ES2347534T3 - LOAD OF LIPOSOMES WITH METAL IONS. - Google Patents

Abstract

A liposomal composition comprising liposomes containing an internal solution, the internal solution comprising copper ions and one or more encapsulated therapeutic agents, an agent that is a drug or an antineoplastic agent5; and in which: (a) the liposomes do not comprise an ionophore; (b) said therapeutic agent has been loaded into preformed liposomes containing said copper ions in said internal solution; (c) the internal solution comprises a solution compatible with copper ions; and (d) the composition comprises the liposomes in a solution external to the liposomes.

Description

Technical Field
This invention relates to the encapsulation of drugs.
and other agents in liposomes.

Background of the Invention

Liposomes are microscopic particles that are constituted by one or more lipid bilayers that enclose a internal compartment Liposomes can be classified into multilamellar vesicles, multivesicular liposomes, Unilamellar vesicles and giant liposomes. Liposomes multilamellar (also known as vesicles multilamellar or "MLV" (contain for its acronym in English)) multiple concentric bilayers within each particle liposomal, which resemble the "onion layers". The multivesicular liposomes consist of lipid membranes that enclose multiple non-concentric aqueous chambers. The liposomes that enclose a single internal aqueous compartment include small unilamellar vesicles (SUVs) in English) and large unilamellar vesicles (LUV, for their acronym in English). LUVs and SUVs vary in size of approximately 50 to 500 nm and 20 to 50 nm, respectively. Giant liposomes typically vary in size of 5000 nm to 50,000 nm and are mainly used to study the mechanochemical and interactive characteristics of vesicles of in vitro lipid bilayer (Needham et al., Colloids and Surfaces B: Biointerfaces (2000) 18: 183195).

Liposomes have been studied and used widely as vehicles for a variety of agents such as drugs, Cosmetics, diagnostic reagents and genetic material. Since liposomes consist of non-toxic lipids, they generally have low toxicity and therefore are useful in a variety of pharmaceutical applications. In particular, liposomes are useful for increasing life in circulation of agents that have a short half-life in the blood flow The drugs encapsulated in liposomes they often have biodistributions and toxicities that differ A lot of the free drugs. For the in vivo contribution specific, sizes, electric charges and

surface properties of these vehicles can be changed

to the

to vary the methods from preparation Y  to the adjust the

Constitution

lipid of the vehicle. By example, the

liposomes

they can elaborate for break free a drug plus

rapidly by decreasing the length of the acyl chain of a lipid that constitutes the vehicle.

Liposomes containing metal ions encapsulated in The inside of the gallbladder have been used in applications Diagnostics For example, liposomes have been used for contribution of contrast agents in order to accumulate a contrast agent in a desired area within the body of a subject. In the last application, liposomes have mainly used for the contribution of radionuclides and diagnostic paramagnetic metal ions in obtaining gamma and magnetic resonance images, respectively. This includes liposomal encapsulation of radionuclides. such as 111In, 99mTc and 67Ga and paramagnetic ions such as Gd, Mn and manganese oxide. Typically, two are used methods to prepare liposomes for purposes of obtaining of pictures. In the first method, the metal becomes a soluble chelate and then introduced inside aqueous of a liposome. In the second method, an agent chelator derived with a lipophilic group is anchored to the liposome surface during or after preparation of the liposome.

Manganese and non-transition metal ions they have also been involved in methods for encapsulation of ionizable agents in liposomes that contain an ionophore inserted into the liposome membrane (see U.S. Patent No. 5837282 and Fenske et al., Biochim Biophys Minutes (1998) 1414: 188-204). In this method, the ionophore translocates the metal ion through the membrane of the liposome in a proton exchange, establishing thus a pH gradient. The establishment of a gradient of proper pH through the liposome bilayer allows the ionizable agent is encapsulated, since the agent can easily cross the liposomal bilayer neutrally and subsequently it is encapsulated and trapped inside the

aqueous liposome interior due to conversion in the electrically charged form (see Mayer et al., Patents from the USA UU. 6083530, 5616341, 5795589 and 5744158; Mayer et al., Biochimica et Biophysica Acta (1986) 857: 123). This work arises from mechanical studies completed by Deamer et al. (Biochimica et Biophysica Acta (1476) 455: 269-271) that showed that liposomes effectively concentrated several

catecholamines

(dopamine, norepinephrine Y epinephrine) in

response to a pH gradient of transmembrane).

The

presence from a inside liposomal acid Y a

outside environment from basic to neutral allows agents to they are mainly in neutral form at neutral to basic pH and mainly in electrically charged form at acidic pH be easily trapped inside a liposome. Drugs that contain ionizable moieties, such as amine groups, are easily encapsulated and retained in liposomes that They contain an acidic interior. This method, in which a ionophore (A23187) to generate a pH gradient through a liposome that contains manganese, has been used to load topotecan in cholesterol-free liposomes that comprise a PEG-lipid conjugate inserted into the membrane (see WO / 0185131). However, loading and retention satisfactory using a transmembrane pH gradient will achieved even if the internal pH of the liposome is maintained. Since the pH gradient can only be maintained for short periods of time, the clinical formulation of drugs in liposomes it requires the generation of a pH gradient in liposomes just before drug loading. A second disadvantage of this method results from instability of the lipid, and some drugs, at acidic pH, which prevents need for long-term liposome storage loaded with drug. Freezing formulations liposomal slows the rate of hydrolysis but the Conventional liposomal formulations often add and they lose components during defrosting unless they use appropriately selected cryoprotectants.

There have been complexes between drugs such as doxorubicin or ciprofloxacin and divalent metal ions

such as Mn2 + (Bouma, J., et al. (1986) Pharm. Weekbl. Sci. Edn. 16: 109-133; Riley, C.M., et al. (1993) J. Pharm. Biomed Anal. 11: 49-59; and Fenske, D.B. (1998) Biochim. Biophys Minutes. 1414: 188-204). Recently, it was presented that the uptake of doxorubicin (but not ciprofloxacin) within LUV of sphingomyelin / cholesterol could be carried out with manganese in the internal charging medium without the presence of a ionophore (Cheung et al., Biochimica et Biophysica Acta (1998) 1414: 205). It was suggested that a procedure involving both complex formation between doxorubicin and ions manganese as the protonation of doxorubicin within the Liposome resulted in the uptake of this drug particular in the presence of manganese ions. The stable doxorubicin entrapment, but this work is based on the use of sphingomyelin / cholesterol liposomes, a formulation designated for optimal drug retention. The methodology presented by Cheung, et al., Which implied The use of MnSO4 in pH 7.4 HEPES buffer is not reproducible because the metal precipitates in such a buffer.

Various groups have investigated ion interaction Metallic with liposomes in order to evaluate the effects of metal cations on vesicle membranes (Steffan et al. (1994) Chem. Phys. Lipids 74 (2): 141-150).

Ca2 +

Divalent metal cations such as have been related to unfavorable crosslinking formation induced by liposome metals containing phosphatidylglycerol (PG) due to negative electrical charge of the liposome surface. Metal ions are also have related to the temperature increase of phase transition of modeled membrane systems negatively (Borle, et al., (1985) Chemistry and Physics of Lipids 36: 263-283; Jacobson, et al., (1975) Biochemistry 14 (l): 152-161). These studies revealed that the addition of calcium to dipalmitoylphosphatidylglycerol membranes (DPPG) resulted in an increase in the temperature of phase transition of approximately 50 ° C.

These results indicate that the use of loaded lipids negative electric along with metal ions will give as

-5 results liposomes exhibiting inferior characteristics for in vivo applications. Summary of the Invention

This invention is based on the discovery that loading and retention efficiency properties of a liposome using metal based procedures carried out in absence of an ionophore in the liposome depend surprisingly of the metal used in the constitution Liposome lipid. When selecting the constitution lipid and a metallic composition, the loading properties or retention can be adjusted to reach a load or desired release of a selected agent from a liposome Moreover, the undesirable precipitation of metal in solutions used in the formulation of liposomes encapsulated with metal ions can be avoided by use of compatible solutions with metals, and the load also can be improved by withdrawal or complexation rigorous metal ions of an external solution that It contains such liposomes.

This invention thus provides a composition. liposome comprising liposomes containing a solution internal, comprising the internal solution copper ions and one

or more encapsulated therapeutic agents, agent that is a drug or an antineoplastic agent; and in which:

(to)

liposomes do not comprise an ionophore;

(b)

said therapeutic agent has been loaded into liposomes preformed containing said copper ions in said internal solution;

(C)

the internal solution comprises a solution compatible with copper ions; Y

(d)

the composition comprises the liposomes in a external solution to the liposomes. Preferably, after encapsulation of the

drug, a liposome of this invention or used in methods of this invention has an extraliposomal pH that is substantially similar to intraliposomal pH. Plus preferably, the extraliposomal and intraliposomal pH is at

approximately pH 3.5 to pH 9.0, more preferably, is between approximately pH 6.0 and pH 8.5, even more preferably, it is between about 6.5 and 8.5, and, more preferably, it is between about pH 6.5 and pH 7.5. The invention also provides a method of loading a therapeutic agent in a liposome in a composition Liposomal, comprising the method:

i) provide a liposomal composition comprising

a liposome in an external solution, containing the

liposome an internal solution comprising copper ions

encapsulated;

ii) add one or more therapeutic agents to the solution

external; Y

iii) keep the agent in the external solution for

long enough to load the agent in the

liposome,

in which the liposomes do not contain an ionophore, and

the internal solution comprises a compatible solution

with copper ions.

Preferably, there will be little or no pH difference. between the inside and outside of the liposome. Plus preferably, the pH will be comparable to the pH of the fluids physiological or an approximately neutral pH. Preferably, the external solution will have less, more preferably substantially less, metal ions. Preferably, the external solution and the surface of the liposomes will be essentially free of metal ions in non-state complexed

Additionally, the present invention provides compositions that are prepared according to this method as well as liposomes containing suitable encapsulated metals for use in this method. In particular, the invention provides a liposomal composition comprising liposomes comprising one or more therapeutic agents produced by the method of the invention.

Also described herein are methods for loading agents into liposomes, which comprise the steps of:

i) prepare a liposome comprising a metal ion

encapsulated transition and

ii) add to the external solution of said liposome a agent so that said agent is encapsulated in the liposome

The transition metal ion can be selected from one

or more of Fe, Co, Ni, Cu, Zn, V, Ti, Cr, Rh, Ru, Mo and Pd and

may

encapsulate in a liposome in he that too be

encapsulates Mn.

Too

be describe in the Present memory

compositions that are prepared according to this method as well as liposomes that contain a transition metal ion or two or more such different ions, suitable for use in the method.

A method is also described herein. to charge liposomes using a metal ion in a solution "Compatible with metal", as described herein memory, to minimize metal precipitation and to keep it in solution for long enough to Prepare the liposome. Thus, it is also described herein. memory a method to load an agent in a liposome, said method comprising the steps of:

i) prepare a liposome that has an encapsulated medium comprising a metal ion and a compatible solution with the metal; Y ii) add to the external solution of said liposome a agent so that said agent is encapsulated in the liposome Additionally, they are described herein.

compositions that are prepared according to this method as well as liposomes that contain a metal ion and solutions compatible with metal suitable for use in this method.

This invention is further based on the finding that liposomes prepared to be low cholesterol have unexpected load and retention properties when a metal based charge is used. So, I also know describes herein a method to encapsulate a agent in a liposome, the liposome being present in a

external solution, said methods comprising the steps of:

i) prepare a liposome comprising:

a) one or more lipids that form vesicles, with such

that the liposome is low in cholesterol; Y

b) a metal encapsulated in a compatible solution

with the metal; Y

ii) add an active agent to the external solution

so that the agent is encapsulated in the liposome.

In one case, the solution compatible with metal includes a transition metal.

A method is also described herein. to encapsulate an agent in a liposome, comprising the Method the stages of:

i) provide a liposome of this invention in a

external solution, where the liposome does not have a

pH gradient of transmembrane; Y

ii) add an agent to the external solution so that

The agent is encapsulated in the liposome.

Also described herein are methods for administer liposomes to a mammal and methods to treat a mammal affected by or prone to or suspected of is affected by a disorder (eg cancer). In In particular, a method for administer a liposome to a subject, which comprises administering a pharmaceutical composition comprising Liposomes of the invention. It will be generally understood that treatment or administration methods comprise administer the pharmaceutical composition in a dosage enough to improve said disorder or its symptoms. In a aspect, this invention is based on the finding that liposomes loaded with active agent using a metal encapsulated have load and retention properties that They are different from those presented by manganese.

Also described herein is a liposomal composition comprising a liposome containing an internal solution comprising one or more metal ions encapsulated transition and one or more therapeutic agents, provided that if the liposome has a lipid composition

consisting of sphingomyelin and cholesterol or if the one or more therapeutic agents is only doxorubicin, the one or more Encapsulated ions are not just manganese. This invention also provides the mentioned liposomal composition previously in which the liposomes are in a solution external

A method is also described herein. to load liposomes with an agent, in which the composition Liposomal is a liposomal composition as described previously, comprising the method: select an agent that is able to cross liposome membranes in the composition when present in the external solution of the composition but unable to cross these membranes when is in a complex with one or more metal ions in the internal solution, add the selected agent to the solution external composition, and keep the agent in the external solution for a sufficient time to charge the agent.

This invention also provides methods for prepare, select or design liposomes, which comprise select a metal ion for encapsulation in a liposome to achieve a desired retention of an encapsulated agent in the liposome Thus, a method to select a liposomal composition that has a loading property or preferred retention for an agent according to this invention comprises:

(to)

provide a first liposomal composition that comprises a liposome in an external solution, containing the liposome an internal solution that comprises one or more transition metal ions;

(b)

add the agent to the external solution of the composition of (a) over a period of time to load the agent into the liposome of the first composition;

(C)

provide a second liposomal composition that comprises a liposome in an external solution, containing the liposome an internal solution that comprises one or more transition metal ions;

(d)

add the agent to the external solution of the composition of (c) over a period of time to load the agent into the liposome of the second composition;

(C)

compare the amount of agent charged or the amount of agent retained over time, for the liposome of the resulting composition in (b) with the liposome of the resulting composition in (d); Y

(F)

select the resulting liposomal composition in

(b)

or (d) that has a larger amount of agent loaded

or retained; in which:

(I)

the liposomal compositions of (a) and (c) differ by one or more of: (i) metal ions present in the internal solution; (ii) lipids in the liposome of the liposomal composition; and (iii) the metal ion concentration present in the internal solution;

(II)

the period of time in (b) is different from period of time in (d); or

(III) the load in (b) is carried out at a temperature different than the load in (d).

Brief Description of the Drawings

FIGURE 1A: A graph showing the load of Irinotecan in DSPC / DSPG liposomes (80:20 molar ratio) as a function of time using 100 mM Cu (II) gluconate buffered to pH 7.4 with triethanolamine (ASD) as the medium 300 mM internal and sucrose, 20 mM HEPES, 30 mM EDTA (SHE), pH 7.4, as the external medium. Charging was carried out at 50 ° C at a molar ratio of drug to lipid of 0.1: 1.

FIGURE 1B: A graph showing the load of daunorubicin in DSPC / DSPG liposomes (molar ratio 90:10) as a function of time using 150 mM CuSO4, 20 mM histidine adjusted to pH 7.4 with ASD as the medium internal and SHE, pH 7.4, as the external medium. The load is conducted at 60 ° C at a molar ratio of drug to lipid 0.1: 1

FIGURE 2: A graph showing the load of irinotecan in DPPC / Col liposomes (55:45 molar ratio) as a Time function using 100 mM Cu (II) gluconate adjusted up to pH 7.4 with ASD as the internal medium and SHE, pH 7.4, As the external medium. Charging was carried out at 50 ° C at a Molar ratio of drug to lipid of 0.1: 1.

FIGURE 3: A graph showing the load of epirubicin in liposomes of DSPC / DSPE-PEG2000 (ratio 95: 5 molar) as a function of time using 300 mM MnSO4, 20 mM imidazole, pH 7.4, as the internal medium and SHE, pH 7.4, As the external medium. Charging was carried out at 60 ° C at a Molar ratio of drug to lipid of approximately 0.2: 1.

FIGURE

4A: A graph that show the load from

irinotecan

in liposomes from DSPC / DSPG that they contain

floxuridine

(FUDR) in a relationship cool 85:15 how a

Time function using 100 mM Cu (II) gluconate, TEA 220 mM, pH 7.4, as the internal medium and 300 mM sucrose, HEPES 20 mM, pH 7.4, as the external solution. The FUDR was encapsulated passively and irinotecan loading was carried out at 50 ° C at a molar ratio of drug to lipid of 0.1: 1.

FIGURE 4B: A graph showing irinotecan loading in DSPC / Col / DSPG liposomes (70:10:20 molar ratio) containing FUDR as a function of time using 100 mM Cu (II) gluconate, 220 mM ASD, pH 7.4, as the internal medium and either 20 mM HEPES, either 150 mM NaCl (HBS), pH 7.4, (�) or 300 mM sucrose, 20 mM HEPES, pH 7.4, (�) as The external buffer. The FUDR was passively encapsulated and the irinotecan loading was carried out at 50 ° C at a drug-to-lipid molar ratio of 0.1: 1.

FIGURE 5: A graph showing the load of irinotecan in DSPC / DSPG liposomes (80:20 molar ratio) that contain carboplatin as a function of time using 150 mM CuSO4 adjusted to pH 7.4 with ASD as the medium internal and SHE, pH 7.4, as the external buffer. He carboplatin was passively encapsulated and the burden of irinotecan was carried out at 60 ° C at a molar ratio of 0.1: l lipid drug.

FIGURE 6: A graph showing the load of

daunorubicin in DSPC / Col liposomes (molar ratio 55:45) containing cisplatin as a function of time using 150 mM CuCl2 adjusted to pH 7.4 with NaOH as the internal medium and HBS, pH 7.4, as the external medium. He cisplatin was passively encapsulated and the burden of daunorubicin was carried out at 60 ° C at a molar ratio of 0.1: 1 lipid drug.

FIGURE 7: A graph showing the loading of doxorubicin in DPPC / DSPE-PEG2000 liposomes (95: 5 molar ratio) as a function of time using 300 mM MnSO4 (�) or 300 mM citrate, pH 3.5, (� ) as the internal environment. Doxorubicin loading was carried out at drug-to-lipid weight ratios of 0.1: 1 (Panel A), 0.2: 1 (Panel B)

or 0.3: 1 (Panel C) at 37 ° C. The data points represent the average of three repeated experiments and error bars They represent the standard deviation.

FIGURE 8A: A graph showing the loading of doxorubicin in DMPC / Col liposomes (55:45 molar ratio) as a function of time using 300 mM MnSO4 (�), 300 mM citrate, pH 3.5, (�) or MnCl2 300 mM (�). Doxorubicin was loaded at a molar ratio of drug to lipid of 0.2: 1 at 60 ° C. The data points represent the average of three repeated experiments and the error bars represent the standard deviation.

FIGURE 8B: A histogram showing pH gradients of transmembrane measured before and after loading doxorubicin under various conditions. The samples include those based on the citrate loading method before of (column 1) and after loading doxorubicin (column 2 the loading method of MnSO4 before (column 3) and after loading doxorubicin (column 4); and the method loading of MnCl2 before (column 5) and after loading of doxorubicin (column 6). The results represent the average pH gradient of three separate experiments and the Error bars indicate the standard deviation.

FIGURE 9: A graph showing the load of irinotecan in DSPC / DSPE-PEG2000 liposomes (95: 5 molar ratio) using either 300 mM MnSO4 (�) or 300 mM CuSO4 (�) as

The internal charging medium. The irinotecan was charged at 60 ° C at a molar ratio of drug to lipid of 0.1: 1.

FIGURE 10A: A graph showing the load of daunorubicin in DSPC / DSPE-PEG2000 liposomes (95: 5 molar ratio) as a function of time using 300 mM MnSO4 as the internal medium. Charging was carried out at 23 ° C (�), 37 ° C (�) and 60 ° C (�) at an initial weight to drug-to-lipid ratio of 0.1: 1.

FIGURE 10B: A graph showing the load of daunorubicin in DSPC / DSPE-PEG2000 liposomes (95: 5 molar ratio) as a function of time using 150 mM CoCl2, as the internal medium. Charging was carried out at 23 ° C (�), 37 ° C (�) and 60 ° C (�) at a molar ratio of drug to lipid of 0.1: 1.

FIGURE 10C: A graph showing the load of daunorubicin in liposomes of DSPC / DSPE-PEG2000 (ratio 95: 5 molar) as a function of time using 300 mM NiSO4 As the internal environment. Charging was carried out at 60 ° C at a Molar ratio of drug to lipid of 0.2: 1.

FIGURE 11: A graph showing the load of epirubicin in liposomes of DSPC / DSPE-PEG2000 (ratio 95: 5 molar) as a function of time using 300 mM CuSO4 at 60 ° C The epirubicin was loaded to reach a relationship in 0.2: 1 drug to lipid weight.

FIGURE 12A: A graph showing the load of doxorubicin in DSPC / Col liposomes (55:45 molar ratio) as a function of time using 300 mM CoCl2 as the medium internal and SHE, pH 7.5, as the external buffer. The load is

I wear

to cape to 60 ° C to a relationship in weight from drug to

0.1: 1 lipid.

FIGURE

12B: A graph that sample the load from

daunorubicin in DSPC / Col liposomes (55:45 molar ratio) as a function of time using 300 mM CuSO4 as the internal medium and HBS, pH 7.4, as the external buffer. The daunorubicin was charged at 60 ° C at a weight ratio of drug to lipid of 0.1: 1 (�), 0.2: 1 (�) and 0.4: 1 (�).

FIGURE 12C: A graph showing the load of Topotecan in liposomes of DSPC / DSPE-PEG (95: 5 molar ratio)

as a function of time at 37 ° C. A solution of CuSO4 300 It was used as the internal charging medium. The topotecan was loaded at a molar ratio of drug to lipid of 0.1: 1.

FIGURE 13A: A graph showing the load of daunorubicin in liposomes of DSPC / DSPE-PEG2000 (ratio 95: 5 molar) containing cisplatin as a function of time using 150 mM MnCl, as the internal medium, and HBS, pH 7.4, as the external solution. The cisplatin was encapsulated passively and daunorubicin loading was carried out at 60 ° C at a drug to lipid ratio of 0.1: 1.

FIGURE 13B: A graph showing the load of daunorubicin in DMPC / Col liposomes (molar ratio 55:45) containing cisplatin as a function of time using 150 mM CuCl2 as the internal medium and HBS, pH 7.4, as The external solution. The cisplatin was passively encapsulated and

the

load from daunorubicin be I wear to cape to 60 ° C to a

cool relationship

r from drug to lip C of 0.1: 1.

FIGURE

13C: A graph that sample the load from

daunorubicin in DPPC / Col liposomes (molar ratio 55:45) containing carboplatin as a function of time using 300 mM NiSO4 as the internal medium and 300 mM sucrose, 20 mM HEPES, pH 7.4, as the external solution. He carboplatin was passively encapsulated and the burden of daunorubicin was carried out at 37 ° C at a molar ratio of 0.1: 1 lipid drug.

FIGURE 13D: A graph showing the load of Irinotecan in DPPC / Col liposomes (55:45 molar ratio) containing cisplatin as a function of time using 75 mM CuCl2 + 150 mM CuSO4 as the internal medium and SHE, pH 7.4, as the external solution. The cisplatin was encapsulated passively and irinotecan loading was carried out at 60 ° C at a drug to lipid ratio of 0.1: 1.

FIGURE 14: A graph showing the ratios of vincristine / lipid and doxorubicin / lipid at various time points during vincristine loading at 50 ° C in DSPC / Col liposomes (55:45 molar ratio) preloaded with doxorubicin. Liposomes containing 300 mM MnSO4 were preloaded with doxorubicin (�) at 50 ° C at a drug-to-lipid ratio of 0.2: 1 w / w. The vincristine charge (�) was carried out with the aid of the A23187 ionophore at a drug-to-lipid ratio of 0.05: 1 w / w. Error bars represent the standard deviation between three repeated experiments.

FIGURE 15: A histogram showing the sequential load with irinotecan and doxorubicin metal in liposomes of DSPC / Col (55:45 molar ratio) containing 300 mM CuSO4 As the internal environment. Liposomes were preloaded with irinotecan at 60 ° C at a molar ratio of drug to lipid of 0.2: 1 to about 100% followed by encapsulation of doxorubicin, charged to a drug / lipid molar ratio of 0.15: 1. As a control, the uptake was measured separately liposomal of each drug in loaded liposomes individually. Error bars represent the standard deviation between three repeated experiments.

FIGURE 16: A histogram showing the relationship of DSPC / DSPE-PEG2000 liposome plasma lipid drug (95: 5 molar ratio) containing daunorubicin 24 hours after intravenous administration to Balb / c mice. The daunorubicin was loaded at a drug-to-lipid ratio of 0.1: 1 at 60 ° C in liposomes that well comprised 300 mM CuSO4; well 150 mM citrate, pH 4; or MnSO4 300 mM as the medium

internal.

The bars from error represent the deviation

standard among three repeated experiments.

FIGURE

17: A graph that sample the load from

Irinotecan in DSPC / DSPG liposomes (80:20 molar ratio) in response to encapsulated CuSO4 after passage of the liposomes through a Chelex-100 ™ column equilibrated with 150 mM NaCl. The liposomes were subsequently exchanged in 300 mM sucrose, 20 mM HEPES, pH 7.4. Charging was carried out by incubation at 37 ° C (�), 50 ° C (�) and 60 ° C (�).

FIGURE 18: A graph showing the load of irinotecan in DSPC / DSPG liposomes (80:20 molar ratio) in response to CuSO4 encapsulated at 37 ° C (�), 50 ° C (�) and 60 ° C (�) . The external liposome solution was subjected to buffer exchange for saline and was further exchanged for 300 mM sucrose, 20 mM HEPES,

pH 7.4 (without external EDTA) before loading.

FIGURE 19: A graph showing the load of irinotecan in DSPC / DSPG liposomes (80:20 molar ratio) in response to encapsulated copper gluconate after buffer exchange of the external solution for 300 mM sucrose, 20 mM HEPES, EDTA 30 mM, pH 7.4. Charging was carried out by incubation at 37 ° C (�), 50 ° C (�) and 60 ° C (�).

FIGURE 20: A graph showing the load of irinotecan in DSPC / DSPG liposomes (80:20 molar ratio) in response to encapsulated copper gluconate after the passage of liposomal preparations through a Chelex-100 ™ column balanced with sucrose 300 mM, 20 mM HEPES, pH 7.4. Irinotecan loading was carried out by incubation at 37 ° C (�), 50 ° C (�) and 60 ° C (�).

FIGURE 21: A graph showing plasma lipid levels of DSPC / DSPG liposomes (80:20 molar ratio), DSPC / SM / DSPG (75: 5: 20 molar ratio) and DSPC / SM / DSPG (70 molar ratio : 10: 20) co-charged with daunorubicin and carboplatin represented by �, � and �, respectively. Carboplatin was passively trapped and daunorubicin was actively charged in response to encapsulated CuSO4.

FIGURE 22: A graph showing plasma daunorubicin levels of DSPC / DSPG liposomes (80:20 molar ratio), DSPC / SM / DSPG (75: 5: 20 molar ratio) and DSPC / SM / DSPG (70 molar ratio : 10: 20) co-charged with daunorubicin and carboplatin represented by �, � and �, respectively. Carboplatin was passively trapped and daunorubicin was actively charged in response to encapsulated CuSO4.

FIGURE 23: A graph showing plasma carboplatin levels of DSPC / DSPG liposomes (80:20 molar ratio), DSPC / SM / DSPG (75: 5: 20 molar ratio) and DSPC / SM / DSPG (70 molar ratio : 10: 20) co-charged with daunorubicin and carboplatin represented by �, � and �, respectively. Carboplatin was passively trapped and daunorubicin was actively charged in response to encapsulated CuSO4.

Detailed description of the invention

Liposome Preparation

The term "liposome," as used herein. memory, means vesicles comprised by one or more concentricly ordered lipid bilayers that encapsulate an aqueous phase The formation of such vesicles requires the presence "vesicle-forming lipids" that are lipids amphipathic capable of either forming or incorporating into a bilayer structure. The last term includes lipids they are able to form a bilayer by themselves or when They are in combination with another lipid or lipids. A lipid amphipathic is incorporated into a lipid bilayer by having its hydrophobic moiety in contact with the inner hydrophobic region of the membrane bilayer and its polar head rest oriented towards an outer polar surface of the membrane. Hydrophilicity arises from the presence of functional groups such as hydroxyl, phosphate, carboxyl, sulfate groups, amino or sulfhydryl. Hydrophobia results from the presence of a long chain of aliphatic hydrocarbon groups.

It will be appreciated that any vesicle-forming lipid suitable can be used in the practice of this invention, to trial of an expert in the specialty. This includes phospholipids such as phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), acid phosphatidic (PA), phosphatidylethanolamine (PE) and phosphatidylserine (PS); glycolipids; and sphingolipids such such as sphingosine, ceramides, sphingomyelin, and glycosphingolipids (such as cerebrosides and gangliosides). Preferred phospholipids comprise two acyl chains from 6 to 24 independently selected carbon atoms each other and with varying degrees of unsaturation.

Liposomes prepared according to this invention can be generated by conventional techniques used to Prepare vesicles. These techniques include the method of ether injection (Deamer et al., Acad. Sci. (1978) 308: 250), the surfactant method (Brunner et al., Biochim. Biophys Acta (1976) 455: 322), the method of freezing- defrosting (Pick et al., Arch. Biochim. Biophys. (1981)

212: 186), the reverse phase evaporation method (Szoka et al., Biochim. Biophys Minutes (1980) 601: 559-71), the method of ultrasonic treatment (Huang et al., Biochemistry (1969)

8: 344), the ethanol injection method (Kremer et al., Biochemistry (1977) 16: 3932), the extrusion method (Hope et al., Biochim. Biophys Minutes (1985) 812: 55-65) and the method of the French press (Barenholz et al., FEBS Lett. (1979)

99: 210). All the above procedures are technologies basic for the formation of liposomal vesicles and these Procedures can be used in combinations. Preferably, the small unilamellar vesicles (SUV) are prepared by the method of ultrasonic treatment, the ethanol injection method and the press method French Preferably, multilamellar vesicles (MLV) they are prepared by the reverse phase evaporation method

or by simply adding an aqueous solution to a lipid film followed by dispersion by stirring mechanics (Bangham et al., J. Mol. Biol. (1965) 13: 238-252).

Particularly suitable liposomal preparations that can be used in the practice of this invention are vesicles large unilamellar (LUV). LUVs can be prepared by the ether injection method, the method of surfactant, freeze-thaw method, the reverse phase evaporation method, the press method French or extrusion method. Preferably, the LUVs They are prepared according to the extrusion method. The method extrusion involves first combining lipids in chloroform to give a desired molar ratio. A marker lipid can optionally be added to the preparation lipid The resulting mixture is dried under a stream of gaseous nitrogen and put in a vacuum pump until The solvent is substantially removed. The samples are then hydrate in an appropriate aqueous solution, which It may contain a mixture of therapeutic agent or agents. The mixture is then passed through an apparatus extrusion (eg a Northern Lipids apparatus, Vancouver, Canada) to get liposomes of one size definite. The average size of liposomes can

determined by a variety of methods including quasi-elastic diffusion of light using, for example, a NICOMP ™ 370 submicrometer particle sizing machine a a wavelength of 632.8 nm.

In some aspects of this invention, liposomes are They prepare to be "low cholesterol." Such liposomes "substantially contain no cholesterol" or "They essentially contain no cholesterol." The term "Substantially without cholesterol" allows the presence of a amount of cholesterol that is insufficient to alter significantly phase transition characteristics of the liposome (typically less than 20 mol% of cholesterol). 20 mol% or more of cholesterol expands the temperature range at which the transition occurs phase, disappearing phase transition to levels of higher cholesterol (eg more than 30 mol%). Preferably, a liposome that does not have substantially cholesterol will be about 15 or less and more preferably about 10 mol% or less of cholesterol. The term "essentially cholesterol free" means about 5 mol% or less, preferably about 2 mol% or less and even more preferably about 1 mol% or less of cholesterol. Most preferably, it will not be present or will add cholesterol when liposomes are prepared “with low cholesterol content. "

The liposomes of this invention may comprise a hydrophilic-lipid polymer conjugate such as a conjugate polyalkyl ether lipid. The graft of a hydrophilic polymer such as a polyalkyl ether in the liposome surface to "sterically stabilize" the liposomes, thereby increasing longevity in Liposome circulation. This results in a increased blood stability and circulation time increased, reduced uptake in healthy tissues and a increased contribution to diseased areas such as tumors solids (see: U.S. Patents 5013556 and 5593622; and Patel et al., Crit Rev Ther Drug Carrier Syst (1992) 9: 39

90). Typically, the polymer is conjugated to a component

lipid

of the liposome He finished "conjugate polymer

hydrophilic-lipid ”

be refer to a lipid trainer from

vesicles

connected covalently in its rest from head

polar to a hydrophilic polymer, and is typically made to from a lipid that has a reactive functional group in the rest of the polar head to bind to the polymer. Groups Suitable reactive functionalities are, for example, groups amino, hydroxyl, carboxyl or formyl. The lipid can be any lipid described in the specialty for use in such conjugates. Preferably, the lipid is a phospholipid having two acyl chains comprising between about 6 and about 24 atoms of Long carbon with varying degrees of unsaturation. The more preferably, the lipid in the conjugate is a PE, preferably in a distearoyl manner. The polymer is a biocompatible polymer characterized by a solubility in water that allows polymer chains to extend effectively beyond the surface of the liposome with sufficient flexibility for coverage to occur uniform surface of a liposome. Preferably such polymer is a polyalkyl ether, including polyethylene glycol (PEG), polymethylene glycol, polyhydroxypropylene glycol, polypropylene glycol, poly (lactic acid), poly (acid glycolic), poly (acrylic acid) and their copolymers, as well as those disclosed in US Pat. UU. 5,013,556 and

5,395,619. Preferably, such a polymer has a weight molecular between approximately 350 and 5000 daltons. He conjugate can be prepared to include a releasable lipid polymer bond such as a peptide bond, ester or disulfide The conjugate may also include a ligand of orientation. Conjugate mixtures can be incorporated into Liposomes for use in this invention.

Lipids with negative electrical charge that described below can be incorporated into formulations liposomes that encapsulate metals to increase Longevity in circulation of the vehicle. These lipids can be used instead of hydrophilic polymer conjugates

lipid as surface stabilizing agents. Embodiments of this invention may make use of liposomes. cholesterol-free containing such charged lipids negative electrical to avoid aggregation, increasing in that way the time of permanence in blood of the vehicle. Such embodiments are ideally loaded after the rigorous removal of metal ions from the surface of the Liposome and the external solution of liposomes.

The term "lipid with negative electrical charge" is refers to a vesicle-forming lipid that has one or more negative electrical charges at physiological pH, including phospholipids and sphingolipids. Loaded lipids Negative electrical can be incorporated into a liposome of this invention in 5 to 95 mol%, more preferably in 10 to 50 mol% and most preferably 15-30 mol%.

Preferably, a lipid having an electric charge pH negative for use in this invention will comprise a "non-zwitterionic moiety", which refers to a rest that has no electric charges opposite pH physiological. Such lipids impart liposome properties Circulation desirable for in vivo uses. Load Negative electrical net on the lipid may arise only from the presence of the negative electric charge on the lipid (eg of a phosphate group) or, when the lipid has more than one electric charge, the electric charge Additional refusal may be due to the presence of a remainder non-zwitterionic with negative electrical charge. Preferably, however, the negative electrical charge arises only from the lipid component, in which case the Non-zwitterionic moiety is a neutral group. Preferably, the non-zwitterionic moiety comprises 2 to 6 carbon atoms.

Suitable non-zwitterionic residues contain groups eager functional electrons that impart to the group of Hydrophilic head features. Such functional groups can be selected from the group consisting of alcohols, acids, ketones, esters, ethers, amides and aldehydes. They can non-zwitterionic residues of the following should be used formulas:

Alcohols

P-R or POR or PO (CH2) 2NHR where R is - (CH2) v (CH) w (C) x (OH) and (CH3) z where the number of carbons (v + w + x + z) is 2-6, the most preferably 3-5 where the number of OH groups is 1-3 (y = 1-3), p. ex. DPPG

Ketones

P-R or POR or PO (CH2) 2NHR where R is - (CH2) v (C) x (CO) and (CH3) z where the number of carbons (v + x + y + z) is 2-6, the most preferably 3-5 where the number of ketone groups is 1-2 (y = 1-2), p. ex. N-Butyryl-DPPE, N-Valeryl-DPPE

Carboxylic acids

P-R or POR or PO (CH2) 2NHR where R is - (CH2) or (CH) v (C) x (COOH) and (CH3) z where the number of carbons (u + v + x + y + z) is 2-6, the most preferably 3-5 where the number of carboxylic acid groups is 1-2 (y = 1-2)

Esters

P-R or POR or PO (CH2) 2NHR where R is - (CH2) v (C) x (COO) and (CH3) z where the number of carbons (v + x + y + z) is 2-6, the most preferably 3-5 where the number of ester groups is 1-2 (y = 1-2)

Ethers

P-R or POR or PO (CH2) 2NHR where R is - (CH2) v (C) x (O) and (CH3) z where the number of carbons (v + x + z) is 2-6, the most preferably 3-5 where the number of ether groups is 1-2 (y = 1-2)

Amines

Primary Amines: P-R or POR or PO (CH2) 2NHR where R is - (CH2) v (C) w (CH) x (NH3) and (CH3) z -where the number of carbons (v + w + x + z) is 2-6, the most

preferably 3-5

-where the number of amino groups is 1-2 (y = 1-2) Secondary Amines: P-R or POR or PO (CH2) 2NHR where R is - (CH2) v (C) w (CH) x (NHx) and (CH3) z -where the number of carbons (v + w + x + z) is 2-6, the most

preferably 3-5 -where the number of amine groups is 1-2 (y = 1-2) Tertiary Amines: P-R or POR or PO (CH2) 2NHR where R is - (CH2) v (CH) w (C) x (N) y (CH3) z

-

where he number from carbons (v + w + x + z) is 2-6, the plus

preferably 3-5

-

where the number of amine groups is 1 (y = 1)

He

rest no zwitterionic too may to be

comprised of combinations of functional groups; by example a compound of formula:

Carboxylic Acids and Ketones

P-R or POR or PO (CH2) 2NHR where R is

- (CH2) u (CH) v (C) w (COOH) x (CO) y (CH3) z where the number of carbons (u + v + w + x + y + z) is 2-6, the most preferably 3-5 where the number of carboxylic acid groups is 1-2 (x = 1-2) where the number of ketone groups is 1-2 (y = 1-2), p. ex. N-succinyl-DPPE, N-glutaryl-DPPE

P-R or POR or PO (CH2) 2NHR where R is - (CH2) s (CH) t (C) or (COOH) v (CO) x (OH) and (CH3) z

Carboxylic Acids, Ketones and Alcohols

where the number of carbons (s + t + u + v + x + z) is 2-6, the most preferably 3-5 where the number of carboxylic acid groups is 1-2 (v = 1-2) where the ketone group number is 1-2 (x = 1-2) where the number of hydroxyl groups is 1-2 (y = 1-2), p. ex. N-tartaryl-DPPE

Annular Structures

P-R or POR or PO (CH2) 2NHR where R is a 5 or 6 member ring

which contains 1-5 or 1-6 alcohol groups (cyclitols), respectively (eg phosphatidylinositol).

Carbohydrates

Monosaccharides that can be used in the practice of this invention include arabinose, fucose, galactose, glucose, lyxose, ribose and xylose. Disaccharides include sucrose, lactose, trehalose, cellobiose, gentiobiosa and maltose. With the in order to prolong the circulating life of the liposome, it they prefer monosaccharides and disaccharides that do not bind to cell receptors (eg mannose).

In case the non-zwitterionic residue is neutral, the head group consists of groups that are neutral at pH physiological, including alcohols, ketones, esters, ethers, Amides and aldehydes.

In preferred embodiments of the invention, the remainder does not Zwitterionic is a short chain alcohol, containing a Preferred alcohol two or more hydroxyl groups. The alcohol it can be a linear chain polyol of which it is an example Glycerol Glycerol may constitute the head group of a phosphosphingolipid or a phospholipid through the bond from one of the hydroxyl groups to the lipid phosphate group. Most preferably, glycerol is linked to phosphate a through a terminal hydroxyl group of the molecule of glycerol, called the resulting molecule phosphatidylglycerol (PG). Preferably, the chains of Phosphatidylglycerol fatty acid are selected independently of each other from the group consisting of caproyl (6: 0), octanoyl (8: 0), capril (10: 0), lauroyl (12: 0), myristoyl (14: 0), palmitoyl (16: 0), stearoyl (18: 0), arachidido (20: 0), behenoyl (22: 0), lingnoceroyl

(24: 0) and phyanoyl, including unsaturated versions of these fatty acid chains in cis configurations or trans, such as oleoyl (18: 1), linoleoyl (18: 2), arachidonoyl (20: 4) and docosahexaenoyl (22: 6). They prefer phospholipids that have two acyl chains from 14 to 18 carbon atoms

In another preferred embodiment of the invention, the rest

Non-zwitterionic is an annular structure. The most preferably, the annular structure is a cyclitol, which is a cycloalkane containing a hydroxyl group in each of Three or more ring atoms. Such compounds can derive with various groups to impart to the molecule a desired water solubility. Preferably, the cyclitol is an inositol linked to a phospholipid through the group phosphate, the resulting compound being phosphatidylinositol (PI). Preferably, the fatty acid chains of the phosphatidylinositol are independently selected from each other from the group consisting of caproyl (6: 0), octanoyl (8: 0), goat (10: 0), lauroyl (12: 0), myristoyl (14: 0), palmitoyl (16: 0), stearoyl (18: 0), arachidoyl (20: 0), behenoyl (22: 0), lingnoceroyl (24: 0) and phyanoyl, including unsaturated versions of these chains of fatty acid in cis or trans configurations, such as oleoyl (18: 1), linoleoyl (18: 2), arachidonic (20: 4) and docosahexaenoyl (22: 6). Phosphatidylinositols are preferred which have two acyl chains of 14 to 18 carbon atoms.

Lipids with negative electrical charge can obtained from natural sources or can be synthesized chemically Methods for covalently binding compounds to head group of a lipid are well known in the specialty and usually involve reacting groups functional of the terminal portion of the head group of lipid with functional groups of the rest to be bound. Lipids suitable for chemical ligation of a residue hydrophilic include lipids that have a head group polar ending with a reactive functional group such as an amine or a carboxylic acid. An example of a lipid Particularly suitable is phosphatidylethanolamine, since It contains a reactive amino group. Methods to prepare phosphatidylethanolamine derivatives have been described in Ahl, P., et al. (1997) Biochimica et Biophysica Act 1329: 370-382. Examples of lipids with negative electrical charge obtained from Natural sources include phosphatidyl glycerol and phosphatidylinositol obtained from egg and plant sources, respectively.

Encapsulation of active agents and metals in liposomes

This invention provides a method for charging a agent in a liposome comprising a transition metal encapsulated Within this specification, the term "Agent" refers to substances that are capable of being encapsulated in liposomes according to this invention. Preferably, such an agent will be a "therapeutic agent" able to exert an effect on an objective, in vitro or in alive. Suitable active agents include, for example, prodrugs, diagnostic agents, therapeutic agents, pharmaceutical agents, drugs, organic molecules synthetic, proteins, peptides, vitamins, steroids and steroid analogues. The agent, at least when he is not complexed with a transition metal, it must be permeable to through a liposomal membrane to reach the load.

Transition metals described herein include the elements of Groups 1B, 2B, 3B, 4B, 5B, 6B, 7B and 8B (groups 3-12). Preferred metals include the selected from the group consisting of Fe, Co, Ni, Cu, Zn, V, Ti, Cr, Rh, Ru, Mo, Mn and Pd. More preferably, the metal it is Fe, Co, Ni, Cu, Mn or Zn. Even more preferably, the metal is Zn, Mn, Co or Cu. Even more preferably, the metal is Zn, Co or Cu.

Transition metal ions can be encapsulated in liposomes according to known conventional techniques in the specialty. This includes the techniques of passive encapsulation known in the specialty and described later.

Preferably, the liposomes are formed in a solution comprising a transition metal in a concentration of about 20 mM to about 1 M, preferably from about 50 mM to about 800 mM and more preferably from about 100 to about 350 mM.

Various metal salts can be used in the practice of this invention. Preferably, the salt is pharmaceutically acceptable and soluble in a solvent aqueous. Preferred salts can be selected from the group that

It consists of chlorides, sulfates, tartrates, citrates, phosphates, nitrates, carbonates, acetates, glutamates, gluconates, glycinates, histidinates, lysinates and the like.

Preferably, a therapeutic agent to be encapsulating in a liposome of this invention is one that is able to coordinate with a metal encapsulated in the liposome.

Agents that are able to coordinate with a metal of transition typically comprise coordination sites such as amines, carbonyl groups, ethers, ketones, groups acyl, acetylenes, olefins, thiols, hydroxyl, halides or other suitable groups capable of donating electrons to metal of transition thus forming a complex with the metal. Examples of agents that bind to transition metals include quinolones such as fluoroquinolones, quinolones such as nalidixic acid, anthracyclines such as doxorubicin, daunorubicin idarubicin and epirubicin, aminoglycosides such as kanamycin and other antibiotics such as bleomycin, mitomycin C and tetracycline and mustards nitrogen such as cyclophosphamide, thiosemicarbazone, indomethacin and nitroprusside, camptothecins such as topotecan, irinotecan, lurtotecan, 9-aminocamptothecin, 9-nitrocamptothecin and 10-hydroxycamptothecin and podophyllotoxins such as etoposide. The agents used in this invention may be able to donate electrons of different atoms from the agent and to different sites of the geometric structure of the complex. Such agents able to donate more than a couple of Non-binding electrons are also known as multidented.

Preferably,

a therapeutic agent for use in is

Invention is an antineoplastic agent.

Examples

no limiting from agents assets that be

complex with transition metals are provided in the Table I.

TABLE I EXAMPLES OF METAL-BASED ACTIVE AGENTS

METAL (S) AGENT (S) REFERENCE

Cu etoposide Tawa et al. (1997) Biol. Pharm. Bull. 20: 1002-1005 Fe (III) dexrrazoxane, Hasinoff et al. (1999) Journal of

Inorganic Biochemistry 77: 257-259 losoxantrone, pyroxantrone

Zn, bleomycin Wenbao et al (2001) Biochemistry 40: Cu (II), 7559-7568 Faith (III), Co (III) Fe (III) anthracyclines Fiatlo et al. (1999) Journal of

Inorganic Biochemistry 75: 105-115 Bi (III) quinolones Turel et al. (1997) Journal of Inorganic Biochemistry 66: 241-245 Cu (II) L-lysine Chikira et al. (1997) Journal of Inorganic Biochemistry 66: 131-139

L-arginine Cu (II), desfemoxamine Farkas et al. (1997) Journal of Ni (II), B Inorganic Biochemistry 65: 281-286 Zn (II), Mo (VI) Cu (II) cinnamon Bontchev et al, (1997) Journal of

Inorganic Biochemistry 65: 175

derived from rafamycin

Fe (III) adriamycin Capolongo et al. (1997) Journal of Inorganic Biochemistry 65: 115-122

Cu (II), cinoxacin Ruiz et al. (1997) Journal of Ni (II) Inorganic Biochemistry 65: 87-96

Methods to determine if coordination occurs between

a

agent Y  a metal from transition include techniques

conventional

good known by the experts in the

specialty.

Techniques preferred imply to size the

absorption spectra or use NMR as described by Greenaway and Dabrowiak (J. Inorg. Biochem. (1982) 16 (2): 91). If desired, an active agent can be tested before the encapsulation to determine if coordination occurs and the Optimum pH for complexation.

A preferred technique for preparing liposomes with a encapsulated metal involves combining lipid first in chloroform to give a desired molar ratio. A marker lipid can optionally be added to the preparation lipid The resulting mixture is dried under a stream of gaseous nitrogen and put in a vacuum pump until The solvent is removed. Subsequently, the samples are hydrate in a solution comprising a transition metal (which can comprise more than one metal, for example Cu and Mn, or a metal, but different salts of the metal). The mixture is made then pass through an extrusion apparatus to obtain a liposome preparation of a defined size. The average size of the liposomes can be determined by quasi-elastic diffusion of light using a NICOMP ™ 370 submicrometer particle sizing machine a a wavelength of 632.8 nm. Subsequently to the extrusion, the external solution can be treated or replaced in order to remove metal ions from the external solution and the surface of the liposomes.

This invention preferably makes use of liposomes with an encapsulated or "internal" medium comprising a metal of transition into a "solution compatible with metal". The use of a solution compatible with metal prevents metal precipitation or minimizes precipitation up to enough point to allow the pharmaceutical use of liposomes

A solution compatible with metal is defined as a which consists of a metal in solution that does not make it produce unacceptable precipitation for at least the time required to formulate liposomes.

Preferably, the metal solution should be transparent and soluble, free of aggregation, precipitation or flocculation for at least about 4 hours. As an example, a 300 mM solution of MnSO4 in pH 7.4 HEPES buffer as described in Cheung, et al. [previously] is not a solution compatible with metal since it produces a obvious brown precipitate of Mn (OH) 2 comprising approximately 6-7% molar manganese added to the solution.

Various methods are known in the art, and may be used to determine if the metal solution is forming a precipitate, such as centrifugation of the solution and an evaluation of whether a pellet is formed or an observation of turbidity in the solution. The absorbance of the solution it can also be verified by spectroscopy (e.g. increase in absorbance at 690 nm), where an increase substantial absorbance is indicative of instability of the solution and precipitation. The simplest method is filter the solution and look for the presence of a precipitate on the filter For example, a 50 ml sample can impersonate through Whatman (tm) No. 2 filter paper and the filter be observed with respect to a visible sediment.

A preferred method to determine if a solution is Compatible with metal is to verify the absorbance at 690 nm. The addition of metal should not result in an increase of more than about 0.1 absorption units and preferably no more than about 0.05 units.

An alternative preferred method to determine if a metal solution is compatible with metal is by centrifugation (e.g., a 100 ml sample at 1000 rpm for 10 minutes) to collect any precipitate, measure the amount of precipitate collected and determine the proportion of the metal added to the original solution present in the precipitate. The amount of metal in the precipitate does not must exceed approximately 1 molar% of the amount of metal added to the original solution.

Solutions compatible with preferred metals are those are also pharmaceutically acceptable, such

such as those comprising triethanolamine (TEA), sodium chloride, sodium acetate / acetic acid, sodium citrate / citric acid or sugars such as sucrose, dextrose and lactose. The phosphate and carbonate based solutions (although pharmaceutically acceptable) will have limited use, except at pH outside normal physiological ranges, due to the probability of precipitation of the metal. Preferably, the metal compatible solution is buffered and has a pH in a physiological range.

In the practice of this invention, it can be advantageous that the external solution of the liposomal preparation is replace or treat so that the resulting external solution does not contain substantially uncomplexed metal ions before loading an agent. For the purposes of this Descriptive report, "uncompleted metal ions" includes free metal ions in the external solution and ions metal bonded to (or otherwise associated with) the outer surface of the liposomes. Conversely, an ion complexed metallic is one that is no longer free to interact with the therapeutic agent or liposome surface due to which it is present in the external solution in a complex with a remainder such as a chelating agent. Thus, it is preferable that the surface of the liposomes and the external solution are substantially free of metal ions or, if they are present metal ions, which are complexed with a chelating agent Examples of cationic chelating agents that may be employed include: EDTA and derivatives; EGTA and derivatives; histidine; Chelex (tm); TPEN and derivatives; BAPTA and derivatives; bisphosphonate; o-phenanthrolene (phenanthroline); citrate; InsP6; Diazo-2; and DTPA isocyanate (acid diethylenetriaminepentaacetic acid).

The replacement of the external solution to remove ions Metallic can be done by several other techniques, such as by chromatography of the preparation liposomal through a gel filtration column intensive balanced with a second aqueous solution buffered, by centrifugation, intensive dialysis or repeated, exchange of the external environment, treatment of

external solution with chelating agents or by techniques related. A single solution exchange or a round of dialysis without the use of a chelating agent is typically insufficient to remove metal ions from the surface of liposomes with negative electrical charge.

The external solution is also preferably a buffered solution However, it is appreciated that any suitable solvent can be used in the practice of this invention. A preferred external solution has a pH at pH approximately physiological and comprises a buffer that has a buffering interval that includes physiological pH. Non-limiting examples of buffers suitable for External solution are HBS, pH 7.4 (150 mM NaCl, 20 mM HEPES) and SHE, pH 7.4 (300 mM sucrose, 20 mM HEPES, 30 mM EDTA).

The recruitment of an agent can be established by incubation of the mixture at a suitable temperature after the addition of the agent to the external medium. Depending on the liposome composition, temperature and pH of the medium internal, and the chemical nature of the agent, the uptake of agent can occur over a period of time of minutes or hours Charging can be carried out at temperatures of, for example, 20 ° C to about 75 ° C, preferably from about 30 ° C to about 600

C.

The removal of non-encapsulated agent can lead to out by passing a liposomal preparation through a gel filtration column balanced with a second buffered aqueous solution, or by centrifugation, dialysis or related techniques. Preferably, the second solution is one that is physiologically compatible but not It needs to be "compatible with metals." After withdrawal of the non-encapsulated active agent, the extent of the burden of agent can be determined by measuring levels of drug and lipid according to conventional techniques. Lipid and drug concentrations can be determined. employing techniques such as scintillation counting, testing spectrophotometric, fluorescent assays and chromatography High resolution liquid. The choice of analysis depends

of the nature of the drug and if the liposomes contain a

marker

lipid Radiolabeling A example from

quantification

using a marker radiolabeling be

indicates

in the Examples from the present memory, though be

appreciate that any suitable method can be used to Determine the extent of the load.

Before loading an agent in a liposome using a encapsulated transition metal, the liposome can co-encapsulate passively with an agent and a metal. Using This system, two or more agents can be incorporated into the Liposome by combining passive and active loading methods.

Subsequently to the loading of an agent in a liposome, an ionophore can be incubated with the mixture so that the insertion of the ionophore into the bilayer occurs. He term "ionophore" refers to a compound that forms a complex with a metal ion and helps the ion to cross the lipid bilayer while also helping to transport H + in the opposite direction. Examples of suitable ionophores for the present invention include nigericin, monensin, dianemycin, A23187, 4-BrA23187, ionomycin and X-537A. The ionophores can be specific for metal ions monovalent or divalent. Examples of specific ionophores for monovalent metal ions include nigericin, monensin and dianemycin. The ionophore uptake is set by adding the ionophore to the mixture and the incubation at a temperature suitable for incorporation from the ionophore to the liposomal bilayer. The amount of ionophore used will typically depend on the nature and type of the liposomal formulation The addition of the ionophore to the liposome After loading the agent can be carried out to subsequently impose a pH gradient across the liposomal bilayer to alter retention properties of the agent in the liposome or to protect agents that are affected by neutral or alkaline environments such as Topotecan and Irinotecan.

Solutions compatible with preferred metals can include components such as buffers that can be used between pH 6.0 and 8.5. Preferably, the buffer does not precipitate

substantially over a period of time of two days at 4 ° C with an encapsulated metal ion at pH 6.0 to 8.0 and more preferably pH 6.5 to 7.5. Capacity can be tested of a buffer to prevent precipitation when inspecting visually the appearance of turbidity in the solution, which is indicative of the formation of a precipitate. An example of a method to determine if a buffer is compatible with a Particular transition metal is outlined in Example 3. After the encapsulation of a transition metal in a solution compatible with metal, an agent can be added to the external medium so that the agent is encapsulated in the liposome Liposomes that encapsulate a transition metal and a solution compatible with metal can be prepared from according to conventional techniques known in the specialty including the techniques described above. However, it is appreciated that any metal can be used in this aspect. Preferably, the liposome with the agent or the encapsulated agents have an extraliposomal pH that is substantially similar to intraliposomal pH. The most preferably, the extraliposomal and intraliposomal pH is approximately pH 6.0 to pH 8.0, most preferably, is between approximately pH 6.5 and pH 7.5.

A method is also described herein. to design liposomes, said method comprising select a metal ion for encapsulation in a liposome to achieve a desired retention of an agent encapsulated It will be appreciated that any liposome and agent Suitable can be used in the practice of this aspect. Other preferred features and conditions of this Aspects are as described in general above.

To determine the release rate of an agent of a liposome, the liposome can be administered intravenously and plasma levels of agent and lipid measured after administration. For example, him lipid component can be radioactively labeled and the Plasma is counted by liquid scintillation. The amount of drug can be determined by assays spectrophotometric, HPLC or others. Similarly, the

test regarding the retention of the agent in the liposome it can be carried out in vitro in plasma or a buffer suitable. By way of example, a liposome comprising a encapsulated transition agent and metal can be tested in vitro or in vivo with respect to agent retention. Yes

no

be achieves a retention desired of the agent, a metal

different can be selected and tested

with respect to its

ability to retain the agent of interest.

Liposome Administration

Also described herein are methods for administer liposomes to a mammal and methods to treat a mammal affected by or prone to or suspected of is affected by a disorder (eg cancer). It will be understood generally that the methods of treatment or of administration will understand administer the composition pharmaceutical in a dosage sufficient to improve said disorder or its symptoms.

For the treatment of human ailments, you can expect a qualified doctor to determine how they should the compositions of the present invention be used with regarding dose, schedule and route of administration using established protocols. Such applications too they can use a dose scale increase if the active agents encapsulated in vehicle compositions of contribution of the present invention exhibit reduced toxicity for the healthy tissues of the subject.

Preferably, the pharmaceutical compositions are administer parenterally, that is, intraarterially, intravenously, intraperitoneally, subcutaneously or intramuscularly or through a spray. Methods of aerosol administration include administration intranasal and pulmonary. More preferably, the compositions pharmaceuticals are administered intravenously or intraperitoneally by injection or infusion into bolus. For example, see Rahman et al., Pat. USA UU. No. 3,993,754; Sears, Pat. USA UU. No. 4,145,410; Papahadjopoulos et al., Pat. USA UU. No. 4,235,871; Schneider, Pat. USA UU. No.

4,224,179; Lenk et al., Pat. USA UU. No. 4,522,803; and Fountain et al., Pat. USA UU. No. 4,588,578. Particular formulations that are suitable for this use are in Remington’s Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).

EXAMPLES

The

following examples be give with purposes from

illustration

Y no They are from no mode a limitation of the

scope of the invention. TO

no be that be specify other

thing, the pH was adjusted using triethanolamine (TEA) and the Results shown in the drawings are from a single example representative. Examples 3, 4, 5, 7 and 11 are provided for comparative purposes only.

Methods for the preparation of large unilamellar liposomes

The lipids were dissolved in chloroform solution and subsequently dried under a stream of nitrogen gas and put in a vacuum pump to remove the solvent Unless otherwise specified, it added trace levels of 3H-CHE radioactive lipid to quantify lipid during the procedure of formulation. The resulting lipid film was put under high vacuum for a minimum of 2 hours. Lipid film was hydrated in the indicated solution to form vesicles multilamellar (MLV). The resulting preparation was extruded 10 times through polycarbonate filters stacked with a Extrusion apparatus (Lipex Biomembranes, Vancouver, BC) for reach an average liposome size between 80 and 150 nm. All constituent lipids of liposomes are present in% in moles.

Methods for quantifying drug loading

At various time points after the start of the drug loading, aliquots were removed and passed through a rotating column Sephadex G-50 to separate encapsulated free drug. At a specified volume of eluent Triton X-100 or N-octyl-beta-D was added

glucopyranoside (OGP) to solubilize liposomes. After of the addition of detergent, the mixture was heated until turbidity point of the detergent and allowed to cool to room temperature before absorbance measurement or fluorescence Drug concentrations are calculated by comparison with a standard curve. The lipid levels were measured by scintillation counting of liquid

Example 1

Charging with metal can occur in the absence of a

pH gradient

The ability to load drug from liposomes containing metal with internal solutions and external buffered to pH 7.4. These studies were performed to determine if the drug-based metal load could occur regardless of the presence of a gradient pH Conventional techniques to actively load Liposome drugs often require the presence of a pH gradient of transmembrane.

To determine if copper charging could occur of irinotecan in the absence of a pH gradient using a Cholesterol-free formulation, DSPC / DSPG liposomes (80:20 molar ratio) containing copper (II) gluconate they were prepared with an external and internal pH of 7.4. Films lipids of DSPC / DSPG at a molar ratio of 80:20 se prepared as described above in the section of methods The lipid films were hydrated in gluconate 100 mM Cu (II) adjusted to pH 7.4 with triethanolamine (TEA) and extruded at 70 ° C. The liposomes underwent 300 mM sucrose buffer exchange, 20 mM HEPES, EDTA 30 mM (SHE buffer), pH 7.4, by flow dialysis tangentially and subsequently washed three times in 6 ml SHE, pH 7.4, to remove any copper (II) gluconate of the extraliposomal solution. Irinotecan was added to the liposomal preparation at a molar ratio of drug to 0.1: 1 lipid and incubated at 50 ° C. The extent of the Drug loading was determined as described in the methods

when measuring absorbance at 370 nm and lipid levels are determined by liquid scintillation counting.

The results represented in Figure 1A show that the load of irinotecan in liposomes of DSPC / DSPG

(relationship

cool 80:20) without gradient from pH to 50 ° C was

essentially

complete approximately to the 5 minutes

After the beginning of the charge.

The daunorubicin load was also investigated in DSPC / DSPG liposomes (90:10 molar ratio) containing Encapsulated CuSO4 buffered to pH 7.4. They prepared lipid films according to the methods, except that DSPG was dissolved in chloroform / methanol / water (50: 10: 1 v / v). A solution of 150 mM CuSO4, 20 mM histidine (adjusted to pH 7.4 using ASD) was used as the hydration medium and MLV were extruded at 70 ° C. The liposomes underwent SHE exchange, pH 7.4, using a dialysis column of manual tangential flow. Daunorubicin was charged to a drug / lipid weight ratio of 0.1: 1. One was determined drug to lipid ratio at various time points during loading when measuring absorbance at 480 nm after solubilization in detergent to quantify the daunorubicin as described; lipid levels are determined by liquid scintillation counting.

As summarized in Figure 1B, the uptake of daunorubicin in DSPC / DSPG liposomes (molar ratio 90:10) in the absence of a pH gradient was 100% in all the measured time points.

Charging with irinotecan copper in liposomes that contain cholesterol that do not exhibit pH gradient is investigated using DPPC / Col liposomes (molar ratio 55:45). Liposomes were prepared as described in the methods when hydrating lipid films in a solution 100 mM copper (II) gluconate adjusted to pH 7.4 with TORCH. The liposomes were extruded at 65 ° C and the external buffer of the liposomes was exchanged for SHE, pH 7.4, by tangential flow dialysis. Liposomes were incubated with irinotecan at a weight ratio of drug to lipid of 0.1: 1 at 50 ° C and the extent of drug loading is

determined as described by measuring absorbance at 370 nm after solubilization by detergent.

The irinotecan load in DPPC / Col liposomes (55:45 molar ratio) in the absence of a pH gradient revealed that an almost complete load was observed after approximately 60 minutes incubation (Figure 2).

In addition to charging with copper, the drug loading using liposomes containing MnSO4 in absence of a pH gradient. Liposomes of DSPC / DSPE-PEG2000 (95: 5 molar ratio) with a solution of Internal MnSO4 up to pH 7.4 and a buffered external solution up to pH 7.4 with SHE. Lipid films were prepared according to described and hydrated in 300 mM MnSO4 buffered until pH 7.4 with 20 mM imidazole (the initial pH was adjusted to 7.4 with concentrated HCl) and extrusion was carried out at 70 ° C. The samples were passed down a column Sephadex G-50 to exchange the outer buffer for SHE, pH 7.4. Epirubicin was loaded at a molar ratio of drug at 0.2: 1 lipid and the loading was carried out at 60 ° C. The Drug load extent was measured as described in the methods when measuring the absorbance of drug at 480 nm.

The results summarized in Figure 3 reveal that the loading with epirubicin manganese in liposomes of DSPC / DSPE-PEG2000 (95: 5 molar ratio) at 60 ° C does not require presence of a pH gradient since encapsulation Effective drug was produced at each measured time point.

Example 2 Metal loading of a second drug in buffered liposomes containing a first passively encapsulated drug

Although the examples above describe the load Metal-induced drug in liposomes, the technique can be used to load two or more drugs in a single liposome One technique involves first catching passively at least one drug along with a metal during the liposome preparation followed by active loading with metal of another drug. In this example, the liposomes are they prepared so that there was no pH gradient through

the membrane, thus ensuring the loading of the second drug by the process of this invention.

The irinotecan load in DSPC / DSPG liposomes and DSPC / Col / DSPG, containing encapsulated floxuridine (FUDR) passively, it was investigated using various conditions as well such as irinotecan loading in liposomes that contain carboplatin and daunorubicin loading in liposomes that They contain cisplatin.

DSPC / DSPG liposomes (molar ratio) 85:15) that contain FUDR when dissolving DSPC in chloroform and DSPG in chloroform / methanol / water (50: 10: 1 v / v). TO then the lipids were combined with each other in a 85:15 molar ratio and were labeled with trace amounts of 14C-CHE. The samples were hydrated in copper gluconate (II) 100 mM, 220 mM TEA, pH 7.4, containing 24.62 mg / ml (100 mM) of FUDR with trace levels of 3H-FUDR at 70 ° C. MLV resulting were extruded at 70 ° C, then underwent buffer exchange first by saline solution and then by SHE, pH 7.4, using a manual tangential flow dialysis column. TO This sample was then exchanged in 300 mM sucrose, 20 mM HEPES, pH 7.4, to remove any EDTA from the buffer Exterior.

Irinotecan was added to the liposomal preparation resulting at a molar ratio of drug to lipid of 0.1: 1 at 50 ° C. A drug-to-lipid relationship was generated for the eluent of the rotating column using scintillation counting of liquid to determine lipid concentrations and FUDR, and absorbance at 370 nm to determine Irinotecan concentrations. Before the measurement of the absorbance, liposomes were solubilized in a solution which contained Triton X-100. The initial molar ratio of FUDR lipid drug was 0.09: 1, and 0.06: 1 after it produced the burden of irinotecan.

Figure 4A shows that the irinotecan charge in DSPC / DSPG liposomes (85:15 molar ratio) free of cholesterol containing FUDR and non encapsulated metal requires the presence of a pH gradient since the load

Effective drug was produced throughout the entire experiment.

Liposomes of DSPC / Col / DSPG (70:10:20 molar ratio) that contained FUDR and copper (II) gluconate were prepared as described above. To measure the effects of the buffer external on the load, half of the resulting LUVs are subjected to buffer exchange for SHE, pH 7.4, and at then by 20 mM HEPES, 150 mM NaCl (HBS), pH 7.4, while the other half was additionally exchanged for 300 mM sucrose, 20 mM HEPES, pH 7.4, using a column of manual tangential flow dialysis. Irinotecan was added to liposomes containing FUDR, and subsequently measured, as described above. The relationships Initial FUDR to lipid drug molars were 0.1: 1 and 0.09: 1 for samples containing respectively HBS (closed circles) or 300 mM sucrose, 20 mM HEPES (circles open) as the external buffer. After loading irinotecan, the same samples had drug relationships FUDR / lipid of 0.09: 1 and 0.08: 1, respectively.

The results summarized in Figure 4B show that the irinotecan is effectively loaded in liposomes with low cholesterol content with encapsulated FUDR regardless of the external buffer used. Load on absence of a pH gradient further supports that this degree of irinotecan uptake occurs through the Metal loading technique of this invention.

The present inventors have also shown several other drugs capable of loading with metal in the absence of a pH gradient in liposomes containing drug passively encapsulated; Examples are detailed as follows:

The load of irinotecan in liposomes of DSPC / DSPG (80:20 molar ratio) with encapsulated carboplatin Passively measured using prepared liposomes as described above, except that lipid films were hydrated in 150 mM CuSO4 (adjusted to pH 7.4 using ASD), which contained 25 mg / ml carboplatin. The samples are extruded and external buffers exchanged for SHE, pH 7.4, using a tangential flow dialysis column

manual. Irinotecan 60 ° C was added to a molar ratio of 0.1: 1 lipid drug and uptake was measured as described previously. Absorption spectrometry was used atomic (AA) to determine the concentrations of carboplatin and absorbance at 370 nm was measured to Determine the concentrations of irinotecan. The relationship in Weight of carboplatin drug to initial lipid was 0.030, and 0.025 after the irinotecan charge occurred.

As shown in the graph in Figure 5, the Irinotecan is loaded to a high degree in liposomes of DSPC / DSPG (80:20 molar ratio) containing carboplatin and metal in the absence of a pH gradient.

To measure the load of daunorubicin in liposomes that contain encapsulated cisplatin, liposomes of DSPC / Col (55:45 molar ratio) as described for the Figure 4B, except that the lipid films were hydrated with a cisplatin solution. Solid cisplatin dissolved (40 mg / ml) in 150 mM CuCl2, pH 7.4 (pH adjusted with NaOH) with the addition of 4% DMSO at 80 ° C then added to the lipid films and allowed to hydrate at 80 ° C with frequent turbulence When cooling, the samples are centrifuged on a tabletop centrifuge to nodulize any non-encapsulated cisplatin, and the supernatant was collected. Liposomes were applied to then to a pre-balanced Sephadex G-50 column with HBS, pH 7.4, to remove metal ions in excess of outside of the liposomes.

Daunorubicin was added to the liposomes at a ratio by weight 0.1: 1 and the loading was carried out at 60 ° C. Be aliquots were removed at various time points and applied to a rotating column Sephadex G-50. Measures Absorbance were carried out at 480 nm and used to determine daunorubicin concentrations and Cisplatin levels were measured using AA. The relationship of Cisplatin drug to initial lipid was 0.044: 1.

Figure 6 shows that DSPC / Col liposomes that contain passively encapsulated cisplatin loaded effectively daunorubicin in the absence of a pH gradient.

This additionally supports the loading of a second agent, in liposomes, through complexation with a load of metal.

5 Example 3 The effect of buffer composition on precipitation metal ion Solutions of cobalt, nickel, manganese, Cadmium, zinc and copper at concentrations of 150 and 300 mM in

10 histidine 20 mM. Triethanolamine (1.13 g / ml) was added dropwise until the resulting solution was at pH 7.4 or until the solution had a cloudy appearance (over an observation period of 10 minutes). Typically, less than 500 ml of 1.13 g / ml of triethanolamine was added.

15 Subsequently to the addition of triethanolamine, the solutions were visually inspected to determine if the precipitation of the metal had occurred. An appearance cloudy solution indicated the presence of a precipitate while the transparency of the solution

20 indicated lack of precipitation. The results are shown. in Table 2.

Metal Concentration (mM) Sulfate Chloride Nitrate

Cobalt 300 without ppt without ppt ---- 150 without ppt without ppt ---- Nickel 300 without ppt without ppt ---- 150 without ppt without ppt ---- Manganese 300 without ppt ppt ---- 150 without ppt ppt ----- Cadmium 300 without ppt ppt ---- 150 without ppt ppt ---- Zinc 300 ppt ppt ---- 150 ppt ppt ----

Copper 300 without ppt without ppt without ppt 150 without ppt without ppt ----

ppt represents the formation of a precipitate after the addition of triethanolamine in a period of 10 minutes

5 without ppt: represents that the formation of a precipitate did not occur after of the addition of triethanolamine to reach a pH of 7.4 and in a space of 10 minutes time. dotted line: not measured The concentrations of the indicated metal are concentrations before the addition

10 triethanolamine.

Example 4 The metal charge is different from the citrate based charge The ability of doxorubicin to compare

15 accumulate in DPPC / DSPE-PEG2000 liposomes (molar ratio 95: 5) in accordance with loading procedures based on MnSO4 and citrate. The lipid films were hydrated with 300 mM MnSO4 solution or 300 mM citrate, pH 3.5, and they passed through an extrusion apparatus at 55 ° C.

20 The resulting liposomes were passed down a Sephadex G-50 column balanced with a solution SHE buffer, pH 7.5, for liposomes containing MnSO4 and HBS, pH 7.5, for liposomes containing citrate. After buffer exchange, the liposomes are

25 combined with doxorubicin to give a weight ratio of drug: final lipid of approximately 0.1: 1, 0.2: 1 or 0.3: 1.

The resulting mixture was incubated at 37 ° C for 80 minutes. The Drug load extent was determined as described in the methods when measuring absorbance at 480 nm for quantify the drug; lipid levels were measured by liquid scintillation counting.

The results summarized in Figure 7 show that reached loading efficiencies of doxorubicin of> 95%,> 90% and> 80% in cholesterol-free liposomes containing MnSO4 (300 mM) when the initial drug / lipid ratios were 0.1: l (panel A), 0.2: 1 (panel B) and 0.3: 1 (panel C), respectively. In contrast, cholesterol-free liposomes loaded with doxorubicin according to the procedure of citrate loading (300 mM citrate, pH 4.0) with pH gradient under the same conditions they presented a reduction substantial in encapsulation efficiency since the relationship by weight doxorubicin / lipid increased from 0.1 to 0.3. The last method could reach a weight ratio of maximum lipid drug of <0.075. This results show that cholesterol-free liposomes can effectively charge with doxorubicin until ratios of lipid drug as high as 0.3: 1 (w / w) using load with metal while loading procedures based on Citrate can only reach drug to lipid ratios 0.1: 1 maximum (w / w). These data show that Metal-based loading mechanisms are different from those They hope to maintain a stable pH gradient. The data points represent the relationship of drug to lipid mean and error bars represent the deviation standard.

Example 5

The loading of unbuffered metal causes a collapse of the

transmembrane pH gradient

The effect of doxorubicin loading on liposome transmembrane pH gradient of DMPC / Col se compared using loading techniques with citrate and manganese at measure pH gradients before and after the drug loading Lipid films of DMPC / Col (ratio

molar 55:45) were hydrated with 300 mM citrate buffer, pH 3.5, 300 mM MnSO4 or 300 mM MnCl2. The resulting MLVs are underwent 5 cycles of freezing and thawing (freezing in liquid nitrogen and defrosting at 40 ° C) followed by extrusion at 40 ° C.

To exchange the external solutions of the liposomes, samples were fractionated in Sephadex columns G-50 For liposomes with encapsulated citrate, the buffer external was exchanged for HBS and for liposomes with MnSO4 and Encapsulated MnCl2, the external buffer was exchanged for SHE, pH 7.5. After buffer exchange, it was added doxorubicin in a weight ratio of 0.2: 1 at 60 ° C. The absorbance at 480 nm after solubilization in Detergent was evaluated to quantify the drug and the lipid levels were determined by counting by liquid scintillation

The results presented in Figure 8A show that Doxorubicin loading in liposomes containing citrate (squares) and encapsulated MnSO4 (circles) was essentially Complete after 5 minutes of incubation. The accumulation of doxorubicin using MnCl2 (triangles) was less complete in relation to the loading of MnSO4 and citrate. The points of data represent the mean drug to lipid ratios of at least three repeated experiments and error bars indicate the standard deviation.

The transmembrane pH gradients of the formulations before and after loading doxorubicin were measured using [14C] -methylamine. Briefly, it was added [14C] -methylamine (0.5 (Ci / ml) to liposomal solutions prepared previously. After 15 minutes, parts 150 ml aliquots were passed down 1 ml Sephadex G-50 columns balanced in HBS for remove uncapsulated methylamine. The concentrations of lipid and methylamine before and after chromatography in column were determined by scintillation counting. He pH gradient of transmembrane was calculated according to the relationship:

pH = log {[H +] inside / [H +] outside = log {[methylamine] inside / [methylamine] outside}.

As shown in Figure 8B, after setting the pH gradient, but before loading of doxorubicin, citrate formulations (column 1), MnSO4 (column 3), encapsulated MnCl2 (column 5) exhibited pH gradients measured 3.4, 1.6 and less than 0.18, respectively. These results indicate that the gradients of transmembrane pH are lower when used manganese solutions in relation to citrate. After of the addition of doxorubicin to liposomes containing encapsulated citrate, the pH gradient decreased from 3.4 (column 1) up to 2.3 (column 2). This result is from according to previous reports demonstrating the collapse Doxorubicin-mediated pH gradient in these formulations After loading doxorubicin, the liposomes containing manganese did not exhibit a gradient of Measurable pH (columns 4 and 6) demonstrating that you are formulations lost their pH gradient during loading drug. Data points represent the pH gradient means of three separate experiments and error bars indicate the standard deviation.

Example 6

The charging efficiency depends on the metal ion used

The load of irinotecan in liposomes of DSPC / DSPE-PEG2000 (95: 5 molar ratio) encapsulating sulfate solutions of Manganese or copper sulfate was carried out to compare the loading efficiency of the two different metals.

The lipid films were hydrated in a solution either of 300 mM MnSO4 or 300 mM CuSO4. Vesicles The resulting multilamellar (MLV) were extruded at 60 ° C and the LUV underwent buffer exchange for SHE, pH 7.4. The Drug loading was started by adding irinotecan to the resulting solution at a weight ratio of drug to lipid 0.1: 1 at 60 ° C. The extent of drug loading it was measured as described and the absorbance was measured at 370 nm.

The results in Figure 9 demonstrate that the load with irinotecan manganese it was only 10% complete at the point 30 minutes temporary while irinotecan charge in liposomes containing copper it resulted in more than 95% charge after 5 minutes. These results illustrate that the loading properties of liposomes depend a lot on The identity of the metal ion.

Example 7

Drug loading in cholesterol-free liposomes using

encapsulated manganese, cobalt and nickel

Cholesterol-free daunorubicin uptake (DSPC / DSPEPEG2000) was investigated using solutions internal levels of MnSO4, CoCl2 and NiSO4 at various temperatures of load.

Liposomes (DSPC / DSPE-PEG2000, 95: 5 molar ratio) cholesterol-free encapsulating manganese were prepared by hydrating lipid films in MnSO4 300 mM and extrusion was carried out at 75 ° C. The samples are exchanged for HBS using a dialysis column of manual tangential flow. The external buffer contained 1.67 EDTA mM to remove any divalent cation. Take care daunorubicin at a drug-to-lipid molar ratio of 0.1: 1 and charging was carried out at 23 ° C, 37 ° C or 60 ° C. The Drug load extent was measured by solubilizing the liposomes in detergent followed by measuring absorbance at 480 nm

The results of Figure 10A show that the load of daunorubicin in liposomes containing MnSO4 of DSPC / DSPEPEG2000 (95: 5 molar ratio) is the most effective at 60 ° C, while the load at 23 ° C and 37 ° C occurred in a minor extension Relationships of daunorubicin to lipid (mol: mol) of 0.07 when the Charging temperature is 60 ° C.

DSPC / DSPE-PEG2000 liposomes were prepared (ratio molar 95: 5) containing cobalt by hydration of 150 mM CoCl2 lipid films. MLVs were extruded to 75 ° C and the outer buffer was then exchanged at

dialyze in front of HBS overnight. Following, the liposomes were further exchanged for HBS using a manual tangential flow dialysis column to remove any residual CoCl2. Take care daunorubicin at 23, 37 and 60 ° C at a weight ratio of 0.1: 1 drug / lipid. The extent of the burden of daunorubicin was determined by measuring absorbance at 480 nm after solubilization of liposomes. The levels of Lipids were determined by scintillation counting of liquid.

Daunorubicin was effectively loaded into liposomes of DSPC / DSPE-PEG2000 (95: 5 molar ratio) containing CoCl2 a 60 ° C (see Figure 10B). At 60 ° C, the load looked like result> 95% daunorubicin encapsulation at 5 minutes The load at 23 ° C and 37 ° C was less efficient and required a 60 minute incubation at 37 ° C to reach 80% drug encapsulation.

Liposomes containing DSPC / DSPE-PEG2000 (molar ratio 95: 5) and which encapsulated NiSO4 were prepared as described in the previous examples. The lipid films are hydrated in 300 mM NiSO4 and the external buffer of the Liposomes were exchanged by passing through a Sephadex G-50 column equilibrated with SHE, pH 7.4. The daunorubicin was added so that the weight ratio of initial lipid drug (before loading) out 0.2 to 1 and The loading was carried out at 60 ° C. The loading efficiencies of

daunorubicin

be measured how be described previously

by absorption.

The

results from the Figure 10C show that the

incubation of daunorubicin with DSPC / DSPEPEG2000 liposomes containing NiSO4 at 60 ° C resulted in more than 75% drug encapsulation at 5 minutes.

Example 8 Drug loading in cholesterol-free liposomes using encapsulated copper

The copper charge of epirubicin was also examined in DSPC / DSPE-PEG2000 liposomes (95: 5 molar ratio).

DSPC / DSPE-PEG2000 liposomes (95: 5 molar ratio) that they contained copper were prepared as described in the previous examples. The lipid films were hydrated in 300 mM CuSO4 and extrusion was carried out at 70 ° C. Buffer external was replaced by SHE, pH 7.4, by passing the liposomes through a balanced Sephadex G-50 column with SHE buffer before loading. Epirubicin was added to copper-containing liposomes at a molar ratio of lipid drug of approximately 0.2: 1 and the load was carried out at 60 ° C. Epirubicin and lipid levels are tested by spectrometry and scintillation counting,

respectively.

For quantify the epirubicin, the

absorbance

be measured to 480 nm after from solubilize the

Liposomal preparation with detergent.

Figure 11

sample that epirubicin load in

DSPC / DSPE-PEG2000 liposomes (95: 5 molar ratio) gave as a result> 95% drug accumulation at 5 minutes when the uptake occurred at 60 ° C.

Example 9 Metal loading of liposomes containing cholesterol

The uptake of doxorubicin, daunorubicin and Topotecan in liposomes of DSPC / Col (55:45 molar ratio) investigated using liposomes prepared to encapsulate copper and cobalt.

DSPC / Col liposomes (55:45 molar ratio) that encapsulated cobalt were prepared as described previously by hydrating the films lipids in a solution of 300 mM CoCl2. External buffer was exchanged by column chromatography for SHE, pH 7.5. Charging was initiated by the addition of doxorubicin at a drug-to-lipid molar ratio of approximately 0.1: 1. Then, the liposomes were incubated at 60 ° C to facilitate drug loading. The extent of the burden of drug was measured as previously described by the solubilization of the samples with detergent followed by absorbance measurement at 480 nm.

The results in Figure 12A show that at 10

minutes,> 90% of the added drug was encapsulated.

DSPC / Col liposomes (55:45 molar ratio) that contained copper sulfate were prepared by hydration of a lipid film in 300 mM CuSO4. MLV resulting were extruded at 70 ° C and the external solution was exchanged for HBS by passing through a column Swivel Sephadex G-50. Liposomes subjected to buffer exchange were charged at 60 ° C with daunorubicin at a drug to lipid weight ratio of 0.1: 1, 0.2: 1 or 0.4: 1. The liposomes were solubilized in detergent before determine drug levels by measuring absorbance at 480 nm

The results in Figure 12B indicate that the load of DSPC / Col liposome drug (55:45 molar ratio) loaded using encapsulated CuSO4 was effective with> 90% of added drugs encapsulated at 5 minutes at 60 ° C.

DSPC / Col liposomes (55:45 molar ratio) that encapsulating 300 mM CuSO4 were prepared as described for Figure 12B, except that the external buffer is exchanged for SHE, pH 7.4. Then liposomes they were incubated with topotecan at a weight ratio of drug / lipid 0.1: 1 at 37 ° C. The extent of the load is checked for 2 hours at the indicated time points by quantifying the absorbance of the drug at 380 nm and the lipid by liquid scintillation counting. The drug It was quantified by measuring absorbance at 380 nm.

Figure l2C indicates that the topotecan load in DSPC / Col liposomes (55:45 molar ratio) was essentially 100% (> 95%) complete after 30 minutes.

Example 10

Metal loading of a number of different drugs in

unbuffered liposomes containing encapsulated drug

passively

The load of daunorubicin or irinotecan in various liposomes that contain a passively encapsulated drug will investigated under a number of conditions.

Daunorubicin uptake in liposomes that

contained cisplatin was measured according to the following procedures DSPC / DSPE-PEG2000 liposomes (ratio 95: 5 molar) or DMPC / Col (55:45 molar ratio) were prepared according to the materials and methods of the examples precedents Lipid films were hydrated in MnCl2 150 mM or 150 mM CuCl2, respectively, with 8.5 mg / ml of cisplatin at 80 ° C. The MLV were extruded at 75 ° C. He precipitated cisplatin was removed by centrifugation and the samples were then dialyzed against HBS overnight. Samples containing CuCl2 were additionally exchanged for HBS using a column of manual tangential flow dialysis to remove any CuCl2 or residual cisplatin. The daunorubicin was loaded in liposomes containing cisplatin / MnCl2 and cisplatin / CuCl2 a a drug / lipid weight ratio of 0.1: 1 to a incubation temperature of 60 ° C. The weight ratio of initial cisplatin / lipid drug was 0.01: 1 for both liposomal compositions The extent of the burden of drug was measured as previously described by the solubilization of the samples with detergent followed by absorbance measurement at 480 nm.

Figures 13A and 13B show that liposomes of DSPC / DSPE-PEG2000 (95: 5 molar ratio) and DMPC / Col (ratio molar 55:45) preloaded with cisplatin can be loaded with a second drug (daunorubicin) when a load is used active based either on manganese or copper, respectively. Moreover, the encapsulation of daunorubicin was not as effective using MnCl2 compared with CuCl2.

The load of daunorubicin or irinotecan in liposomes of DPPC / Col (55:45 molar ratio) containing well carboplatin or cisplatin trapped passively, respectively, it was analyzed using nickel or copper charge. Lipid films were hydrated in 300 mM NiSO4 or CuCl2 75 mM + 150 mM CuSO4 with 40 mg / ml carboplatin or 8.5 mg / ml of cisplatin, respectively. The MLV were extruded at 70 ° C. Samples containing nickel were dialyzed during night in front of 1 l of 300 mM sucrose, 20 mM HEPES, pH 7.4,

while samples containing copper were subjected to SHE exchange, pH 7.4, by chromatography on Sepharose columns containing CL4B resin. The daunorubicin was charged at 37 ° C at a molar ratio of 0.1: 1 lipid drug. The irinotecan was loaded in the liposomes as previously described at 60 ° C at weight ratio of drug to lipid of 0.1. The levels of drug and lipid were measured using described procedures previously.

The results summarized in Figures 13C and l3D illustrate that DPPC / Col liposomes (55:45 molar ratio) prepared with ion solutions either nickel or copper that contain a platinum drug effectively load a Second drug

Example 11 Metal loading combined with mediated loading techniques by ionophores results in the encapsulation of multiple agents

Combine the load with metal with a loading mechanism additional active results in effective encapsulation both doxorubicin and vincristine in a single liposome The loading with doxorubicin metal followed by the vincristine ionophore-mediated charge is detailed later.

DSPC / cholesterol liposomes (55:45 molar ratio) are prepared as described in the preceding examples, except that the lipid films were hydrated in MnSO4 300 mM and the 14C-CHE lipid marker was used. MLV resulting were extruded at 65 ° C and then made pass through a Sephadex G-50 column that had pre-balanced with 300 mM sucrose, 20 mM HEPES and 15 mM EDTA (pH 7.5). Doxorubicin was then added in a 0.2: 1 drug-to-lipid weight ratio and incubated additionally at 60 ° C for 60 minutes.

After loading doxorubicin, the cation divalent ionophore A23187 (1 g ionophore / mol lipid) se added to the liposomes and the mixture was incubated at temperature

environment for 3 minutes to facilitate the incorporation of A23187 in the bilayer. Subsequently, vincristine was added to the mixture and incubated at 50 ° C for 100 minutes. A small amount of radiolabeled vincristine was added to Drug preparation to facilitate quantification of the drug. Drug uptake was performed at a Vincristine to lipid weight ratio of 0.05: 1. The vincristine and lipid were quantified by counting by scintillation after solubilization of liposomes with Detergent. The absorbance at 480 nm was used to quantify Doxorubicin levels.

Figure 14 shows that liposomes preloaded with doxorubicin (circles) through metal loading they have an almost maximum encapsulation of load mediated by vincristine ionophore (squares) after 40 minutes of incubation at 50 ° C, without significant loss of doxorubicin during vincristine encapsulation. Data points represent mean drug to lipid ratio of three separate experiments and error bars indicate the standard deviation.

Example 12 The metal loading of two drugs in the absence of ionophore gives as a result an effective encapsulation of the two agents

The preceding examples have made use of procedures of charge either passive or ionophore mediated in combination with active load with metal to give as result the encapsulation of two drugs in liposomes of various compositions The following example shows that the Single metal charging can be used to actively charge Two drugs in a single liposome. Doxorubicin e was loaded Irinotecan in liposomes of DSPC / Cholesterol as described previously.

DSPC / Col liposomes (55:45 molar ratio) are prepared as previously detailed with 300 mM CuSO4 encapsulated Extruded liposomes were passed to through a Sephadex G-50 column that had pre-balanced with SHE, pH 7.5. First, it was loaded

irinotecan at a molar ratio of drug to lipid of 0.2: 1 at 60 ° C up to about 100% encapsulation. After of this, doxorubicin was incubated at 60 ° C at a molar ratio of drug to lipid of 0.15: 1 with the liposomal formulation which contains irinotecan to allow a sufficient load of doxorubicin Irinotecan levels were measured by measuring absorbance at 370 nm using a standard curve prepared in the presence of doxorubicin to account for its absorbance at 370 nm. Similarly, concentrations of doxorubicin were determined by measuring the absorbance at 480 nm using a standard curve prepared in the presence of Irinotecan to account for its absorbance at 480 nm. How One control, the individual uptake of each drug was measured separately in liposomes of the same composition.

The results summarized in Figure 15 illustrate that the doxorubicin and irinotecan can be effectively loaded into a single liposome using the active loading procedure with metal of the invention. The results represent the relationship from drug to average lipid from three separate experiments and Error bars indicate the standard deviation.

Example 13

In vivo drug release rates depend on

the nature of the metal ion

The capacity of different internal charging means to control the release of daunorubicin from liposomes of DSPC / DSPE-PEG2000 (95: 5 molar ratio) in vivo was investigated using 150 mM citrate, pH 4.0, 300 mM CuSO4 and 300 mM MnSO4. DSPC / DSPE-PEG2000 liposomes were prepared as described and extruded at 75 ° C. The external solution is exchanged for HBS by dialysis against HBS. Take care daunorubicin at a drug-to-lipid molar ratio of approximately 0.1: 1 and the loading was carried out at 60 ° C. The Daunorubicin load was measured as described in the preceding examples using EDTA in the buffer of solubilization Next, liposomes loaded with drug were administered intravenously to Balb / c mice a 100 mg / kg lipid dose and 10 daunorubicin dose

mg / kg Blood was recovered 24 hours after administration by cardiac puncture (3 mice per point temporary) and was collected in tubes containing EDTA. The plasma lipid concentrations were determined by liquid scintillation count. The daunorubicin was extracted of plasma as follows:

A defined volume of plasma was adjusted to 200 µl with distilled water followed by the addition of 600 µl of distilled water, 100 µl of 10% SDS and 100 µl of 10 mM H2S04. The resulting mixture was mixed and 2 ml of isopropanol / chloroform

1: 1 were added followed by turbulence. The samples are frozen at -20 ° C overnight or -80 ° C for 1 hour to promote protein aggregation, they took up room temperature, they underwent turbulence and centrifuged at 3000 rpm for 10 minutes. Organic layer lower was removed and tested for intensity of fluorescence at 500 nm as the wavelength of excitation (bandpass 2.5 nm) and 550 nm as a length

from

wave  from issue (He passed  from band 10 nm) Y using a

absorbance wavelength

480 nm

The

Figure 16 shows that liposomes from DSPC / DSPE

PEG2000

(relationship cool 95: 5) loaded with daunorubicin

using encapsulated citrate, pH 4.0, CuSO4 and MnSO4 present different drug to lipid ratios in plasma 24 hours after intravenous administration. This results show that drug release can be controlled at through the selection of an appropriate metal ion. The results represent the ratio of drug to mean lipid of at least three separate experiments and error bars indicate the standard deviation.

Example 14 Liposome loading in the presence and absence of metal ions not complexed

Metal-based drug loading was examined in presence and absence of metal ions on the surface external liposomes containing phosphatidylglycerol and the Results are represented in Figures 17 and 18.

Liposomes composed of DSPC / DSPG (molar ratio 80:20) were prepared following the procedures that were described in Example 1. The DSPC and DSPG lipids were dissolved in chloroform and chloroform / methanol / water (50: 10: 1 v / v), respectively. Then the lipids are combined in appropriate amounts for each formulation. He solvent was removed under a steady stream of N2 gas while maintaining the temperature at 70 ° C and set under vacuum for 5 minutes. Lipid films resulting were redissolved in chloroform to remove additionally any methanol or water and then the solvent was removed as before and dried under vacuum to remove any residual solvent. The samples are subsequently rehydrated in 150 mM CuSO4, pH 7.4 (pH adjusted with ASD) and the resulting MLVs were extruded at 70 ° C. Liposomal samples were well passed down a Chelex-100 ™ column (BioRad) of 15 ml equilibrated with 150 mM NaCl at 0.5 ml / min or subjected to buffer exchange for saline solution and additionally they were exchanged for sucrose 300 mM, 20 mM HEPES, pH 7.4, using tangential flow. Subsequently, the liposomes that were passed through of the Chelex-100 ™ column underwent exchange for 300 mM sucrose, 20 mM HEPES, pH 7.4, using flow tangential.

Both liposomal preparations were loaded at then at 37, 50 and 60 ° C with irinotecan at a relationship in 0.1: 1 drug to lipid weight as described previously. Drug uptake was tested using liquid scintillation count to determine lipid concentrations and absorbance at 370 nm for determine irinotecan concentrations after solubilization in detergent.

The results represented in Figure 17 reveal that the irinotecan load in DSPC / DSPG liposomes (ratio molar 80:20) was enhanced when the liposomal preparation was passed through a Chelex-100 ™ column to remove external metal ions. In contrast, the

Results shown in Figure 18 show that the load of irinotecan in liposomes undergoing exchange for a solution that did not contain a chelating agent was produced at a reduced speed Although not wanting to limit yourself by one particular theory, the removal of associated metal ions with the liposomal surface negatively charged at complexing the ions with a chelating agent can reduce the metal-drug interactions on the outer surface of the membrane thereby increasing the amount of drug free that can cross the membrane to get caught in the internal liposome compartment.

Example 15

Methods for removing metal ions from the solution

external liposomes

The copper-based charge of irinotecan was investigated in DSPC / DSPG liposomes (80:20 molar ratio) after removal of Cu2 + from the external solution using two techniques different, both based on the chelation of the external metal. The first technique involved the removal of copper by the passage through a Chelex ™ column and the second technique it involved subjecting the liposomes to exchange for a buffer which contains EDTA.

DSPC / DSPG (80:20 molar ratio) were prepared as in Example 14, except that the samples were rehydrated in 150 mM copper gluconate, pH, 7.4 (pH adjusted with ASD). He External copper was removed by: i) passing through a Chelex-100 15 ml column equilibrated in 300 mM sucrose, 20 mM HEPES, pH 7.4; or ii) by buffer exchange for saline solution and then by 300 mM sucrose, HEPES 20 mM, 30 mM EDTA, pH 7.4 (SHE buffer) using flow tangential.

Both liposomal preparations were loaded at then at 37, 50 and 60 ° C with irinotecan at a relationship in 0.1: 1 drug to lipid weight as described in the Example 14. Aliquots (100 µL) were removed in various time points after the beginning of the load were applied to a rotating column Sephadex G-50. TO

then the samples were solubilized in detergent and the quantification of drug and lipid was performed as described previously in Example 14.

The metal-based irinotecan charge after the removal of the external metal by passing through a chelation column (Figure 20) is comparable to the load after subjecting the liposomes to exchange for a solution containing EDTA (Figure 19). This results demonstrate that various means can be used to remove outer metal ions from charged membranes negatively without significantly affecting the ability to load.

Example 16 Pharmacokinetics of liposomes containing phosphatidylglycerol cocargados with daunorubicin and carboplatin

Daunorubicin and carboplatin retention coencapsulated in liposomes containing PG was investigated by passive carboplatin loading followed by loading with daunorubicin metal.

Daunorubicin and carboplatin were encapsulated in DSPC / DSPG liposomes (80:20 molar ratio), DSPC / SM / DSPG (75: 5: 20 molar ratio) and DSPC / SM / DSPG (molar ratio 70:10:20). Liposomes were prepared following the procedures described in the preceding examples. The DSPG was dissolved in a solution of

chloroform / methanol / water

50: 10: 1 (v / v) Y he marker

radioactive

14C-CHE be added to the preparation for

quantify

he lipid The films lipid be

rehydrated in 150 mM CuSO4, 20 mM histidine, pH 7.4, which contained 80 mg / ml carboplatin (with 4% DMSO for improve the solubility of carboplatin). After the extrusion, samples were centrifuged to remove carboplatin not encapsulated. Liposomes subjected to SHE buffer exchange was loaded with 3Hdaunorubicin. Liposomes were given to mice at a dose of 100 mg / kg of lipid. Count was used by liquid scintillation to quantify daunorubicin and the

lipid Plasma carboplatin levels were determined by atomic absorption.

The results in Figure 21 indicate that liposomes of DSPC / DSPG (80:20 molar ratio), DSPC / SM / DSPG (ratio molar 75: 5: 20) and DSPC / SM / DSPG (molar ratio 70:10:20) doubly charged have plasma lipid levels boosted at various time points after the intravenous administration, although prepared liposomes with 10 mol% sphingomyelin exhibited lipid levels lower in relation to liposomes with 5 mol% of sphingomyelin Liposomes effectively altered the Pharmacokinetics of the drug as demonstrated by senior levels of daunorubicin and carboplatin that remained in the blood compartment after administration (see Figures 22 and 23). Liposomes prepared with DSPC / DSPG (80:20 molar ratio) and DSPC / SM / DSPG (ratio molar 75: 5: 20) exhibited daunorubicin levels and higher carboplatin in relation to liposomes of DSPC / SM / DSPG (70:10:20 molar ratio).

Although the preceding invention has been described with some detail by way of illustration and examples with motives of clarity and understanding, will be readily apparent to experts in the specialty in light of the teachings of this invention that changes and modifications can be made in it without departing from the spirit and scope of attached claims. All patents, applications and Patent publications mentioned herein are incorporated herein by reference.

Claims (21)

l. A liposomal composition comprising liposomes containing an internal solution, comprising the solution internal copper ions and one or more therapeutic agents encapsulated, agent that is a drug or agent antineoplastic; and in which:

(to)

liposomes do not comprise an ionophore;

(b)

said therapeutic agent has been loaded into liposomes preformed containing said copper ions in said internal solution;

(C)

the internal solution comprises a solution compatible with copper ions; Y

(d)

the composition comprises the liposomes in a external solution to the liposomes.

2.

The composition according to claim 1, wherein said therapeutic agent is a quinolone, a anthracycline, an antibiotic, a nitrogen mustard, a camptothecin or a podophyllotoxin.

3.

The composition according to claim 1 or 2, in which the external solution and external surfaces of the liposomes do not contain substantially metal ions not complexed

Four.

The composition according to claim 3, in which the external solution comprises a chelating agent of metals

5.

The composition according to any one of the claims 1-4, wherein the external solution and the internal solution have substantially the same pH in the range of 6.0-8.5.

6.

The composition according to any one of the claims 1-5, wherein the liposomes contain less of 20% cholesterol.

7.

The composition according to any one of the claims 1-6, wherein the liposomes comprise one

or more lipids that have a negative pH electrical charge physiological.

8.

The composition according to claim 7, in the one or more lipids with negative electrical charge is

select from phosphatidylglycerol (PG) and phosphatidylinositol (PI).

9.

A method of loading a therapeutic agent in a liposome in a liposomal composition, comprising the method:

i) provide a liposomal composition comprising a liposome in an external solution, containing the liposome an internal solution comprising copper ions encapsulated; ii) add one or more therapeutic agents to the solution external; Y iii) keep the agent in the external solution for long enough to load the agent in the liposome, in which the liposomes do not contain an ionophore, and the internal solution comprises a compatible solution with copper ions.

10.

The method according to claim 9, in the that the external solution and the external surfaces of the Liposome does not contain uncomplexed metal ions; I

uncomplexed metal ions are removed from the external solution before stage (ii) by chromatography or by intensive solution exchange or dialysis; I the internal solution and the external solution have substantially the same pH; I liposomes contain less than 20 mol% of cholesterol.

eleven.

The method according to claim 10, in the that the external solution comprises a chelating agent of metals

12.

The method according to claim 10, in the that liposomes comprise one or more lipids that have negative electrical charge at physiological pH.

13.

The method according to claim 12, in the that the one or more lipids with negative electrical charge is select from PG and PI.

14.

The method according to any one of the claims 9-13, wherein a second agent therapeutic is loaded by means of a pH gradient of transmembrane formed after stage (iii).

fifteen.

The method according to any one of the claims 9-14, wherein a second agent therapeutic is loaded by means of a gradient of transmembrane established with an ionophore presented in the liposome, in which the transmembrane gradient is set up after stage (iii).

16.

A liposomal composition comprising liposomes comprising one or more therapeutic agents produced by the method according to any one of the claims 9-15.

17.

A method to select a composition liposome that has a load or retention property preferred for an agent, comprising:

(to)

provide a first liposomal composition that comprises a liposome in an external solution, containing the liposome an internal solution that comprises one or more transition metal ions;

(b)

add the agent to the external solution of the composition of (a) over a period of time to load the agent into the liposome of the first composition;

(C)

provide a second liposomal composition that comprises a liposome in an external solution, containing the liposome an internal solution that comprises one or more transition metal ions;

(d)

add the agent to the external solution of the composition of (c) over a period of time to load the agent into the liposome of the second composition;

(C)

compare the amount of agent charged or the amount of agent retained over time, for the liposome of the resulting composition in (b) with the liposome of the resulting composition in (d); Y

(F)

select the resulting liposomal composition in

(b)

or (d) that has a larger amount of agent loaded

or retained; in which:

(I)

the liposomal compositions of (a) and (c) differ by one or more of: (i) metal ions present in the internal solution; (ii) lipids in the liposome of the liposomal composition; and (iii) the metal ion concentration present in the internal solution;

(II)

the period of time in (b) is different from period of time in (d); or

(III) the load in (b) is carried out at a temperature different than the load in (d).

18.

The method according to claim 17, in the that one of the first and second compositions comprises liposomes containing cholesterol and the other one of said compositions comprises liposomes of low content of cholesterol.

19.

The method according to claim 17 or 18, in which at least one of the first and second compositions comprises one or more metal ions selected from Cu, Co and Zn

twenty.

The method according to any one of the claims 17-19, wherein the internal solution of each one of the first and second compositions comprises a metal compatible solution.

twenty-one.

The method according to any one of the claims 17-20, comprising the additional step of loading an agent into liposomes of the composition that is Note that you have the highest amount of agent loaded or detained.