adjusting agents include acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid and phosphoric acid; bases such as sodium carbonate or sodium hydroxide; or buffers such as potassium dihydrogenphospate or sodium bicarbonate. The amount of pH adjusting agent, when used, will vary depending on the targeted pH. hi a preferred embodiment, the pH adjusting agent is added in an amount sufficient to provide a pH of from about 2 to about 6; more preferably of from about 3 to about 6; most preferably about 4.

In one embodiment, the invention relates to a second composition prepared by activating a first composition, said first comprising a lipid, a surfactant, and a platinum complex having the formula (I) or (II), wherein R₁-R₄ or R₆-R₉ are as defined above; wherein said lipid is capable of forming a liposome.

In certain embodiments, the pH adjusting agent promotes activation of the platinum complex of formula (I) or (II). For instance, in embodiments wherein R₆ and R₇ are alkyl carboxylato groups in platinum complex of the formula (I), or wherein R₃ and R₄ are - alkyl carboxylato groups in the platinum complex of the formula (II), use of a pH adjusting agent that supplies a source of chloride ion is desirable. In a preferred embodiment, the pH adjusting agent is hydrochloric acid.

Non-limiting methods for activating the first composition include those described in Section 5.2 such as heating, adding a chloride source, acidifying, or any combination thereof.

5.2. METHODS FOR MAKING THE COMPOSITIONS OF THE INVENTION

The present invention also relates to methods for making compositions of the present invention.

In one embodiment, the invention relates to a method for making a composition comprising mixing a lipid, a surfactant, and a platinum complex of formula (I) or (II) with a diluent to form a liquid composition; wherein R₁-R₄ or R₆-R₉ are as defined above, and said lipid is capable of forming a liposome.

The present invention relates to methods for making a lyophilate comprising a lipid, a surfactant, and a platinum anticancer agent. In one embodiment, the invention relates to a method for making a composition comprising:

(a) mixing a lipid, a surfactant, and a platinum complex of formula (I) or (II) with a first diluent to form a liquid composition; wherein R₁-R₄ or R₆-R₉ are as defined above, and said lipid is capable of forming a liposome; and (b) lyophilizing the liquid composition to form a lyophilate. The invention further relates to a method for making a reconstituted lyophilate, comprising contacting the lyophilate formed in step (b) with a second diluent comprising water to form a reconstituted composition.

(a) mixing a lipid and a platinum complex of formula (I) or (II) with a first diluent to form a liquid composition lacking a surfactant; wherein R₁-R₄ or R₆-Rg are as defined above, and said lipid is capable of forming a liposome;

(d) contacting the reconstituted composition with a surfactant. In another embodiment, the invention relates to a method for making a composition comprising: (a) mixing a lipid and a platinum compound with a first diluent to form liquid composition lacking a surfactant, the platinum compound of formula (I) or (II); wherein R₁- R₄ or R6-R₉ are as defined above, and said lipid is capable of forming a liposome;

(b) lyophilizing the liquid composition to form a lyophilate; and

(c) contacting the lyophilate with a second diluent comprising water and a surfactant to form a reconstituted composition.

The type and amount of lipid, surfactant, and, complex of formula (I) or (II), and aqueous diluent include those described above in Section 5.1.

The first diluent comprises a solvent that can be lyophilized. Non-limiting examples of useful solvents that can be lyophilized include those described in Section 5.1 above. In one embodiment, the first diluent comprises t-butanol. In another embodiment, the first diluent comprises t-butanol and water.

The second diluent comprises water. Typically, the second diluent comprises water in an amount sufficient to allow the lipids to form liposomes.

The method used to form the liquid composition of step (a) ("the pre- lyophilate composition") is not critical. Typically, the components are combined in any order and admixed for a time and at a temperature sufficient to form a solution or suspension. Preferably, the pre-lyophilate composition is a solution. Any method can be used to admix the pre-lyophilate composition including, e.g., stirring, shaking, ultrasonicating, pumping, and the like. Preferably, the pre-lyophilate composition is admixed by stirring.

A temperature sufficient for forming the pre-lyophilate composition will depend on the amount and solvating power of the solvent. In one embodiment, a temperature sufficient for forming the pre-lyophilate composition is from about 20°C to about the boiling point of the solvent; in another embodiment, from about 20°C to about 50°C; in another embodiment from about 20°C to about 45°C; and in another embodiment from about 25°C to about 45 °C. A time sufficient for forming the pre-lyophilate composition is from about 0.1 hours to about 24 hours; in another embodiment, from about 2 hours to about 12 hours; and in another embodiment, from about 3 hours to about 10 hours. The skilled artisan recognizes that the time needed to lyophilize a sample depends on the amount of material to be lyophilized. Thus, the skilled artisan recognizes that the time may exceed 24 hours depending on the size of the sample and scale of lyophilization process.

Preferably, the pre-lyophilate composition is a homogenous solution. Non- limiting methods for making a homogeneous pre-lyophilate solution include heating the pre- lyophilate solution for a time and at a temperature sufficient to dissolve any insoluble material as described above; physical separation methods including, e.g., filtering and centrifuging; or any combination thereof.

The pre-lyophilate composition is then lyophilized to form a lyophilate. Any method useful for lyophilizing can be used (see Perez-Soler et ah, Cancer Res. 52:6341-6347 (1992)); and U.S. Patent No. 6,696,079 B2 to Perez-Soler et al, each of which is incorporated by reference herein in its entirety). Typically, the pre-lyophilate composition is frozen (e.g., in a dry-ice acetone bath or cooled shelf) and the solvent removed by lyophilization to form a lyophilate. In one embodiment, the lyophilate is a powder.

Once formed, the lyophilate may be contacted (reconstituted) with the second diluent comprising water to form a reconstituted lyophilate. In certain embodiments, the reconstituted lyophilate comprises liposomes. In certain other embodiments, the reconstituted lyophilate consists essentially of liposomes. The amount of aqueous diluent used is from about 1000% to about 10000% by weight based on the total weight of the lyophilate. In another embodiment, the amount of aqueous diluent is from about 2000% to about 6000% by weight based on the total weight of the lyophilate. In another embodiment, the amount of aqueous diluent is from about 3000% to about 4000% by weight based on the total weight of the lyophilate.

In a preferred embodiment, the lyophilate is reconstituted with a diluent which also serves to activate the platinum complex. For instance, in a specific embodiment the diluent is acidified saline.

Typically, the lyophilate is contacted with a sufficient amount of an aqueous diluent for a time and a temperature sufficient to form a reconstituted lyophilate. Non- limiting methods for contacting the lyophilate with the aqueous diluent include e.g., stirring, shaking, ultrasonicating, pumping, extruding and the like. Preferably, the contacting of the lyophilate with the aqueous diluent is carried out by shaking.

A temperature sufficient to admix a reconstituted lyophilate is from about 5°C to about 50°C, preferably from about 20°C to about 50°C, more preferably about 37°C.

A time sufficient to form a reconstituted lyophilate, is from about 0.5 min to about 240 min; from about 30 min to about 180 min; or from about 75 min to about 145 min. In another embodiment, the reconstituted composition comprises liposomes formed by the lipids, and the platinum complex of formula (I) or (II) is entrapped in said liposomes.

In certain embodiments, the reconstituted composition comprises an activated platinum complex. Without being limited by theory, it is believed that activated platinum complex is a complex of formula (I) or (II) wherein R₁-R₄ or R₆-R₉ are as defined with the proviso that at least one OfR₁ and R₂ is not an -alkyl-carboxylato bearing a hydrophobic radical function. Without being limited by theory, it is also believed that the reconstituted composition comprises liposomes formed from the lipids, and the activated platinum complex is entrapped in the liposomes (see MacLean et ah, J. Microencapsul. Yh 307-322 (2000), which is incorporated by reference herein in its entirety).

In one embodiment, the composition of the invention formed by contacting an aqueous diluent comprising a surfactant with a lyophilate lacking a surfactant is an activated composition.

In another embodiment, the composition of the invention formed by adding a surfactant to a reconstituted lyophilate forms an activated composition.

In another embodiment, the method of the invention further comprises activating the reconstituted composition. Any method useful for activating jconventional liposomal platinum compositions can be used to activate the composition of the present invention. Non-limiting methods for carrying out the activation step include, e.g., heating, treating with a chloride source such as saline, treating with acid, admixing such as ultrasonication or vigorous shaking, or any combination thereof.

Heating, when used, is carried out for a time and at a temperature sufficient to activate the composition of the invention. In one embodiment, a temperature sufficient for activating the compositions of the invention is from about 15°C to about 45°C. In another embodiment, a temperature sufficient for activating the compositions of the invention is from about 30°C to about 40°C. hi another embodiment, a temperature sufficient for activating the compositions of the invention is about 37°C.

A time sufficient for activating the compositions of the present invention is from about 0.1 hours to about 48 hours; in another embodiment, from about 0.1 hours to about 30 hours; in another embodiment, from about 0.1 hours to about 20 hours; in another embodiment, from about 0.1 hours to about 15 hours; in another embodiment, from about 0.1 hours to about 10 hours; in another embodiment, from about 0.1 hours to about 5 hours; in another embodiment, from about 0.25 hours to about 3 hours; and ir» another embodiment, from about 0.25 hours to about 2 hours. A chloride source is also useful for activating the compositions of the invention (see MacLean et ah, J. Microencapsul. Yh 307-322 (2000)). Non-limiting examples of useful chloride sources include saline and hydrochloric acid.

A chloride source is also useful for activating the compositions of the invention (see MacLean et ah, J. Microencapsul. Yh 307-322 (2000)). Non-limiting examples of useful chloride sources include saline and hydrochloric acid.

In one embodiment, the chloride source is saline. In a preferred embodiment, the amount of saline, when used, is from about 0.001% to about 2% by weight based on the total weight of the aqueous diluent. In another embodiment, the amount of saline, when used, is from about 0.1% to about 1.5% by weight based on the total weight of the aqueous diluent. In another embodiment, the amount of saline, when used, is from about 0.75% to about 1% by weight based on the total weight of the aqueous diluent. In another embodiment, the amount of saline, when used, is about 0.9% to about 1% by weight based on the total weight of the aqueous diluent.

When acidifying the reconstituted composition, the acid is added in an amount sufficient to provide a composition of the invention having a pH from about 2 to about 6 (e.g., about 2.4); in another embodiment, from about 3 to about 6; in another embodiment, about 4; and in another embodiment, about 5. Without being limited by theory, it is believed that compositions of the invention having an acidic pH, e.g., from about 1 to about 6, will undergo activation more rapidly than compositions of the invention having a pH of about 7 or above. Without being limited by theory, it is believed that the activation process can be inhibited or stopped by increasing the pH of the compositions of the invention to a pH of about 7.

In another embodiment, the composition of the present invention is substantially free of chloride. In one embodiment, the composition of the invention comprises less than about 0.1% chloride; in another embodiment, less than about 0.05% chloride by weight; and in another embodiment, less than about 0.02% chloride by weight. As noted above, the aqueous diluent can further comprise one or more of a surfactant, an isotonic agent, an anti-cancer agent other than the platinum complex, a pH- adjusting agent, a therapeutic agent other than the platinum complex, an immunotherapeutic agent, or any combination thereof. In one embodiment, the aqueous diluent comprises a surfactant.

Non-limiting examples of pH-adjusting agents include those described above. In another embodiment, a surfactant, an isotonic agent, a pH-adjusting agent, an anti-cancer agent other than the platinum complex, a therapeutic agent other than the platinum complex, an immunotherapeutic agent, or any combination thereof, can be added to the composition after addition of the aqueous diluent.

In another embodiment, a surfactant, an isotonic agent, a pH-adjusting agent, an anti-cancer agent other than the platinum complex, a therapeutic agent other than the platinum complex, an immunotherapeutic agent, or any combination thereof, can be added to the lyophilate before addition of the aqueous diluent.

The present invention also comprises methods for making a composition as described above, wherein the composition further comprises a therapeutic agent as described below in Section 5.5.6. The therapeutic agent, when used, can be included in the pre- liposomal solution; added to the lyophilate prior to contacting with the aqueous diluent; and/or included in the aqueous diluent.

5.3. PHARMACEUTICAL COMPOSITIONS AND THERAPEUTIC

ADMINISTRATION

In other aspects, the present invention provides a pharmaceutical composition comprising an effective amount of a composition of the invention and a pharmaceutically acceptable carrier or vehicle. The pharmaceutical compositions are suitable for veterinary or human administration. The pharmaceutical compositions of the present invention can be in any form that allows for the composition to be administered to a subject, the subject preferably being an animal, including, but not limited to a human, mammal, or non-human animal, such as a cow, horse, sheep, pig, fowl, cat, dog, mouse, rat, rabbit, guinea pig, etc., and is more preferably a mammal, and most preferably a human.

Pharmaceutical compositions of the invention include the lyophilate of the invention that comprises a surfactant, and the compositions of the invention. The pharmaceutical compositions of the invention comprise an effective amount of the platinum agent such that a suitable dosage form will be delivered.

The pharmaceutical compositions of the invention can be in the form of a solid, liquid or gas (aerosol). Pharmaceutical compositions of the invention can be formulated so as to allow the platinum agent of the invention to be bioavailable upon administration of the composition to a subject. Compositions can take the form of one or more dosage units, where for example, a tablet can be a single dosage unit, and a container of a pharmaceutical composition of the invention in aerosol form can hold a plurality of dosage units. Materials used in preparing the pharmaceutical compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of subject (e.g., human), the overall health of the subject, the type of cancer the subject is in need of treatment of, the use of the composition as part of a multi-drug regimen, the particular form of the pharmaceutical composition of the invention, the manner of administration, and the composition employed.

The pharmaceutically acceptable carrier or vehicle may be a particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) can be liquid, with the compositions being, for example, an oral syrup or injectable liquid. In addition, the carrier(s) can be gaseous, so as to provide an aerosol composition useful in, e.g., inhalatory administration.

The composition may be intended for oral administration, and if so, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid. As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as ethyl cellulose, carboxymethylcellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent. When the pharmaceutical composition is in the form of a capsule, e.g., a gelatin capsule, it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The pharmaceutical composition can be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid can be used for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included. The liquid compositions of the invention, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral composition can be enclosed in an ampoule, a disposable syringe, or a multiple-dose vial made of glass, plastic or other material. Physiological saline is a preferred diluent. An injectable composition is preferably sterile.

The amount of the pharmaceutical composition of the invention that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. As noted above, the pharmaceutical comprise an effective amount of a platinum complex such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of platinum by weight of the pharmaceutical compositions. When intended for oral administration, this amount can be varied to be between 0.1% and 80% by weight of platinum by weight of the pharmaceutical compositions. Preferred oral compositions can comprise from between 4% and 50% of platinum by weight of the pharmaceutical composition. Preferred pharmaceutical compositions of the present invention are prepared so that a parenteral dosage unit contains from between 0.01% and 2% by weight of the platinum. The pharmaceutical compositions of the invention can be administered in a single dose or in multiple doses.

In one embodiment, the pharmaceutical compositions of the invention are administered in multiple doses. When administered in multiple doses, the pharmaceutical compositions are administered with a frequency and in an amount sufficient to treat the condition. In one embodiment, the frequency of administration ranges from once a day up to about once every eight weeks. For example, the pharmaceutical compositions can be administered once a week, once every two weeks, once every three weeks or once every four weeks. In another embodiment, the frequency of administration ranges from about once a week up to about once every six weeks. In another embodiment, the frequency of administration ranges from about once every two weeks up to about once every four weeks. In certain embodiments, the daily, weekly, or multi-weekly administration may be continued for several cycles as determined by the physician and the nature of the cancer. In certain embodiments, the number of cycles may be about 1, 2, 5, 8, 10, 15, 20, 25 or 30.

Generally, the dosage of the pharmaceutical composition of the invention administered to a subject is in the range of about 0.1 to 450 mg of Pt /kg, e.g., 0.1 to 400 mg of Pt /kg, 0.1 to 350 mg of Pt /kg, 0.1 to 300 mg of Pt /kg, 0.1 to 250 mg of Pt /kg, 0.1 to 200 mg of Pt /kg, 1 to 125 mg of Pt /kg, 5 to 100 mg of Pt /kg or 10 to 75 mg of Pt /kg, of the subject's body weight. In other embodiments, the dosage administered to a subject is in the range of about 0.1 mg of Pt /kg to 50 mg of Pt /kg, or 1 mg of Pt /kg to 50 mg of Pt /kg, of the subject's body weight, more preferably in the range of 0.1 mg of Pt /kg to 25 mg of Pt /kg, or 1 mg of Pt /kg to 25 mg of Pt /kg, of the subject's body weight.

The pharmaceutical compositions of the invention can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. Various delivery systems are known, e.g., liposomes, microparticles, microcapsules, capsules, etc., and may be useful for administering a pharmaceutical composition of the invention. In certain embodiments, more than one pharmaceutical composition of the invention is administered to a subject.

Typical routes of administration may include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, and intranasal. Parenteral administration includes subcutaneous injections, intradermal, subcutaneous; intranasal, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, transdermal, rectally, by inhalation, intravenous, intramuscular, intraperitoneal, intrapleural, intrasternal injection or infusion techniques. The preferred mode of administration is left to the discretion of the practitioner, and will depend in-part upon the site of the medical condition (such as the site of cancer, a cancerous tumor or a pre-cancerous condition).

In some embodiments, the pharmaceutical compositions of the invention are administered using an intravenous, intra-peritoneal, or subcutaneous drug delivery device. Such devices include, but are not limited to, syringes, i.v. drip bags and catheters. In one embodiment, the pharmaceutical compositions of the invention are administered parenterally.

In a preferred embodiment, the pharmaceutical compositions of the invention are administered intravenously.

In specific embodiments, it can be desirable to administer one or more pharmaceutical compositions of the invention locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery by injection; by means of a catheter; by means of a suppository; or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumor, or precancerous tissue. In certain embodiments, it can be desirable to introduce one or more pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, the pharmaceutical compositions of the invention can be formulated as a suppository, with traditional binders and carriers such as triglycerides. In yet another embodiment, the pharmaceutical compositions of the invention can be delivered in a controlled release system. In one embodiment, a pump can be used (see Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al, Surgery 88:507 (1980); and Saudek et al, N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. ScL Rev. Macromol. Chem. 23:61 (1983); see also Levy et al, Science 228:190 (1985); During et ah, Ann. Neurol. 25:351 (1989); and Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controUed-release system can be placed in proximity of the target of the pharmaceutical compositions of the invention, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled- release systems discussed in the review by Langer (Science 249:1527-1533 (1990)) can be used.

The term "carrier" refers to a diluent, adjuvant or excipient, with which composition of the invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to a subject, the pharmaceutical compositions of the invention and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the pharmaceutical composition of the invention is administered intravenously. Saline solutions and aqueous mannitol, dextrose and glycerol or 1,2-propane-diol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable carrier is a capsule (see, e.g., U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical carriers are described in {see Remington 's: The Science and Practice of Pharmacy (20th ed. 2000)).

Sustained or directed release compositions that can be formulated include, but are not limited to, a pharmaceutical composition of the invention and other formulations where a pharmaceutical compositions of the invention is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc. It is also possible to use the lyophilate of the invention for injection.

Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving complex are also suitable for orally administered compositions of the invention, hi these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving complex, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time-delay material such as glycerol monostearate or glycerol stearate can also be used. Oral compositions can include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such carriers are preferably of pharmaceutical grade. The pharmaceutical compositions of the invention can be intended for topical administration, in which case the carrier can be in the form of a solution, emulsion, ointment or gel base. The base, for example, can comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents can be present in a composition for topical administration. If intended for transdermal administration, the composition can be in the form of a transdermal patch or an ionotophoresis device. Topical formulations can comprise a concentration of a platinum of from between 0.01% and 10% w/v (weight per unit volume of pharmaceutical composition). The pharmaceutical compositions can include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition can include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and can be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients can be encased in a gelatin capsule.

In a preferred embodiment, the pharmaceutical compositions of the invention are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly human beings. Typically, the carriers or vehicles for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the pharmaceutical compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally comprise a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition of the invention is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition of the invention is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration, hi certain embodiments the lyophilate compositions described herein can be formulated according to the teaching of U.S. Patent No. 6,284,277 (see also, EP 858325 and WO 97/17604).

The pharmaceutical compositions can consist of gaseous dosage units, e.g., it can be in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery can be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of the compositions can be delivered in single phase, bi- phasic, or tri-phasic systems in order to deliver the composition. Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, Spacers and the like, which together can form a kit. Preferred aerosols can be determined by one skilled in the art, without undue experimentation.

Whether in solid, liquid or gaseous form, the pharmaceutical compositions of the present invention can comprise an additional therapeutically active agent other than the platinum complex of formula (I) or (II) selected from among those including, but not limited to, an additional anticancer agent, an immunotherapeutic agent, an antiemetic agent, a hematopoietic colony stimulating factor, an anti-depressant and an analgesic agent.

The pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical art.

In one embodiment, the pharmaceutical compositions of the present invention may comprise one or more known therapeutically active agents.

In another embodiment, the pharmaceutical compositions of the present invention can be administered prior to, at the same time as, or after an additional anticancer agent, or on the same day, or within 1 hour, 2 hours, 12 hours, 24 hours, 48 hours, 72 hours, 1 week, 2 weeks, 3 weeks or 4 weeks of each other.

In another embodiment, the pharmaceutical compositions of the present invention can be administered prior to, at the same time as, or after an immunotherapeutic agent, or on the same day, or within 1 hour, 2 hours, 12 hours, 24 hours, 48 hours or 72 hours of each other.

In another embodiment, the pharmaceutical compositions of the present invention can be administered prior to, at the same time as, or after an antiemetic agent, or on the same day, or within 1 hour, 2 hours, 12 hours, 24 hours, 48 hours or 72 hours of each other. In another embodiment, the pharmaceutical compositions of the present invention can be administered prior to, at the same time as, or after a hematopoietic colony stimulating factor, or on the same day, or within 1 hour, 2 hours, 12 hours, 24 hours, 48 hours, 72 hours, 1 week, 2 weeks, 3 weeks or 4 weeks of each other.

In another embodiment, the pharmaceutical compositions of the present invention can be administered prior to, at the same time as, or after an opioid or non-opioid analgesic agent, or on the same day, or within 1 hour, 2 hours, 12 hours, 24 hours, 48 hours or 72 hours of each other.

In another embodiment, the pharmaceutical compositions of the present invention can be administered prior to, at the same time as, or after an anti-depressant agent, or on the same day, or within 1 hour, 2 hours, 12 hours, 24 hours, 48 hours or 72 hours of each other.

5.4. KITS The invention encompasses kits that can simplify the administration of a composition, pharmaceutical composition, or lyophilate of the invention to a subject.

A typical kit of the invention comprises a unit dosage of a lyophilate of the invention, hi one embodiment, the unit dosage form is in a first container, which can be sterile, containing an effective amount of a lyophilate of the invention comprising a surfactant, and a second container which contains a solution useful for the reconstitution of the lyophilate, such as saline or phosphate buffered saline. In another embodiment, the unit dosage form is in a first container as described above containing an effective amount of a lyophilate of the invention which does not comprise a surfactant, and a second container which contains an aqueous diluent as described above and further comprising a surfactant. In another embodiment, the unit dosage form is in a first container as described above containing an effective amount of a lyophilate of the invention comprising a surfactant, and a second container which contains an aqueous diluent comprising a surfactant. The kit can also comprise a label or printed instructions for use of a pharmaceutical composition of the invention.

In alternative embodiments, the kits may contain the pharmaceutical composition of the invention in reconstituted form. For instance, the reconstituted composition may be in a unit dosage form in a syringe or ampoule.

The kit can further comprise a unit dosage form of another therapeutically active agent. In one embodiment, the kit comprises a container containing an amount of an additional anticancer agent effective to treat cancer. Such kits may contain the anticancer agents in synergistically effective amounts. In another embodiment the kit comprises a container containing a therapeutically active agent such as an antiemetic agent, a hematopoietic colony-stimulating factor, an analgesic agent an anxiolytic agent, imniunotherapeutic agent or vaccine.

In one embodiment, the kit comprises a unit dosage form of a pharmaceutical composition of the invention.

In one embodiment, the kit comprises a unit dosage form of a lyophilate comprising a surfactant of the invention. In another embodiment, the kit comprises unit dosage forms of a pharmaceutical composition or lyophilate in a first container and a therapeutic agent other than the platinum complex in a second container. In certain embodiments the kit may also comprise a solution useful for the reconstitution of the lyophilate, such as saline or phosphate buffered saline. Kits of the invention can further comprise a device for heating the reconstituted composition to activate it. The heating element may be a disposable unit such as a heating pack relying on an exothermic chemical reaction, or a heating mantle, or a heating block. Kits of the invention can further comprise a device that is useful for administering the unit dosage forms of a pharmaceutical composition or lyophilate comprising a surfactant of the invention. A skilled artisan understands tha the unit dosage may vary according to the cancer to be treated and the physiology of the subject. Examples of such devices include, but are not limited to, a syringe, drip bag, or enema, which optionally contain the unit dosage forms.

5.5. THERAPEUTIC USES

5.5.1. TREATMENT OF CANCER

Cancer or a neoplastic disease, including, but not limited to, neoplasms, tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth, can be treated, suppressed, delayed, inhibited or prevented by administration of an amount of a composition of the invention effective to treat cancer. The invention as it applies to cancer encompasses the treatment, suppression, delaying, inhibiting of growth and/or progression, and prevention of cancer or neoplastic disease as described herein.

5.5.2. THERAPEUTIC METHODS In a preferred embodiment, the present invention provides methods for treating cancer, including: killing a cancer cell or neoplastic cell; inhibiting the growth of a cancer cell or neoplastic cell; inhibiting the replication of a cancer cell or neoplastic cell; inhibiting metastases; delaying the progression of cancer, or ameliorating a symptom thereof, the methods comprising administering to a subject in need thereof an amount of a pharmaceutical composition of the invention effective to treat cancer.

In a specific embodiment, the treating results in a delay in the progession of cancer.

The pharmaceutical compositions of the invention can be used accordingly in a variety of settings for the treatment of various cancers. Without being bound by theory, in one embodiment, a platinum complex present in the pharmaceutical compositions of the invention can enter a cell by diffusion and react with DNA to form interstrand and intrastrand cross-links and DNA-protein crosslinks, which can interfere with the ability of the cell to replicate.

In a specific embodiment, the subject in need of treatment has previously undergone treatment for cancer. Such previous treatments include, but are not limited to, prior chemotherapy, radiation therapy, surgery or immunotherapy, such as cancer vaccines.

In another embodiment, the cancer being treated is a cancer which has demonstrated sensitivity to platinum therapy or is known to be responsive to platinum therapy. Such cancers include, but are not limited to, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, breast cancer, bladder cancer, testicular cancer, head and neck cancer, colorectal cancer, Hodgkin's disease, leukemia, osteogenic sarcoma, and melanoma.

In still another embodiment, the cancer being treated is a cancer which has demonstrated resistance to platinum therapy or is known to be refractory to platinum therapy. Such refractory cancers include, but are not limited to, cancers of the cervix, prostate, and esophagus. A cancer may be determined to be refractory to a therapy when at least some significant portion of the cancer cells are not killed or their cell division are not arrested in response to the therapy. Such a determination can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of treatment on cancer cells, using the art-accepted meanings of "refractory" in such a context. In a specific embodiment, a cancer is refractory where the number of cancer cells has not been significantly reduced, or has increased. Such cancers include, but are not limited to, cancers of the cervix, prostate, and esophagus.

In specific embodiments, the cancer being treated is a cancer which is refractory to existing cancer therapies. In such embodiments, the platinum complexes may be used alone or in combination with other cancer therapeutic agents. Other cancers that can be treated with the pharmaceutical compositions of the invention include, but are not limited to, solid tumors, blood-borne cancers, acute and chronic leukemias, and lymphomas.

Non-limiting examples of solid tumors that can be treated with the composition of the invention include acoustic neuroma, adenocarcinoma, angiosarcoma, astrocytoma, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, bone cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma, endotheliosarcoma, ependymoma, epithelial carcinoma, esophageal cancer, Ewing's tumor, fallopian tube cancer, fibrosarcoma, glioblastoma multiforme, glioma, hemangioblastoma, hepatoma, kidney cancer, leiomyosarcoma, liposarcoma, lung cancer, lymphangioendotheliosarcoma, lymphangiosarcoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, myxosarcoma, nasal cancer, neuroblastoma, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinomas, papillary carcinoma, peritoneal cancer, cancer confined to the peritoneal cavity, pinealoma, primary peritoneal carcinoma, prostate cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung carcinoma, squamous cell carcinoma, stomach cancer, sweat gland carcinoma, synovioma, testicular cancer, throat cancer, uterine cancer, and Wilms' tumor.

Non-limiting examples of blood-borne cancers that can be treated with the composition of the invention include acute erythroleukemic leukemia, acute lymphoblastic B- cell leukemia, acute lymphoblastic leukemia "ALL", acute lymphoblastic T-cell leukemia, acute megakaryoblastic leukemia, acute monoblastic leukemia, acute myeloblastic leukemia "AML", acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute promyelocytic leukemia "APL", acute undifferentiated leukemia, chronic lymphocytic leukemia "CLL", chronic myelocytic leukemia "CML", hairy cell leukemia, and multiple myeloma.

Non-limiting examples of acute and chronic leukemias that can be treated with the composition of the invention include lymphoblastic, myelogenous, lymphocytic, and myelocytic leukemias.

Non-limiting examples of lymphomas that can be treated with the composition of the invention include heavy chain disease, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, and Waldenstrom's macroglobulinemia. In one embodiment, the cancer is selected from pancreatic cancer, colorectal cancer, mesothelioma, a malignant pleural effusion, peritoneal carcinomatosis, peritoneal sarcomatosis, renal cell carcinoma, small cell lung cancer, non-small cell lung cancer, testicular cancer, bladder cancer, breast cancer, head and neck cancer, and ovarian cancer.

In a preferred embodiment the cancer is pancreatic cancer, colorectal cancer or mesothelioma.

5.5.3. PROPHYLACTIC METHODS

The pharmaceutical compositions of the invention can also be administered to prevent progression to a neoplastic or malignant state, including but not limited to the solid tumors, blood-borne cancers, acute and chronic leukemias, and lymphomas described above. Such prophylactic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79). Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. For example, endometrial hyperplasia often precedes endometrial cancer and precancerous colon polyps often transform into cancerous lesions. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. A typical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.

Alternatively or in addition to the presence of abnormal cell growth characterized as hyperplasia, metaplasia, or dysplasia, the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a patient, can indicate the desirability of prophylactic/therapeutic administration of the composition of the invention. Such characteristics of a transformed phenotype include morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, and the disappearance of the 250,000 dalton cell surface protein {see also Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 84-90 for characteristics associated with a transformed or malignant phenotype). In a specific embodiment, leukoplakia, a benign-appearing hyperplastic or dysplastic lesion of the epithelium, or Bowen's disease, a carcinoma in situ, are pre-neoplastic lesions indicative of the desirability of prophylactic intervention. In another embodiment, fibrocystic disease (cystic hyperplasia, mammary dysplasia, particularly adenosis (benign epithelial hyperplasia)) is indicative of the desirability of prophylactic intervention.

The prophylactic use of the pharmaceutical compositions of the invention is also indicated in some viral infections that may lead to cancer. For example, human papilloma virus can lead to cervical cancer {see, e.g., Hernandez-Avila et ah, Archives of Medical Research 28:265-271 (1997)), Epstein-Barr virus (EBV) can lead to lymphoma (see, e.g., Herrmann et ah, J. Pathol. 199:140-5 (2003)), hepatitis B or C virus can lead to liver carcinoma (see, e.g., El-Serag, J Clin. Gastroenterol. 35 (5 Suppl 2VS72-8 (2002)), human T cell leukemia virus (HTLV)-I can lead to T-cell leukemia (see, e.g., Mortreux et ah,

Leukemia 17:26-38 (2003)), human herpes virus-8 infection can lead to Kaposi's sarcoma (see, e.g., Kadow et ah, Curr . Opin. Investig. Drugs 3_: 1574-9 (2002)), and Human Immune deficiency Virus (HIV) infection contribute to cancer development as a consequence of immunodeficiency (see, e.g., Dal Maso et ah, Lancet Oncol. 4:110-9 (2003)). In other embodiments, a patient which exhibits one or more of the following predisposing factors for malignancy can be treated by administration of an effective amount of composition of the invention: a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome for chronic myelogenous leukemia, t(14;18) for follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy (a possible forerunner of multiple myeloma), a first degree kinship with persons having a cancer or precancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, neurofibromatosis of on Recklinghausen, retinoblastoma, carotid body tumor, cutaneous melanocarcinoma, intraocular melanocarcinoma, xeroderma pigmentosum, ataxia telangiectasia, Chediak- Higashi syndrome, albinism, Fanconi's aplastic anemia, and Bloom's syndrome; see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 112-113) etc.), and exposure to carcinogens (e.g., smoking, and inhalation of or contacting with certain chemicals).

In another specific embodiment, a pharmaceutical composition of the invention is administered to a human patient to prevent, delay or inhibit the growth and/or progression of breast, colon, ovarian, or cervical cancer. In another specific embodiment, a pharmaceutical composition of the invention is administered to a human patient to delay progression to breast, colon, ovarian, or cervical cancer.

In specific embodiments, the pharmaceutical compositions inhibit progression from pre-malignancy to malignancy.

5.5.4. MULTI-MODALITYTHERAPYFORCANCER

The pharmaceutical compositions of the invention can be administered to a subject that has undergone or is currently undergoing one or more additional anticancer treatment modalities including, but not limited to, surgery, radiation therapy, chemotherapy or immunotherapy, such as cancer vaccines.

In one embodiment, the invention provides methods for treating cancer comprising (a) administering to a subject in need thereof an amount of a composition of the invention effective to treat cancer; and (b) administering to the subject one or more additional anticancer treatment modalities including, but not limited to, surgery, radiation therapy, or immunotherapy, such as a cancer vaccine.

In one embodiment, the additional anticancer treatment modality is radiation therapy.

In another embodiment, the additional anticancer treatment modality is surgery. In still another embodiment, the additional anticancer treatment modality is immunotherapy.

In a specific embodiment, the pharmaceutical composition of the invention is administered concurrently with radiation therapy. In another specific embodiment, the additional anticancer treatment modality is administered prior or subsequent to administration of a pharmaceutical composition of the invention, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e.g., up to three months), prior or subsequent to administration of a pharmaceutical composition of the invention.

When the additional anticancer treatment modality is radiation therapy, any radiation therapy protocol can be used depending upon the type of cancer to be treated. For example, but not by way of limitation, x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers. Gamma-ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements, can also be administered.

Additionally, the invention provides methods for treatment of cancer with a composition of the invention as an alternative to chemotherapy or radiation therapy where the chemotherapy or the radiation therapy has proven or can prove too detrimental, e.g., results in unacceptable or unbearable side effects, for the subject being treated. The subject being treated can, optionally, be treated with another anticancer treatment modality such as surgery or immunotherapy, depending on which treatment is found to be acceptable or bearable. Types of cancer immunotherapies include, but are not limited to: a. heat shock protein based therapies described in U.S. Patent Nos.:

5,837,251; 6,136,315; 6,017,540; 6,383,494; 6,436,404; 6,403,095; 6,383,493; 6,455,048; 5,750,119; 6,017,544; 6,168,793; and International Application Publication Nos.: WO 03/090686; WO 03/092624; b. antibody based vaccines described in U.S. Patent Nos.: 6,764,680; 6,759,049; 6,306,393; or c. cell-based cancer vaccines (see, for example, 6,689,355; and International Application Publication Nos.: WO 93/06867; WO 94/18995; WO 97/24132; WO 90/03183; and WO 91/06866).

In another embodiment, the cancer immunotherapy may comprise strategies to enhance the innate immune system of the cancer patient such as described in U.S. Pat. No. 4,690,915. In certain other embodiments, the cancer immunotherapy is based on attenuating acquired immune response strategies such as active specific immunotherapy or ("ASI") (See, e.g., U.S. Pat. Nos. 5,290,551; 5,484,596; 5,679,356; 5,637,483; or Vermorken et al., Lancet 353:345-350 (1999)) or adoptive cellular immunotherapy or ("ACI") (See, e.g., U.S. Pat. No. 4,690,914; Rosenberg, S. A., et al., 1988, N. England J. Med. 319:1676-1680) cancer antigen immunotherapy ("CAI") (See, e.g., U.S. Pat. No. 6,406,699; Yoshizawa et al., J. Immunol. 147:729-737 (1991); Saxton et al., Blood 89:2529-2536 (1997); Shu et al., J. Immunol. 152: 1277-88 (1994); Baldwin et al., J. Neuro. Oncol. 32:19-28 (1997); and Chang et al., J. Clin. Oncol. 15:79-807 (1997)). The pharmaceutical compositions of the invention can also be used in an in vitro or ex vivo fashion, such as for the treatment of certain cancers, including, but not limited to leukemias and lymphomas, such treatment involving autologous stem cell transplants. This can involve a multi-step process in which the animal's autologous hematopoietic stem cells are harvested and purged of all cancer cells, the patient's remaining bone-marrow cell population is then eradicated via the administration of a high dose of a pharmaceutical composition of the invention with or without additional anticancer agents and/or high dose radiation therapy, and the stem cell graft is infused back into the animal. Supportive care is then provided while bone marrow function is restored and the subject recovers.

5.5.5. MULTI-DRUG THERAPY FOR CANCER

The present invention also provides methods for treating cancer comprising administering to a subject in need thereof an amount of a composition of the invention effective to treat cancer and one or more additional anticancer agents or pharmaceutically acceptable salts thereof, the additional anticancer agents not being pharmaceutical compositions of the invention. The combination of agents can act additively or synergistically. In certain embodiments, dosing regimens based on the synergy of the combinations of agents can be used. Suitable additional anticancer agents include, but are not limited to, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, carboplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, epirubicin, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustine and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, platinum complexes such as cisplatin, carboplatin and oxaliplatin, imatinib mesylate, hexamethylmelamine, topotecan, tyrosine kinase inhibitors, tyrphostins herbimycin A, genistein, erbstatin, and lavendustin A.

In one embodiment, the additional anticancer agent can be, but is not limited to, a drug listed in Table 1. TABLE l

Alkylating agents

Nitrogen mustards: Cyclophosphamide

Ifosfamide Trofosfamide Chlorambucil

Nitrosoureas: Carmustine (BCNU)

Lomustine (CCNU) Alkylsulphonates : Busulfan

Treosulfan

Triazenes: Dacarbazine

Platinum containing complexes: Cisplatin

Carboplatin

Oxaliplatin

Plant Alkaloids Vinca alkaloids: Vincristine

Vinblastine

Vindesine

Vinorelbine

Taxoids: Paclitaxel

Docetaxel

DNA Topoisomerase Inhibitors Epipodophyllins : Etoposide

Teniposide

Topotecan

9-aminocamptothecin

Camptothecin

Crisnatol

Mitomycins: Mitomycin C

Anti-metabolites

Anti-folates: DHFR inhibitors: Methotrexate

Trimetrexate

IMP dehydrogenase Inhibitors: Mycophenolic acid

Tiazofurin

Ribavirin

EICAR

Ribonuclotide reductase Inhibitors: Hydroxyurea

Deferoxamine Pyrimidine analogs: Uracil analogs: 5-Fluorouracil

Floxuridine

Doxifluridine

Ratitrexed

Cytosine analogs: Cytarabine (ara C)

Cytosine arabinoside

Fludarabine

Gemcitabine

Capecitabine

Purine analogs: Mercaptopurine

Thioguanine DNA Antimetabolites: 3-HP

2 '-deoxy-5 -fluorouridine

5-HP alpha-TGDR aphidicolin glycinate ara-C

5-aza-2 '-deoxycytidine beta-TGDR cyclocytidine guanazole inosine glycodialdehyde macebecin II

Pyrazoloimidazole

Hormonal therapies: Receptor antagonists: Anti-estrogen: Tamoxifen

Raloxifene

Megestrol

LHRH agonists: Goserelin

Leuprolide acetate Anti-androgens: Flutamide

Bicalutamide Retinoids/Deltoids

Czs-retinoic acid

Vitamin A derivative: All-trans retinoic acid (ATRA-IV) Vitamin D3 analogs: EB 1089 CB 1093 KH 1060

Photodvnamic therapies: Vertoporfm (BPD-MA) Phthalocyanine Photosensitizer Pc4 Demethoxy-hypocrellin A (2BA -2-DMHA)

Cytokines: Interferon-α Interferon-β Interferon-γ Tumor necrosis factor

Angio genesis Inhibitors: Angiostatin (plasminogen fragment) antiangiogenic antithrombin III Angiozyme ABT-627 Bay 12-9566 Benefin Bevacizumab BMS-275291 cartilage-derived inhibitor (CDI) CAI

CD59 complement fragment CEP-7055 Col 3

Combretastatin A-4 Endostatin (collagen XVIII fragment)

Fibronectin fragment Gro-beta Halofuginone Heparinases

Heparin hexasaccharide fragment HMV833

Human chorionic gonadotropin (hCG) IM-862

Interferon alpha/beta/gamma Interferon inducible protein (IP-IO) Interleukin-12

Kringle 5 (plasminogen fragment) Marimastat

Metalloproteinase inhibitors (TIMPs) 2-Methoxyestradiol MMI 270 (CGS 27023 A) MoAb IMC-ICl 1 Neovastat NM-3 Panzem PI-88

Placental ribonuclease inhibitor Plasminogen activator inhibitor Platelet factor-4 (PF4) Prinomastat

Prolactin 16kD fragment Proliferin-related protein (PRP) PTK 787/ZK 222594 Retinoids Solimastat Squalamine SS 3304 SU 5416 SU6668 SUl 1248

Tetrahydrocortisol-S

Tetrathiomolybdate

Thalidomide

Thrombospondin-1 (TSP-I)

TNP-470

Transforming growth factor-beta

(TGF-b)

Vasculostatin

Vasostatin (calreticulin fragment)

ZD6126

ZD 6474 farnesyl transferase inhibitors (FTI)

Bisphosphonates

Antimitotic agents: Allocolchicine

Halichondrin B

Colchicine colchicine derivative dolstatin 10

Maytansine

Rhizoxin

Thiocolchicine trityl cysteine

Others:

Isoprenylation inhibitors: lonafarnib (SCH66336)

Dopaminergic neurotoxins: l-methyl-4-phenylpyridinium ion

Cell cycle inhibitors: Staurosporine

Actinomycins: Actinomycin D

Dactinomycin

Bleomycins: Bleomycin A2

Bleomycin B2

Peplomycin

Anthracyclines: Daunorubicin Doxorubicin (adriamycin)

Idarubicin

Epirubicin

Pirarubicin

Zorubicin

Mitoxantrone

MDR inhibitors: Verapamil

Ca2+ATPase inhibitors: Thapsigargin

Additional anticancer agents that can be used in the compositions and methods of the present invention include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; folinic acid, fosquidone; fostriecin sodium; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alfa-2 α; interferon alfa-2β; interferon alfa-nl ; interferon alfa-n3; interferon beta-Iα; interferon gamma-Iβ; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride.

Other anticancer drugs that can be used include, but are not limited to: 20-epi-

1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti- dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; carboxamide-amino- triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin

A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-acytidine; dihydrotaxol; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorubicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ihnofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, A-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum complexes; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid

A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anticancer agents; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+peηtazocine; napavin; naphteφin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin

B; plasminogen activator inhibitor; a platinum complex other than the platinum complex of formula (I) or (II) such as a platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinirnex; rubiginone Bl; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfmosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors, e.g., BIBRl 532; temoporfm; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; rurosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. Other anticancer drugs that can be used include, but are not limited to: OncoTICE/TiceBCG, Gemzar, Farmorubicin/Pharmorubicin/Ellence, Calsed, Gossypol, Arimidex, Nolvadex, Faslodex, Zoladex, Modrenal, Megace, Zinecard, Topotecin, Navelbine, Femara, Lentaron, Zavedos/Idamycin, Xeloda, Caelyx, Fareston, Leuplin/Lupron/Enantone, Onconase, Theratope, Enhanzyn, Evista, Arzoxifene, GW572016, IGNlOl, Liposome Encapsulated Doxorubicin (LED), Zometa, Aptosyn, Lasofoxifene, Yondelis, MS-209, Tesmilifene, OvaRex MAb, ATN-224, Oncomyc-NG, BAY 59-8862 (IDN 5109), Javlor, Xyotax, CYC202, SB-715992, SB-743921, Genasense, Dendritic cell/cancer cell fusion vaccine, INGN 225, KRX-0403, KRX-0402, KOS-862/R1492, Targretin, Pipendoxifene, GTI-2040, MDX-010, MT201, Velcade, LE-SN38, Sandostatin, MetXia, Omnitarg, Rl 492, SR 31747, Dacogen, Telcyta, MVA-MUC1-IL2 (TG4010), VB2- 011, Tadquidar (XR9576), Tomudex, Campto, UFT, Camptosar, Celebrex/Celebra/Onsenal, Avastin, Eloxatin, ABX-EGF, Orzel, Thalidomide analogues (ENMD 0995/ENMD 0997), IGNlOl, PTK787, ZK 222584, Avicine, CoFactor, Oncophage (HSPPC-96), G17DT, Cancer vaccines/ALVAC-CEA/B7.1 , BAY 43 -9006, CF 101 , Canvaxin, PG-CPT, Angiozyme, SB- 715092, Epimmune, EP-2101, TNFerade, Collidem, IMMU-101, IMMU-111, Patrin, Vitaxin, HyCAMP, EMD 273066/EMD 273063, TroVax, Combretastatin A4P, Aplidin, MS- 275, Imunace (S-6820), Telik, WX-UKl, Proleukin, Roferon-A, Sumiferon, AEterna Zentaris, Celgene, IL-2, Genta, OS1-461, Thymitaq, T67, IMMU-105, Irofulven, Sandostatin, Kahalalide F, Progen Industries, PI-88, Protherics, NQO Platform Technology/NQO2/NQO1 , Ad-IFNg (TG1042), Lobaplatin, Paraplatin, Etopophos, Hycamtin, Neovastat, RSR13, Javlor, Affmitak, BEC2, Ethyol, BEC2, Xcytrin, PDX, QS-21, AVI BioPharma, BLP25, Karenitecin BNP1350, GVAX cancer vaccines, GVAX Lung Cancer Vaccine, CYC202, Affinitak, huN901-DMl, Onco TCS, Advexin, Panretin, BLP25, DN-IOl, OSI-211, OSI-461, Prinomastat, Cl-1033, CP-547,632, SDX-102, SR 48692, SGN-15, Orathecin/CZ 112, Triapine, Hexalen Capsules, Antisoma, Rl 549, AP5280, GnRH pharmaccine, O-Vax, FolateScan, Phenoxodiol, EMD 72000, Irofulven, OSI-211, CP-547,632, Benzvix, Mitozytrex, DX-8951f, Virulizin, Tarceva, Orathecin/CZ 112, QS-21, GVAX cancer vaccines, GVAX Pancreatic Cancer Vaccine, ISIS 2503, KT5555/CEP-701, Cotara, SDX- 102, Triapine, Lupron Depot/Lucrin, Zoladex LA, Casodex, Aventis, Suprefact/Suprecur, Anandron/Nilandron, Viadur, Novantrone, Estracyt, Plenaxis, Eligard, Androcur, Eulexin, Provenge, Avodart, Satraplatin, DN-101, Glivec/Gleevec, Satraplatin, D-63153, AMD473, „ Abetafen, Actimid, GVAX Prostate Cancer Vaccine, CG7870 (Prostate), BBR 3576, MLN2704, MT201, MLN2704/MLN591RL, MDX-070, DN-IOl, Reolysin, Onyvax-P, OSI- 461, PCK3145, PROSTVAC-VF/TRICOM, MVA-MUC1-IL2 (TG4010), Norelin, and TGFa Vaccine/HER-1 Vaccine.

In a preferred embodiment, the additional anticancer agent is gemcitabine, capecitabine, 5-fluorouracil or leucovorin, or a combination thereof. In another preferred embodiment, the additional anticancer agent is docetaxel, paclitaxel, tamoxifen, cisplatin, carboplatin, oxaliplatin, vinorelbine, etoposide, doxorubicin, raltitrexed, irinotecan or topotecan.. Other specific additional anticancer agents are bevacizumab (AVASTIN®); erlotinib hydrochloride (TARCEV A®); gefitinib (IRESSA®); cyclophosphamide (ENDOXAN®); epidermal growth factor inhibitors, e.g., EKB 569 (pelitinib), PKI-166 (Novartis), canertinib, lapatinib (GSK 572016); monoclonal antibodies, e.g., cetuxhnab (ERBITUX®), matuzumab, trastuzumab (HERCEPTIN®), MDX-210 (Medarex, Inc.), pertuzumab (Genentech), tgDCC-El A (Targeted Genetics), rituximab (Rituxan®) and the like.

Other additional anticancer agents include ibritumomab tiuxetan (ZEV ALIN®); protein kinase C-alpha expression inhibitors, e.g., aprinocarsen; histodone deacetylase inhibitors, e.g., suberoylanilide hydroxamic acid (SAHA); thimidylate synthase and transferase inhibitors, e.g., Alimta.

Moreover, in certain embodiments of the invention, the compositions may be used in certain clinically used regimens such as FOLFOX. This treatment regimen is a chemotherapy regimen for treating colorectal cancer, and includes fluorouracil, leucovorin, and oxaliplatin. The compositions of the invention may be used in place of oxaliplatin.

In one embodiment, pharmaceutical compositions of the invention are used in combination with one or more biological response modifiers to treat cancer or infectious disease. One group of biological response modifiers is the cytokines. In one such embodiment, a cytokine is administered to a subject receiving the pharmaceutical compositions of the present invention. In another such embodiment, the pharmaceutical compositions of the present invention are administered to a subject receiving a cytokine. In various embodiments, one or more cytokine(s) can be used and are selected from the group consisting of IL-lα, IL- lβ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l, IL-12, IFNα, IFNβ, IFNγ, TNFα, TNFβ, G-CSF, GM-CSF, TGF-β, IL-15, IL-18, GM-CSF, INF-γ, INF-α, SLC, endothelial monocyte activating protein-2 (EMAP2), MIP-3α, MIP-3β, or an MHC gene, such as HLA-B7. Additionally, other exemplary cytokines include other members of the TNF family, including but not limited to TNF-α-related apoptosis-inducing ligand (TRAIL), TNF-α-related activation-induced cytokine (TRANCE), TNF-α-related weak inducer of apoptosis (TWEAK), CD40 ligand (CD40L), lymphotoxin alpha (LT-α), lymphotoxin beta (LT-β), OX40 ligand (OX40L), Fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), 41BB ligand (41BBL), APRIL, LIGHT₅ TLl, TNFSF16, TNFSF17, and AITR-L, or a functional portion thereof. See, e.g., Kwon et ah, 1999, Curr. Opin. Immunol. 11 :340-345 for a general review of the TNF family. The pharmaceutical compositions of the present invention may be administered prior to, during, or after the treatment modalities mentioned above. In a specific embodiment, compositions of the invention are administered to a subject receiving cyclophosphamide in combination with IL- 12 for treatment of cancer. In another embodiments, the pharmaceutical compositions of the present invention are used in combination with one or more biological response modifiers which are agonists or antagonists of various ligands, receptors and signal transduction molecules of the immune system. For examples, the biological response modifiers include but are not limited to agoinsts of Toll-like receptors (TLR-2, TLR-7, TLR-8 and TLR-9; LPS; agonists of 41BB ligand, OX40 ligand, ICOS, and CD40; and antagonists of Fas ligand, PDl, and CTLA-4. These agonists and antagonists can be antibodies, antibody fragments, peptides, peptidomimetic compounds, and polysaccharides.

In yet another embodiment, the pharmaceutical compositions of the present invention are used in combination with one or more biological response modifiers which are immunostimulatory nucleic acids. Such nucleic acids, many of which are oligonucleotides comprising an unmethylated CpG motif, are mitogenic to vertebrate lymphocytes, and are known to enhance the immune response. See Woolridge, et ah, 1997, Blood 89:2994-2998. Such oligonucleotides are described in International Patent Publication Nos. WO 01/22972, WO 01/51083, WO 98/40100 and WO 99/61056, each of which is incorporated herein in its entirety, as well as United States Patent Nos. 6,207,646, 6,194,388, 6,218,371, 6,239,116, 6,429,199, and 6,406,705, each of which is incorporated herein in its entirety. Other kinds of immunostimulatory oligonucleotides such as phosphorothioate oligodeoxynucleotides containing YpG- and CpR-motifs have been described by Kandimalla et a in "Effect of Chemical Modifications of Cytosine and Guanine in a CpG-Motif of Oligonucleotides: Structure-Immunostimulatory Activity Relationships." Bioorganic & Medicinal Chemistry 9:807-813 (2001), incorporated herein by reference in its entirety. Also encompassed are immunostimulatory oligonucleotides that lack CpG dinucleotides which when administered by mucosal routes (including low dose administration) or at high doses through parenteral routes, augment antibody responses, often as much as did the CpG nucleic acids, however the response was Th2-biased (IgGl»IgG2a). See United States Patent Publication No. 20010044416 Al, which is incorporated herein by reference in its entirety. Methods of determining the activity of immunostimulatory oligonucleotides can be performed as described in the aforementioned patents and publications. Moreover, immunostimulatory oligonucleotides can be modified within the phosphate backbone, sugar, nucleobase and internucleotide linkages in order to modulate the activity. Such modifications are known to those of skill in the art. In a preferred embodiment, pharmaceutical compositions of the present invention are used in combination with a toll like receptor agonist such as an immunostimulatory oligonucleotide comprising a CpG motif or an aminoalkyl glucosaminide 4-phosphate, or a combination thereof.

In yet another embodiment, the pharmaceutical compositions of the present invention are used in combination with one or more adjuvants. In one embodiment, the adjuvants provide a generalized stimulation of the immune response. The adjuvant(s) can be administered separately or they may be present in a composition in admixture with complexes of the invention. A systemic adjuvant is an adjuvant that can be delivered parenterally. Systemic adjuvants include adjuvants that creates a depot effect, adjuvants that stimulate the immune system and adjuvants that do both. An adjuvant that creates a depot effect as used herein is an adjuvant that causes the antigen to be slowly released in the body, thus prolonging the exposure of immune cells to the antigen. This class of adjuvants includes but is not limited to alum (e.g., aluminum hydroxide, aluminum phosphate); or emulsion- based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in-water-in oil emulsion, oil-in- water emulsions such as Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720, AirLiquide, Paris, France); MF-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.; and PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micelle-forming agent; IDEC, Pharmaceuticals Corporation, San Diego, Calif.).

Other adjuvants stimulate the immune system, for instance, cause an immune cell to produce and secrete cytokines or IgG. This class of adjuvants includes but is not limited to immunostimulatory nucleic acids, such as CpG oligonucleotides; saponins purified from the bark of the Q. saponaria tree, such as QS21 ; poly[di(carboxylatophen- oxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides (LPS) such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.).

Other systemic adjuvants are adjuvants that create a depot effect and stimulate the immune system. These compounds are those compounds which have both of the above- identified functions of systemic adjuvants. This class of adjuvants includes but is not limited to ISCOMs (Immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2 which is an oil-in-water emulsion containing MPL and QS21: SmithKline Beecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB₃ Belgium); non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxpropylene flanked by chains of polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex Adjuvant Formulation (SAF, an oil-in-water emulsion containing Tween 80 and a nonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.). The mucosal adjuvants useful according to the invention are adjuvants that are capable of inducing a mucosal immune response in a subject when administered to a mucosal surface in conjunction with complexes of the invention. Mucosal adjuvants include but are not limited to CpG nucleic acids (e.g. PCT published patent application WO 99/61056), Bacterial toxins: e.g., Cholera toxin (CT), CT derivatives including but not limited to CT B subunit (CTB) (Wu et al., 1998, Tochikubo et al., 1998); CTD53 (VaI to Asp) (Fontana et al., 1995); CTK97 (VaI to Lys) (Fontana et al., 1995); CTKl 04 (Tyr to Lys) (Fontana et al., 1995); CTD53/K63 (VaI to Asp, Ser to Lys) (Fontana et al., 1995); CTH54 (Arg to His) (Fontana et al., 1995); CTN107 (His to Asn) (Fontana et al., 1995); CTEl 14 (Ser to GIu) (Fontana et al., 1995); CTEl 12K (GIu to Lys) (Yamamoto et al., 1997a); CTS61F (Ser to Phe) (Yamamoto et al., 1997a, 1997b); CTS 106 (Pro to Lys) (Douce et al., 1997, Fontana et al., 1995); and CTK63 (Ser to Lys) (Douce et al., 1997, Fontana et al., 1995), Zonula occludens toxin, zot, Escherichia coli heat-labile enterotoxin, Labile Toxin (LT), LT derivatives including but not limited to LT B subunit (LTB) (Verweij et al., 1998); LT7K (Arg to Lys) (Komase et al., 1998, Douce et al., 1995); LT61F (Ser to Phe) (Komase et al., 1998); LTl 12K (GIu to Lys) (Komase et al., 1998); LTl 18E (GIy to GIu) (Komase et al., 1998); LT146E (Arg to GIu) (Komase et al., 1998); LT192G (Arg to GIy) (Komase et al., 1998); LTK63 (Ser to Lys) (Marchetti et al., 1998, Douce et al., 1997, 1998, Di Tommaso et al., 1996); and LTR72 (Ala to Arg) (Giuliani et al., 1998), Pertussis toxin, PT. (Lycke et al., 1992, Spangler BD, 1992, Freytag and Clemments, 1999, Roberts et al., 1995, Wilson et al., 1995) including PT-9K/129G (Roberts et al., 1995, Cropley et al., 1995); Toxin derivatives (see below) (Holmgren et al., 1993, Verweij et al., 1998, Rappuoli et al., 1995, Freytag and Clements, 1999); Lipid A derivatives (e.g., monophosphoryl lipid A, MPL) (Sasaki et al.,

1998, Vancott et al., 1998; Muramyl Dipeptide (MDP) derivatives (Fukushima et al., 1996, Ogawa et al., 1989, Michalek et al., 1983, Morisaki et al., 1983); lipid A mimetics (e.g., aminoalkyl glucosaminide 4-phosphates; Persing et al. 2002, Trends Microbiology 10:S32), bacterial outer membrane proteins (e.g., outer surface protein A (OspA) lipoprotein of Borrelia burgdorferi, outer membrane protine of Neisseria meningitidis)(Marinaro et al.,

1999, Van de Verg et al., 1996); oil-in-water emulsions (e.g., MF59) (Barchfield et al., 1999, Verschoor et al., 1999, O'Hagan, 1998); aluminum salts (Isaka et al., 1998, 1999); and

Saponins (e.g., QS21) Aquila Biopharmaceuticals, Inc., Worster, Me.) (Sasaki et al., 1998, MacNeal et al., 1998), ISCOMs, MF-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.); the Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720; AirLiquide, Paris, France); PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micell-forming agent; IDEC Pharmaceuticals Corporation, San Diego, Calif.); Syntext Adjuvant Formulation (SAF; Syntex Chemicals, Inc., Boulder, Colo.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA) and Leishmania elongation factor (Corixa Corporation, Seattle, Wash.). In another embodiment, the pharmaceutical compositions of the present invention are administered in conjunction with an adjuvant and immunogen.

5.5.6. OTHER THERAPEUTIC AGENTS

The present methods can further comprise the administration of a composition of the invention and another therapeutically active agent or pharmaceutically acceptable salt thereof. The composition of the invention and the therapeutically active agent can act additively or, more preferably, synergistically. In a preferred embodiment, a composition of the invention is administered concurrently with the administration of one or more other therapeutically active agents, which can be part of the same composition or in a different composition from that comprising the composition of the invention. In another embodiment, a composition of the invention is administered prior to or subsequent to administration of one or more other therapeutically active agents. Kits comprising a composition of the invention and one or more therapeutically active agents, in one or more containers are also provided. In the present methods for treating cancer the other therapeutically active agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, oxyperndyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine, onapristone, and tropisetron.

In a preferred embodiment, the antiemetic agent is granisetron or ondansetron. In another embodiment, the other therapeutically active agent can be an hematopoietic colony-stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alfa.

In still another embodiment, the other therapeutically active agent can be an opioid or non-opioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, normorphine, etorphine, buprenorphine, meperidine, lopermide, anileridine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazocine, pentazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofenac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.

In yet another embodiment, the other therapeutically active agent can be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.

6. EXAMPLES

Unless otherwise stated herein, all reagents and solvents were obtained from AlfaAesar, Ward Hill, MA. The lipids DMPG and DMPC were obtained from Lipoid, GmbH, Ludwigshafen, Germany or NOF America Corporation, White Plains, NY. NDDP was prepared by the method described in U.S. Patent No. 5,186,940 to Khokhar, et a and Perez-Soler et al, J. Micronencap. ϋ:41-54 (1994). The lyophilate compositions of the present invention can be reconstituted by contacting the lyophilate with an aqueous diluent, hi certain embodiments, the reconstituted lyophilate forms a composition substantially comprising liposomes. The reconstituted lyophilate compositions may also undergo post-reconstitution treatments such as heating for a period of time, acidifying, or contacting with chloride ions. The stability of the reconstituted lyophilate compositions during the reconstitution or post-reconstitution treatment processes were evaluated by allowing the compositions to stand in clear glass vials at 25°C or at 37°C and visually observing the vials for solids formation. Compositions that remained free of solids during the reconstitution and/or post-reconstitution process were deemed to be stable. The formation of solids in the compositions was also assessed using turbidimetry as described in Eckhardt et ah, J. Pharm. Sci. TechnoL 48:64-70 (1994)).

The viscosities of compositions were measured using a Gilmont falling ball viscometer.

Liposome sizes were measured by dynamic light scattering using PD2000 DLS detector (Precision Detectors, Inc., Bellingham WA).

6.1. EXAMPLE 1: PREPARATION OF AN NDDP COMPOSITION COMPRISING TWEEN 20 AND SALINE

A lyophilized composition containing NDDP was prepared in a manner similar to that described in Perez-Soler et ah, J. Micronencap. ϋ:41-54 (1994). A solution of DMPC (6.4418 g; 9.5 mmol) in tert-butanol (118 g) and a solution of DMPG (2.7608 g; 4.0 mmol) in tert-butanol (25.2 g) and water (20 g) mixture were combined, and NDDP (0.6624 g; 1.0 mmol) was added. The resultant mixture was then lyophilized to provide the NDDP/lipid lyophilate as a free-flowing powder.

Tween 20 (85 mg) was dissolved in 55 mL of 0.9% saline having a pH of 2.4 (from addition of hydrochloric acid). The resultant solution was combined with the NDDP lyophilate (1.608g; containing 0.108 g of NDDP), and the resultant suspension was shaken for 60 seconds at 25⁰C to provide an NDDP composition containing a surfactant.

For comparison, an NDDP composition was prepared as described above except that no Tween 20 was used. Liposomes were observed in both compositions.

The viscosities of the NDDP compositions prepared with and without Tween 20 are provided in Table 2. No precipitation was observed when the NDDP composition containing a surfactant was allowed to stand at 25°C or at 37°C for at least 72 hours. In contrast, precipitation was observed in the NDDP composition prepared without Tween 20 after standing for 24 hours at 25°C or after standing for 4 hours at 37°C. The results indicate that a surfactant such as Tween 20 is useful for stabilizing an NDDP compositions containing a chloride source such as saline.

6.2 EXAMPLE 2: PREPARATION OF AN NDDP COMPOSITION

COMPRISING TWEEN 20, AND DEXTROSE

Tween 20 (171.5 mg) was dissolved in 55 mL of water containing 5% dextrose and having a pH of 2.4 (from addition of hydrochloric acid). The resultant solution was combined with the NDDP lyophilate prepared as described in Example 1 (1.608 g; containing 0.108 g of NDDP). The resultant suspension was shaken for 60 seconds at 25°C to provide a reconstituted NDDP lyophilate composition.

For comparison, an NDDP composition was prepared as described above except that no Tween 20 was used.

Liposomes were observed in both compositions.

The viscosities of the NDDP compositions prepared with and without Tween 20 are provided in Table 2.

6.3 EXAMPLE 3: PREPARATION OF AN NDDP COMPOSITION COMPRISING TWEEN 20, SALINE, AND SUCROSE

An NDDP composition was prepared by the method described in Example 2, except that the water contained 5% sucrose instead of 5% dextrose.

For comparison, an NDDP composition was prepared as described above except that no Tween 20 was used. The viscosities of the NDDP compositions prepared with and without Tween

20 are provided in Table 2.

6.4 EXAMPLE 4: PREPARATION OF AN NDDP COMPOSITION

COMPRISING TWEEN 20, AND MANNITOL

An NDDP composition was prepared by the method described in Example 2, except that the water contained 5% mannitol instead of 5% dextrose.

For comparison, an NDDP composition was prepared as described above except that no Tween 20 was used. The viscosities of the NDDP compositions prepared with and without Tween 20 are provided in Table 2.

6.5 EXAMPLE 5: PREPARATION OF AN NDDP COMPOSITION COMPRISING TWEEN 20, AND GLYCEROL An NDDP composition was prepared by the method described in Example 2, except that the water contained 2.6% glycerol instead of 5% dextrose.

Table 2. NDDP formulations in saline (0.9%) containing an isotonic agent.

* Concentration based on the total weight of isotonic agent per volume of the water and the agent.

**The viscosities were determined at different times. NA: Not available.

Use of Tween 20 to reduce the viscosity of the NDDP compositions: When used as an injectable composition, the viscosities of NDDP compositions are preferably less than about 8 (Cp). The results of the above studies (Table 2) show that NDDP comprising an organic isotonic agent and no surfactant had viscosities that are undesirable for injectable compositions. However, NDDP compositions of the invention, which comprise the surfactant Tween 20 had viscosities suitable for injection. The results indicate that a surfactant such as, for example, Tween 20, is useful for reducing the viscosity of NDDP composition containing an organic isotonic agent. Moreover, surfactants can lower the size of the liposomes formed to be on average less than 1 micron in diameter. Smaller liposomes are advantageous because they are likely to remain in circulation in vivo for prolonged periods (i.e., they are not taken as avidly by reticulo-endothelial system), thus increasing their chance of crossing the endothelial barrier and reaching the tissue of interest. (See U.S. Patent No. 5,902,604).

6.6 EXAMPLE 6: PREPARATION OF A RECONSTITUTED NDDP COMPOSITION COMPRISING SALINE (FORMULATION A, COMPARATIVE

FORMULATION) A vial containing a pre-liposomal powder formed from 108 mg of NDDP lyophilized with 1050 mg of DMPC and 450 mg of DMPG was shaken to break up the cake and then a saline solution containing 0.9% sodium chloride, USP (55 ml), which was acidified with hydrochloric acid to pH 2.4, was added via a 60 ml syringe. The suspension was shaken vigorously by hand for 1 minute and then on an orbital shaker at 160-180 rpm for 2 hours at ambient temperature. The vial was then kept stationary at ambient temperature for 1 hour to allow any foam to dissipate. This procedure assures delivery of about 50 ml of liposomal suspension at 2 mg per ml of NDDP. This NDDP composition is referred to below as Formulation A.

6.7 EXAMPLE 7: PREPARATION OF A RECONSTITUTED NDDP COMPOSITION COMPRISING TWEEN 20 AND SALINE (FORMULATION B)

A vial containing the pre-liposomal powder containing NDDP, DMPC and DMPG in the quantities as specified in Example 6, was shaken to break up the cake and then a saline solution containing 0.9% sodium chloride, USP (55 ml), containing 170 mg of Tween 20^® (Polysorbate 20), which was acidified to pH 2.4 was added via a 60 ml syringe. The suspension was shaken vigorously by hand for 1 minute and then heated at about 37⁰C for 2 hours. The vial was then allowed to equilibrate to ambient temperature for one hour. This NDDP composition is referred to below as Formulation B.

6.8 EXAMPLE 8: NDDP ACTIVATION ASSAY

In this assay, the rate of conversion of NDDP to DACH-Pt-Cl₂ was compared for Formulations A and B. As described in Han et al. (Cancer Chemother Pharmacol. 1996;39(l-2):17-24; Maclean et al. J Microencapsul. May-Jun 2000;17(3):307-322) and Raymond et al. (Semin Oncol. Apr 1998; 25 (2 Suppl 5):4-12), DACH-Pt-Cl₂ most likely plays a very important role in the biological activity of a liposomal composition comprising NDDP. Under the conditions of activation of liposomal NDDP with acidified saline (i.e., in the presence of high concentration of chloride ions), commensurate with and in proportion to the acid-catalyzed hydrolysis of NDDP, DACH-Pt-Cl₂ is formed as the major conversion product of NDDP. The activation process was quantified as a decrease in the NDDP signal by HPLC.

For each time point, a sample (2 ml) of the reconstituted NDDP suspension (Formulation A or B) was diluted with methanol in a volumetric flask to 25 ml. The resulting solution was analyzed by HPLC on Zorbax SB-Cl 8 column kept at 30 ⁰C and eluted isocratically with methanol. The degree of activation was measured by NDDP disappearance. Samples were prepared 15 to 20 minutes before injection and were kept at 4 ⁰C in the autosampler. The assay was based on the comparison with the NDDP standard. HPLC Model 600 by Waters was used for the analyses.

Figure f shows a time course comparison of activation of the NDDP compositions, shown as NDDP conversion of Formulation A (Example 6) and Formulation B (Example 7). The rate of NDDP conversion for the Formulation B was measured over a period of 24 hours, and compared to that of the Formulation A. The data points represent mean values ± Standard Deviation of 17 experiments for the Formulation B (filled squares) and 4 experiments for the Formulation A (filled diamonds). The hatched vertical bars represent the start and end of the 3-8 hour time period during which the drug can be administered to patients following its reconstitution.

For Formulation B (containing surfactant), the data in Figure 1 shows about 85% NDDP conversion within the 2 hours of incubation, which remained stable from 2 to 24 hours. In contrast, NDDP conversion with Formulation A (without surfactant) proceeded at a slower rate, whereby, at 3 hours, only about 45% of NDDP was converted. Elevated activation temperatures could not be used for Formulation A because rapid NDDP conversion resulted in the precipitation OfDACH-Pt-Cl₂.

DACH-Pt-Cl₂ formation for Formulation B reaches high levels within 2 hours and remains at stable plateau levels from 2 to 24 hours. In contrast, DACH-Pt-Cl₂ formation with Formulation A barely reaches above baseline at 3 hours and continues to increase slowly, taking over 24 hours to reach close to the levels OfDACH-Pt-Cl₂ achieved with Formulation B. By this time, however, the highly insoluble DACH-Pt-Cl₂ precipitates out from Formulation A. 6.9 EXAMPLE 9: IN VITRO ASSAYS FOR TREATMENT OR PREVENTION OF CANCER

The following test was used to assess the in vitro anticancer activity of compositions of the present invention in human and murine cell lines. A liposomal suspension of platinum complex was added to human tumor cell lines (HT 29, HCTl 16 and PACA2) and murine tumor cell lines (L1210, CT26) established in 96-well plates. After 18 hours of incubation, the cells were pulsed with ³H thymidine and washed with phosphate-buffered saline (PBS). The amount of radioisotope incorporation was measured and used to calculate the inhibitory concentration 50 (IC₅₀), which is the concentration that causes a 50% decrease in cell proliferation. The test protocol for the L1210 model is described in Han et ah, Cancer Chemother. Pharmacol. 39: 17-24 (1996). The results of the test are shown in Table 3.

Table 3. In Vitro activity of Platinum Complex Formulations in Human and Mouse Tumor Cell Lines.

6.10 EXAMPLE lO: IN VITRO ASSAY FOR TREATMENT OR PREVENTION OF CANCER WITH NDDP FORMULATIONS, OXALIPLATIN,

CARBOPLATIN AND CISPLATIN

The following test was used to determine the in vitro anticancer activity of NDDP formulations, Formulation A (prepared according to Example 6) and Formulation B (prepared according to Example 7). The in vitro anticancer activity of the NDDP formulations were also compared with the anticancer activity of oxaliplatin, carboplatin and cisplatin.

A previously described protocol, with some modifications, was used to evaluate the compounds (Faivre et al. Cancer Chemother. Phamacol. 1999; 44(2): 117-123). The platinum complexes were added to human tumor cell lines (HT 29, HCTl 16, PC3, A2780, DU145 and PACA2) and murine tumor cell lines (L1210, CT26) established in 96- well plates. After 20 hours of incubation, ³H thymidine was added to each well and incubated for an additional 4 hours. Radioactivity incorporation was measured on a β- counter and used to calculate the inhibitory concentration 50 (IC₅₀), which is the concentration that causes a 50% decrease in cell proliferation. The results of the test are shown in Table 4.

The results show that the surfactant containing formulation (Formulation B) is more active than Formulation A, ranging from a 3 to 18 fold improvement, with the exception of one cell line (DU145). Formulation B is also more active than the other platinum compounds in most cell lines tested.

6.11 EXAMPLE 11: INVIVO ASSAYFORTREATMENT ORPREVENTION OF

LEUKEMIA

The following test was used to assess the in vivo antileukemia activity of liposomal suspensions of platinum complex against L 1210 mouse leukemia.

Groups of 8-10 B6D2/F1 mice were challenged via the intraperitoneal (i.p.) route with 10⁶ L1210 cells. The animals were then treated via i.p. route with 5 mg/kg of the NDDP compositions as prepared in Examples 1, 2 and 4; an acidified formulation containing NDDP, lipid and an acidic diluent; or empty liposomes on days 1, 4 and 9 post-challenge, and the animals were scored once daily to assess abdominal swelling - indicative of leukemia cell activity - and survival. The results of the swelling study and survival are shown in Table 5. Mice treated with the compositions of the invention (saline, mannitol and dextrose) exhibited less swelling and a higher rate of survival (Table 5) than did mice treated only with diluent. In the i.v. model, the mice were challenged in the same manner as described above for the i.p. route except that the mice were treated via i.v. route with 10 mg/kg of drug on days 1, 4 and 9 post-challenge. Mice were then scored as above. The results of the swelling study and of the survival study are shown in Table 5. Mice treated with a composition of the invention comprising mannitol exhibited about the same swelling and rate of survival (Table 5) as did mice treated only with diluent. On the other hand, mice treated with the compositions of the invention comprising saline and dextrose exhibited less swelling and a higher rate of survival (Table 5) than did mice treated only with diluent.

The results of the studies indicate that the compositions of the invention comprising a lipid, a platinum anti-tumor agent, and a surfactant are useful for treating or preventing cancer.

6.12 EXAMPLE 12: IN VIVO ASSAY FOR TREATMENT OR

PREVENTION OF COLORECTAL CANCER

The following test was used to assess the in vivo activity of compositions of the present invention against colorectal cancer using HT29 human colorectal carcinoma cell xenografts. Female BALB/c nude mice aged 6 weeks were injected subcutaneously with

HT29 cells (10⁶). Treatment was started on day 1 and repeated on days 8 and 15. Groups of mice were treated by injection into the tail vein: Group 1 was injected with diluent; group 2 with liposomal formulation of platinum complex at 5 and 10 mg/kg. Tumor size measurements and body weights were taken daily. The test protocol is described in Raymond et ah, Anti-Cancer Drugs 8: 876-885 (1997). The results of the study show that mice treated with the compositions of the invention (saline, mannitol and dextrose) exhibited smaller tumor volumes than did mice treated only with diluent. The results indicate that the compositions of the invention comprising a lipid, a platinum anti-tumor agent, a surfactant and an isotonic agent are useful for treating or preventing cancer. Table 5 shows tabulated results of administering several compositions of the present invention in human and mouse tumor models of Examples 11 and 12. Note that the diluent does not contain any platinum complex, whereas the dextrose, mannitol, and saline formulations contain lipid, surfactant (polysorbate 20), and NDDP. The acidified formulation contains lipid, NDDP, and an acidic diluent. Table 5. In Vivo Activity of Platinum Complex Formulations in Human and Mouse Tumor Models.

* Swelling score reflects the size of the tumor swelling and is evaluated at the day the diluent group reached grade 4.

**Tumor reduction is reported as percentage of tumor size in treated groups with respect to the diluent group at the end of the study. *** The T/C of diluent is by definition 100% **** Not determined.

6.13 EXAMPLE 13: INVIVO ASSAYFORTREATMENT ORPREVENTION OF

LEUKEMIA

The following test was used to assess the in vivo antileukemia activity of liposomal suspensions of platinum complex against L1210 mouse leukemia.

Groups of 9 B6D2/F1 mice were challenged via the intraperitoneal (i.p.) route with 10⁶ L1210 cells. The animals were then treated via i.v. route with the NDDP formulations (Formulations A and B as prepared in Examples 6 and 7, respectively, at 0.2 mg/animal) or Oxaliplatin (0.2 mg/animal) on days 1, 4 and 8 post-challenge. Both drugs were used at Maximum Tolerated Dose (MTD).

The number of animals alive in the L1210 ip/iv model was measured. Two different time points were evaluated: i) day 22 or 23, the day in which all mice from the diluent group died and ii) the end of the study. For survival, Fisher's Exact Tests were used to determine if there were significant differences in the number of mice alive for the Formulation B group compared to the diluent, Formulation A and Oxaliplatin groups. The results of the test are shown in Figure 2.

This data shows that those mice treated with Formulation B (containing surfactant) have a prolonged survival when compared to control untreated animals (diluent). On day 23 when all of the mice in the control group or those treated with Formulation A (without surfactant) have died or have been euthanized, 100% of the mice treated with the Formulation B survive. This also compares favorably with Oxaliplatin which had a more moderate effect (33% survival as of day 23) via this route. Formulation B group showed, at day 23, a statistically significant improvement in survival when compared to the diluent, Formulation A and Oxaliplatin groups (p<.0001, p<.0001 and p<.02 respectively). At the end of the study, the differences were not statistically significant for any of the treatment groups.

6.14 EXAMPLE 14: IN VIVO ASSAY FOR TREATMENT OR PREVENTION OF COLORECTAL CANCER

The following test was used to assess the in vivo activity of compositions of the present invention against colorectal cancer using HT29 human colorectal carcinoma cell xenografts in BALB/c nude mice.

Briefly, female BALB/c nude mice aged approximately 7 weeks were injected subcutaneously with HT29 cells (10⁶). Treatment was started on day 1 and repeated on days 8 and 15. Groups of 9 mice were treated with Formulation B (0.2 mg/animal, prepared as in Example 7) or Oxaliplatin (0.2 mg/animal) by injection into the tail vein. Tumor size measurements were taken twice per week. Both drugs were used at MTD.

Following the injection of tumor cells, tumor growth was monitored over a period of 3-4 weeks. Per protocol rules, the animals are sacrificed once the tumors size reaches approximately 2 cm. Thus, these studies measure the ability of a drug to suppress the growth of the xenograft tumor in the nude mice.

The tumor size (volume) was measured in the study. A one-way analysis of variance using Dunnett's method was used to compare the Formulation B group to the diluent or Oxaliplatin groups. The results of the test are shown in Figure 3.

Formulation B caused 92% reduction in tumor volume, compared to the untreated control mice, whereas mice treated with Oxaliplatin showed a 50% tumor reduction. Statistical analysis at the end of the study (day 38) shows significant difference in tumor size between the Formulation B treatment group and that of the diluent group (p<0.01), but not that of the Oxaliplatin group (p=0.61).

6.15 EXAMPLE 15: IN VIVO ASSAY FOR TREATMENT OR

PREVENTION OF LIVER CANCER

The following test can be used to assess the in vivo activity of compositions of the present invention against M5076 Reticulosarcoma liver metastases.

C57BL/6 mice are inoculated intravenously with M5076 cells (2 x 10⁴). Three groups of mice are treated by injection into the tail vein at day 4, 11 and 18: Group 1 is injected with diluent; Group 2 is injected with therapeutic composition at 3, 5 and 10 mg/kg; and Group 3 is injected with therapeutic composition at 20 mg/kg. The animals are sacrificed on day 30. The livers are dissected and placed in Bouin's solution (15 parts (by volume) of concentrated picric acid, 3 part of 37-40% formalin, and 1 part glacial acetic acid). The test protocol is described in Perez-Solar et al, Cancer Res. 52: 6341-6347 (1992).

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims along with the full scope of equivalents to which such claims are entitled.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a lipid, a surfactant, and a platinum complex having the formula:

Pt R9 R7

(H) wherein R₆ and R₇ each independently represent a leaving group; wherein R₈ and R₉ are each amines substituted by a group selected from a substituted or unsubstituted alkyl, -aryl, -arylalkyl, heteroalkyl, heterocyclyl, heteroaryl, heteroarylalkyl, -alkenyl, -cycloalkyl, or -cycloalkenyl groups having between 4 and 20 carbon atoms; or R₈ and R₉ are amine linked together as represented by formula (Ha): <^•

(Ha) wherein, A is absent or represents a ring containing 4 to 10 ring atoms selected from C,

N, O, and S;

B represents, independently for each occurrence, one or more occurrences of a group selected from H, halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate, — (CH₂)_p>alkyl, — (CH₂)_P'alkenyl, — (CH₂)_P'alkynyl, — (CH₂)_p.aryl, — (CH₂)_P'aralkyl, — O(CH₂)_p.R', — (CH₂)_P'SR', and — (CH₂)_P'NR'₂, wherein R' represents, independently for each occurrence, H or substituted or unsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to 8-membered ring; and p' represents an integer from 0 to 4; V and V, independently, represent a C(H)_m wherein m represents 0 or 1; W is absent or represents (Z(H)_n)_p wherein Z represents C, N, O, or S; and n and p independently represent 0, 1, 2, or 3; and wherein said lipid is capable of forming a liposome.

2. The composition of claim 1, wherein R₆ and R₇ are each independently an -alkyl-carboxylato bearing a hydrophobic radical function having 5 to 24 carbon atoms, -

OH, -OH₂, halo, -NO₃, or -HSO₄; or R₆ and R₇ are linked together to form a -dicarboxylato bearing a hydrophobic radical function.

3. The composition of claim 2, wherein R₆ and R₇ are each independently an -alkyl-carboxylato bearing a hydrophobic radical function having 5 to 24 carbon atoms, - OH, -OH₂, -NO₃, or -HSO₄; or Rg and R₇ are linked together to form a -dicarboxylato bearing a hydrophobic radical function.

4. The composition of claim 1, wherein the platinum complex has the formula:

wherein R₁ and R₂ are each independently an -alkyl-carboxylato bearing a hydrophobic radical function having 5 to 24 carbon atoms, -OH, -OH₂, halo, -NO₃, or -HSO₄; or R₁ and R₂ are linked together to form a -dicarboxylato bearing a hydrophobic radical function; wherein R₃ and R₄ are each amines of the formula:

wherein each R₅ is independently -H, -alkyl, -aryl, -arylalkyl, -alkenyl, -cycloalkyl, or -cycloalkenyl having between 1 and 20 carbon atoms; or R₃ and R₄ are linked to form a cycloalkyl- 1,2-diamino having between 3 and 7 carbon atoms or an alkyl- vicinal- diamino having between 2 and 12 carbon atoms.

5. The composition of claim 1 or 4 further comprising an isotonic agent; •

6. The composition of claim 1 or 4 further comprising a pH-adjusting agent.

7. The composition of claim 1 or 4 further comprising a diluent.

8. The composition of claim 7, wherein the composition is a solution or a suspension.

9. The composition of claim 8, where the composition is a suspension.

10. The composition of claim 9, wherein the suspension is a liposomal suspension.

11. The composition of claim 10, wherein said platinum complex is entrapped in said liposomes.

12. The composition of claim 1 or 4, wherein the composition is in a lyophilized form.

13. The composition of claim 4, wherein R₃ and R₄ are linked to form a cycloalkyl-l,2-diamino having between 3 and 7 carbon atoms or an alkyl-vicinal-diamino having between 2 and 12 carbon atoms.

14. The composition of claim 13, wherein R₃ and R₄ are linked to form trans-R,R- 1 ,2-diaminocyclohexane, trans-S,S- 1 ,2-diaminocyclohexane, cis- 1 ,2- diaminocyclohexane, ethylene diamine, or l,l-bis(aminomethyl)cyclohexane.

15. The composition of claim 4, wherein R₁ and R₂ are each independently -neohexanoato, -neoheptanoato, -neononanoato, -neodecanoato, -neooctanoato, - neopentanoato, -2-ethylhexanoato, -2-ethylbutyrato, -2-propylpropanoato, -2-methyl-2- ethylheptanoato, -2,2-diethylhexanoato, -2,2-dimethyl-4-ethylhexanoato, -2,2-diethyl-4- methylpentanoato, -2,2-dimethyloctanoato, -2-methyl-2-ethylheptanoato, -2,2- diethylhexanoato, -2,3,5-trimethylheptanoato,-2,2-diethyl-4-methylpentanoato or -2,2,4,4- tetramethylpentanoato, or -2,2-dimethyloctanoato.

16. The composition of claim 4, wherein R₁ and R₂ are each independently a hydrophobic radical function having 5 to 24 carbon atoms, -OH, -OH₂, -Cl, -halo, -NO₃, or -HSO₄, with the proviso that R₁ and R₂ are not simultaneously both an -alkylcarboxylate.

17. The composition of claim 4, wherein the surfactant is a non-ionic surfactant, an anionic surfactant, a cationic surfactant, or a combination thereof.

18. The composition of claim 17, wherein the surfactant is a non-ionic surfactant selected from the group consisting of a poloxamer surfactant, a polysorbate surfactant, and a combination thereof.

19. The composition of claim 4, wherein the surfactant is present in an amount from about 1 part by weight to about 100 parts by weight based on the amount of platinum.

20. The composition of claim 1, wherein the lipid is dimyristoyl phosphatidyl choline, egg phosphatidyl choline, dilauryloyl phosphatidyl choline, dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline, l-myristoyl-2-palmitoyl phosphatidyl choline, l-palmitoyl-2-myristoyl phosphatidyl choline, l-palmitoyl-2-stearoyl phosphatidyl choline, l-stearoyl-2-palmitoyl phosphatidyl choline, dioleoyl phosphatidyl choline, dimyristoyl phosphatidyl glycerol, dilauryloyl phosphatidyl glycerol, dioleoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol, distearoyl phosphatidyl glycerol, l-myristoyl-2- palmitoyl phosphatidyl glycerol, l-palmitoyl-2-myristoyl phosphatidyl glycerol, l-palmitoyl- 2-stearoyl phosphatidyl glycerol, l-stearoyl-2-palmitoyl phosphatidyl glycerol, dioleoyl phosphatidyl glycerol, dimyristoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dimyristoyl phosphatidyl serine, dilauryloyl phosphatidyl serine, dipalmitoyl phosphatidyl serine, distearoyl phosphatidyl serine, l-myristoyl-2-palmitoyl phosphatidyl serine, l-palmitoyl-2-myristoyl phosphatidyl serine, l-palmitoyl-2-stearoyl phosphatidyl serine, l-stearoyl-2-palmitoyl phosphatidyl serine, dioleoyl phosphatidyl serine, brain sphingomyelin, dipalmitoyl sphingomyelin, distearoyl sphingomyelin, dimyristoyl phosphatidic acid, dilauryloyl phosphatidic acid, dioleoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, l-myristoyl-2-palmitoyl phosphatidic acid, 1- palmitoyl-2-myristoyl phosphatidic acid, l-palmitoyl-2-stearoyl phosphatidic acid, 1- stearoyl-2-palmitoyl phosphatidic acid, dioleoyl phosphatidic acid, or a combination thereof.

21. The composition of claim 5, wherein the isotonic agent is a carbohydrate-based isotonic agent.

22. The composition of claim 21, wherein the carbohydrate-based isotonic agent is dextrose, sucrose, lactose, mannitol, inositol, saccharose, maltitol or a combination thereof.

23. The composition of claim 5, wherein the isotonic agent is glycerol or 1 ,2-propane-diol.

24. The composition of claim 5, wherein the isotonic agent is an inorganic isotonic agent.

25. The composition of claim 24, wherein the inorganic isotonic agent is sodium chloride.

26. The composition of claim 1 or 4, having a pH from about 2 to about 6.

27. The composition of claim 1 or 4, wherein the composition contains less than about 0.1% chloride by weight.

28. A pharmaceutical composition comprising the composition of claim 1 , wherein the platinum complex is present in an amount effective to treat cancer.

29. The pharmaceutical composition of claims 28 further comprising a pharmaceutically acceptable carrier.

30. A pharmaceutical composition comprising the composition of claim 11, wherein the platinum complex is present in an amount effective to treat cancer.

31. A second composition prepared by activating a first composition, said first composition comprising a lipid, a surfactant, and a platinum complex having the formula

II:

Rs_{χ /Rs}

Pt

Rg F?7

(H) wherein R₆ and R₇ each independently represent a leaving group; wherein R₈ and R₉ are each amines substituted by a group selected from a substituted or unsubstituted alkyl, -aryl, -arylalkyl, heteroalkyl, heterocyclyl, heteroaryl, heteroarylalkyl, -alkenyl, -cycloalkyl, or -cycloalkenyl groups having between 4 and 20 carbon atoms; or Rg and R₉ are linked together as represented by formula (Ha):

(Ha) wherein,

A is absent or represents a ring containing 4 to 10 ring atoms selected from C, N, O, and S;

B represents, independently for each occurrence, one or more occurrences of a group selected from H, halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate, — (CH₂)_p>alkyl, — (CH₂)_p>alkenyl, — (CH₂)_p>alkynyl, — (CH₂)p'aryl, — (CH₂)_P'aralkyl, — O(CH₂)_P'R', — (CH₂)_P>SR\ and — (CH₂)_P>NR'₂, wherein R' represents, independently for each occurrence, H or substituted or unsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to 8-membered ring; and p' represents an integer from 0 to 4;

V and V, independently, represent a C(H)_m wherein m represents 0 or 1; W is absent or represents (Z(H)_n)_p wherein Z represents C, N, O, or S; and n and p independently represent 0, 1, 2, or 3; and wherein said lipid is capable of forming a liposome; and wherein the activating is heating, adding a chloride source, acidifying, or a combination thereof.

32. The second composition of claim 31 , wherein R₆ and R₇ are each independently an -alkyl-carboxylato bearing a hydrophobic radical function having 5 to 24 carbon atoms, -OH, -OH₂, -halo, -NO₃, or -HSO₄; or R₁ and R₂ are linked together to form a - dicarboxylato bearing a hydrophobic radical function.

33. The second composition of claim 31 , wherein said platinum complex-, of said first composition has the formula:

R₃ and R₄ are each amines of the formula:

H

— N

R₅

wherein each R₅ is independently -H, -alkyl, -aryl, -arylalkyl, -alkenyl, -cycloalkyl, or -cycloalkenyl having between 1 and 20 carbon atoms; or R₃ and R₄ are linked to form a cycloalkyl- 1,2-diamino having between 3 and 7 carbon atoms or an alkyl- vicinal- diamino having between about 2 and 12 carbon atoms.

34. The second composition of claim 31 or 33, wherein the activating is heating.

35. The second composition of claim 34, wherein the heating is carried at a temperature from about 3O⁰C to about 45°C.

36. The second composition of claim 31 or 33, wherein the activating is adding a chloride source.

37. The second composition of claim 36, wherein the chloride source is saline.

38. The second composition of claim 31 or 33, wherein the activating is acidifying.

39. The second composition of claim 38, wherein the acidifying is acidifying the first composition to a pH of about 2 to about a pH of 6.

40. The second composition of claim 38, wherein the first composition is acidified with hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, or a combination thereof.

41. A pharmaceutical composition comprising the second composition of claim 31, wherein the platinum complex is present in an amount effective to treat cancer.

42. A method for making a composition comprising:

(a) mixing a lipid, a surfactant, and a platinum complex with a first diluent to form a liquid composition, the platinum complex having the formula:

Pt

K₉ K₇ (II) wherein R₆ and R₇ each independency represent a leaving group; wherein R₈ and R₉ are each amines substituted by a group selected from a substituted or unsubstituted alkyl, -aryl, -arylalkyl, heteroalkyl, heterocyclyl, heteroaryl, heteroarylalkyl, -alkenyl, -cycloalkyl, or -cycloalkenyl groups having between 4 and 20 carbon atoms; or R₈ and R₉ are linked together as represented by formula (Ha):

(Ha) wherein,

B represents, independently for each occurrence, one or more occurrences of a group selected from H, halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate, — (CH₂)_P'alkyl, — (CH₂)_p>alkenyl, — (CH₂)_p>alkynyl,

— (CH₂)p'aryl, — (CH₂)_p>aralkyl, — O(CH₂)_P>R', — (CH₂)_P>SR\ and — (CH₂)_P'NR'₂,- < ' - • • ■ wherein R' represents, independently for each occurrence, H or substituted or unsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to 8-membered ring; and p' represents an integer from 0 to 4; V and V₃ independently, represent a C(H)_n, wherein m represents 0 or 1 ;

W is absent or represents (Z(H)_n)_p wherein Z represents C, N, O, or S; and n and p independently represent 0, 1, 2, or 3; and wherein said lipid is capable of forming a liposome; and (b) lyophilizing the liquid composition to form a lyophilate.

43. The method of claim 42, wherein R₆ and R₇ are each independently an

-alkyl-carboxylato bearing a hydrophobic radical function having 5 to 24 carbon atoms, - OH, -OH₂, -halo, -NO₃, or -HSO₄; or R₁ and R₂ are linked together to form a -dicarboxylato bearing a hydrophobic radical function.

44. The method of claim 42, wherein said platinum complex of (a) has the formula:

R. R,

Pt

R₄ R₂ wherein R₁ and R₂ are each independently an -alkyl-carboxylato bearing a hydrophobic radical function having 5 to 24 carbon atoms, -OH, -OH₂, -halo, -NO₃, or - HSO₄; or R₁ and R₂ are linked together to form a -dicarboxylato bearing a hydrophobic radical function;

R₃ and R₄ are each amines of the formula:

H

—\

R₅

wherein each R₅ is independently -H, -alkyl, -aryl, -arylalkyl, -alkenyl, -cycloalkyl, or -cycloalkenyl having between 1 and 20 carbon atoms; or R₃ and R₄ are linked to form a cycloalkyl- 1,2-diamino having between 3 and 7 carbon atoms or an alkyl-vicinal- diamino having between 2 and 12 carbon atoms.

45. The method of claim 44, wherein R₃ and R₄ are linked to form trans- R₅R- 1 ,2-diaminocyclohexane.

46. The method of claim 44, wherein R₁ and R₂ are each -neodecanoato.

47. The method of claim 42 or 44, where the liquid composition is a solution.

48. The method of claim 42 or 44, wherein the first diluent comprises an organic solvent.

49. The method of claim 42 or 44, wherein the first diluent comprises t- butanol and water.

50. The method of claim 42 or 44, further comprising:

(c) contacting the lyophilate with a second diluent comprising water to form a reconstituted composition.

51. The method of claim 50, further comprising activating the reconstituted composition.

52. The method of claim 51 , wherein the reconstituted composition comprises liposomes formed by said lipids.

53. The method of claim 52, wherein said platinum complex is entrapped in said liposomes.

54. The method of claim 51 , wherein activating comprises heating, adding a chloride source, acidifying, or a combination thereof.

55. The method of claim 50, further comprising acidifying the reconstituted composition by adding an acid in an amount sufficient to provide a reconstituted composition having a pH of from about 2 to about 6.

56. The method of claim 55, wherein the acid is hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, or a combination thereof.

57. The method of claim 50, wherein the second diluent has a pH of from about 2 to about 6.

58. The method of claim 50, further comprising heating the reconstituted composition at a temperature from about 30°C to about 45°C.

59. The method of claim 50, further comprising adding an isotonic agent to the reconstituted composition.

60. A composition prepared by the method of any one of claims 42-59.

61. A method for making a composition comprising: (a) mixing a lipid and a platinum complex with a first diluent to form a liquid composition lacking a surfactant, the platinum compound having the formula:

^RN /^β

R₉ /\ R₇ (II) wherein R₆ and R₇ each independently represent a leaving group; wherein R₈ and R₉ are each amines substituted by a group selected from a substituted or unsubstituted alkyl, -aryl, -arylalkyl, heteroalkyl, heterocyclyl, heteroaryl, heteroarylalkyl, -alkenyl, -cycloalkyl, or -cycloalkenyl groups having between 4 and 20 carbon atoms; or R₈ and R₉ are linked together as represented by formula (Ha):

(Ha) wherein,

B represents, independently for each occurrence, one or more occurrences of a group selected from H, halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate, — (CH₂)_p>alkyl, — (CH₂)_p>alkenyl, — (CH₂)_P'alkynyl, — (CH₂)_p>aryl, — (CH₂)_P'aralkyl, — O(CH₂)_P>R', — (CH₂)_P'SR'₅ and — (CH₂)_P>NR'₂, wherein R' represents, independently for each occurrence, H or substituted or unsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to 8-membered ring; and p' represents an integer from 0 to 4; V and V, independently, represent a C(H)_m wherein m represents 0 or 1 ;

W is absent or represents (Z(H)_n)_p wherein Z represents C, N, O, or S; and n and p independently represent 0, 1, 2, or 3; wherein said lipid is capable of forming a liposome;

(b) lyophilizing the liquid composition to form a lyophilate; and (c) contacting the lyophilate with a second diluent comprising water and a surfactant to form a reconstituted composition.

62. The method of claim 61 , wherein R₆ and R₇ are each independently an -alkyl-carboxylato bearing a hydrophobic radical function having 5 to 24 carbon atoms, -OH, -OH₂, halo, -NO₃, or -HSO₄; or R₁ and R₂ are linked together to form a -dicarboxylato bearing a hydrophobic radical function.

63. The method of claim 61 , wherein said platinum complex of (a) has the formula:

wherein R₁ and R₂ are each independently an -alkyl-carboxylato bearing a hydrophobic radical function having 5 to 24 carbon atoms, -OH, -OH₂, -halo, -NO₃, or - HSO₄; or R₁ and R₂ are linked together to form a -dicarboxylato bearing a hydrophobic radical function;

R₃ and R₄ are each amines of the formula:

wherein each R₅ is independently -H, -alkyl, -aryl, -arylalkyl, -alkenyl,

-cycloalkyl, or -cycloalkenyl having between 1 and 20 carbon atoms; or R₃ and R₄ are linked to form a cycloalkyl-l,2-diamino having between about 3 and 7 carbon atoms or an alkyl- vicinal-diamino having between 2 and 12 carbon atoms.

64. The method of claim 63, wherein R₃ and R₄ are linked to form trans- R₅R- 1 ,2-diaminocyclohexane.

65. The method of claim 63, wherein R₁ and R₂ are each -neodecanoato.

66. The method of claim 63, where the liquid composition is a solution.

67. The method of claim 63, wherein the first diluent comprises an organic solvent.

68. The method of claim 63, wherein the first diluent comprises t-butanol and water.

69. The method of claim 63, further comprising activating the reconstituted composition.

70. The method of claim 63, wherein the reconstituted composition comprises liposomes formed by said lipids.

71. The method of claim 63, wherein said platinum complex is entrapped in said liposomes.

72. The method of claim 69, wherein activating comprises heating, adding a chloride source, acidifying, or a combination thereof.

73. The method of claim 63 , wherein the second diluent has a pH of from about 2 to about 6.

74. The method of claim 63, further comprising heating the reconstituted composition at a temperature from about 30°C to about 45°C.

75. The method of claim 63, further comprising adding an isotonic agent to the reconstituted composition.

76. A composition prepared by the method of any one of claims 61-75.

77. A pharmaceutical composition comprising the composition prepared by the method of any one of claims 42, 50, 61 or 63, wherein the platinum compound is present in an amount effective to treat cancer.

78. A kit comprising, in a first container, the composition produced by the method of claim 42 or 44, and in a second container, a diluent for reconstitution.

79. The kit of claim 78, further comprising a therapeutic agent other than the platinum complex.

80. A method for treating or preventing cancer comprising administering to a patient in need thereof the pharmaceutical composition of any one of claims 28, 30 or 41.

81. The method of claim 80, wherein the cancer is selected from pancreatic cancer, colorectal cancer, mesothelioma, a malignant pleural effusion, peritoneal carcinomatosis, peritoneal sarcomatosis, renal cell carcinoma, small cell lung cancer, non- small cell lung cancer, testicular cancer, bladder cancer, breast cancer, head and neck cancer, ovarian cancer and peritoneal cavity cancer.

82. A method for treating or preventing cancer comprising administering to a patient in need thereof the pharmaceutical composition of claim 77.

83. A method of claim 82, wherein the cancer is selected from pancreatic cancer, colorectal cancer, mesothelioma, a malignant pleural effusion, peritoneal carcinomatosis, peritoneal sarcomatosis, renal cell carcinoma, small cell lung cancer, non- small cell lung cancer, testicular cancer, bladder cancer, breast cancer, head and neck cancer, ovarian cancer and peritoneal cavity cancer.

84. The method of claim 80 or 82, the method further comprising administering a therapeutic agent other than the platinum complex.

85. The method of claim 84, wherein the therapeutic agent other than the platinum complex is an immunotherapeutic agent, an antiemetic agent, a hematopoietic colony stimulating factor, an anti-depressant, an analgesic agent, or a combination thereof.

86. A second composition comprising a lipid, a surfactant, and a water- insoluble substituted diamino platinum (II) X₂ complex represented by formula III in a solubilized form:

Pt

_R/ \

(III) wherein R₈ and Rg are each amines substituted by a group selected from a substituted or unsubstituted alkyl, -aryl, -arylalkyl, heteroalkyl, heterocyclyl, heteroaryl, heteroarylalkyl, -alkenyl, -cycloalkyl, or -cycloalkenyl groups having between 4 and 20 carbon atoms; or R₈ and Rg are linked together as represented by formula (Ha):

(Ha) wherein,

A is absent or represents a ring containing 4 to 10 ring atoms selected from C, N, O, and S; B represents, independently for each occurrence, one or more occurrences of a group selected from H, halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate, — (CH₂)_p>alkyl, — (CH₂)_P'alkenyl, — (CH₂)_P"alkynyl, — (CH₂)_p.aryl, — (CH₂)_p>aralkyl, -0(CH₂VR', -(CH₂VSR', and — (CH₂)_P'NR'₂, wherein R' represents, independently for each occurrence, H or substituted or unsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to 8-membered ring; and p' represents an integer from 0 to 4; V and V, independently, represent a C(H)_m wherein m represents 0 or 1 ;

W is absent or represents (Z(H)_n)_p wherein Z represents C, N, O, or S; and n and p independently represent 0, 1, 2, or 3; and X represents a halo.

87. The second composition of claim 86, wherein the second composition is prepared by activating a first composition, said first composition comprising a lipid, a surfactant and a platinum (II) complex represented by the formula (II):

Pt R₉ R₇ (II) wherein R₈ and R₉ are as defined above;

R₆ and R₇ are each independently an -alkyl-carboxylato bearing a hydrophobic radical function having 5 to 24 carbon atoms, -OH, -OH₂, -NO₃, or -HSO₄; or R₁ and R₂ are linked together to form a -dicarboxylato bearing a hydrophobic radical function; wherein said lipid is capable of forming a liposome; wherein the activating is heating, adding a chloride source, acidifying, or a combination thereof.

88. A composition comprising a lipid, a polysorbate surfactant, an isotonic agent and a platinum complex having the formula:

wherein R₁ and R₂ are both neodecanoato;

R₃ and R₄ are linked to form a 1,2-diaminocyclohexane; and wherein said lipid is capable of forming a liposome.

89. The composition of claim 88, wherein the polysorbate surfactant is polysorbate 20.

90. The composition of claim 88, wherein the lipid comprises a mixture of dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline.

91. The composition of claim 88, wherein the isotonic agent is sodium chloride.

92. The composition of claim 88, wherein the pH of the composition is from about 2 to about 6.

93. The composition of claim 88, wherein: the polysorbate surfactant is polysorbate 20, the lipid comprises a mixture of dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline, the isotonic agent is sodium chloride, and the pH of the composition is from about 2 to about 6.

94. A method for making a composition comprising:

R,_{χ /R},

Pt

R₄ R₂ wherein R₁ and R₂ are both neodecanoato;

R₃ and R₄ are linked to form a 1,2-diaminocyclohexane;

(b) lyophilizing the liquid composition to form a lyophilate;

(c) contacting the lyophilate with a second diluent comprising water, an isotonic agent and a polysorbate surfactant to form a reconstituted composition; and (d) heating the reconstituted composition at a temperature of from about 30°C to about 45°C.

95. The method of claim 93, wherein the first diluent comprises t-butanol and water.

96. The method of claim 93, wherein the polysorbate surfactant is polysorbate 20.

97. The method of claim 93, wherein the lipid comprises a mixture of dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline.

98. The method of claim 93, wherein the isotonic agent is sodium chloride.

99. The method of claim 93, wherein the pH of the reconstituted composition is from about 2 to about 6.

100. The method of claim 93, wherein step (d) comprises heating the reconstituted composition for about 0.25 to about 3 hours.

101. The method of claim 93 , wherein: the first diluent comprises t-butanol and water, the polysorbate surfactant is polysorbate 20, the lipid comprises a mixture of dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline, the isotonic agent is sodium chloride, the pH of the reconstituted composition is from about 2 to about 6, and step (d) comprises heating the reconstituted composition for about 0.25 to about 3 hours.

102. A method for making a composition comprising:

(a) mixing a lipid, a polysorbate surfactant and a platinum complex with a first diluent to form a liquid composition the platinum compound having the formula:

^R3\ /^Ri

Pt

(b) lyophilizing the liquid composition to form a lyophilate;

(c) contacting the lyophilate with a second diluent comprising water and an isotonic agent to form a reconstituted composition; and

(d) heating the reconstituted composition at a temperature of from about 30°C to about 45°C.

103. The method of claim 102, wherein the first diluent comprises t-butanol and water.

104. The method of claim 102, wherein the polysorbate surfactant is polysorbate 20.

105. The method of claim 102, wherein the lipid comprises a mixture of dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline.

106. The method of claim 102, wherein the isotonic agent is sodium chloride.

107. The method of claim 102, wherein the pH of the reconstituted composition is from about 2 to about 6.

108. The method of claim 102, wherein step (d) comprises heating the reconstituted composition for about 0.25 to about 3 hours.

109. The method of claim 102, wherein: the first diluent comprises t-butanol and water, the polysorbate surfactant is polysorbate 20, the lipid comprises a mixture of dimyristoyl phosphatidyl glycerol and dimyristoyl phosphatidyl choline, the isotonic agent is sodium chloride, the pH of the reconstituted composition is from about 2 to about 6, and step (d) comprises heating the reconstituted composition for about 0.25 to about 3 hours.

110. A composition formed by the method of any one of claims 94- 109.