WO2007098968A1 - Triggered release of liposome contents - Google Patents
Triggered release of liposome contents Download PDFInfo
- Publication number
- WO2007098968A1 WO2007098968A1 PCT/EP2007/001877 EP2007001877W WO2007098968A1 WO 2007098968 A1 WO2007098968 A1 WO 2007098968A1 EP 2007001877 W EP2007001877 W EP 2007001877W WO 2007098968 A1 WO2007098968 A1 WO 2007098968A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- liposome
- liposomes
- agent
- boron cluster
- cluster compound
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/69—Boron compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
Definitions
- the present invention relates to drug delivery systems using compositions of lipid carrier, such as liposomes or liposomal formulations or compositions.
- the present invention relates to compounds and methods for triggering the release of agents encapsulated by lipid carriers under pre-determined conditions.
- Such pre-determined conditions may be the release at a pre-determined site or the release in the presence of a pre-determined material, i.e. by the choice of the trigger, or both. More particular, it relates to the release of an agent for therapeutic, diagnostic, or investigative purposes.
- the present invention relates to the use of boron cluster compounds in drug discovery, to preparation of pharmaceutical compositions containing the boron cluster compound and to methods for drug release using said boron cluster compound.
- a common manner of administering agents, such as drugs and dyes to the body is via injection into the bloodstream or a localized area in the body.
- agents such as drugs and dyes
- the injected material When injected into the bloodstream, the injected material is carried throughout the blood system and therefore the entire body, known as systemic delivery.
- systemic delivery The drawbacks to this delivery system are that the concentration of the injected material is extremely diluted and the material acts on most tissues in the body and may be toxic to some of them. In addition, the location and duration of exposure of these injected materials cannot be controlled.
- lipid particles have been shown to be efficient "vehicles” for many in vitro and in vivo applications, this can be realized by encapsulating drugs in lipid vesicles, such as liposomes.
- the other major difficulty is to trigger the release of the liposome's content at the site of activity.
- Several methods of controlled drug release delivery have been employed, for example the release by exposing the liposome composition to a number of different triggers, such as acidic environment (pH- value), to irradiation, to heat, as well as to enzymes or enzyme activating agents, all indeed resulting more or less in the destruction or rearrangement of the liposome membrane, thereby releasing the encapsulated agent, but exhibiting severe drawbacks, as will be described infra.
- tumor specific drug delivery has become of increasing interest, since the use of chemotherapeutics is often limited due to their severe side-effects.
- conventional drug delivery systems have shown low efficiency, alternative drug delivery principles were needed, and therefore liposome- based drug delivery has been introduced on the market, and are still being developed as an alternative way of administering therapeutically active agents.
- one major requirement, among others, for a successful treatment using a liposome- based strategy is that the cells, tissues, or organs challenged with cancer can be exposed to a high concentration of a therapeutically active agent without doing irreversible harm to the host, i.e. without affecting the surrounding healthy cells, tissues, or organs.
- liposomes were suggested as a sort of micro carrier-technology, useful as drug carriers in cancer chemotherapy, the use of which requires first, prolonged blood circulation of the liposomes, second, a sufficient accumulation at the sites of challenged cells, tissues, or organs, i.e. for example tumors, and third, the controlled release of the drug and uptake of the same by the tumor.
- Stealth liposomes contain a "stealth component", such as monosialoganglioside, or polyethyleneglycol-conjugated lipids such as polyethyleneglycol-distearoylphosphatidylethanolamine or -ceramide both having a strong influence on the clearance profile. This is one basic requirement to provide controlled and increased concentration of active agents at or near the desired cells, tissues, or organs.
- the site-specific delivery of the liposomes to the desired cells, tissues, or organs for example by targeting the liposomes to the sites of activity which was discussed as a big improvement for accelerated and selective drug delivery, is of less value, as without either retaining the encapsulated drug until it reaches the tumor, or releasing the encapsulated agents once the liposomes have reached the tumor site, the liposomes are more or less useless.
- one of the major drawbacks of the prior triggering methods is that they all require either the targeting of the trigger (towards the liposomal carrier or desired cell, tissue, or organ) or predetermined (and therefore specially modified) properties of the liposomes used, which can then be exploited by an external stimulus, having the disadvantage that the reach of most of the external triggers is poor and therefore their effect is locally restricted. Thus, they cannot trigger drug release from liposomes at sites which are either not yet known or only hardly (if at all) accessible to the external stimulus. Alternatively, when internal triggering mechanisms such as low pH values or specific enzymatic setup of the target tissue are used, these cannot be manipulated. For that reason, treatment of metastases (as an example in the field of cancer for a tumor which is inaccessible, and/or the location or even existence of which is unknown) is not possible with external triggers, and cannot be controlled when using internal triggers.
- the present invention solves the problems of unsatisfactorily (since insufficient and therefore ineffective) release of liposomal carrier-encapsulated substances, and permits said substances to be released at or at least in the vicinity of the specific and desired target sites by the administration of a boron cluster compound, triggering the release of the liposome- encapsulated substance, allowing the substance to exert its desired effect substantially only on the targeted cells, tissues, or organs.
- Lipid carriers such as liposomes offer considerable promise as vehicles for delivery of agents for use in a variety of applications including biochemical and immunological assays, diagnosis, and also pharmaceutical delivery systems. Unfortunately their application is undermined by the difficulty associated with selectively releasing their contents at a specific time or location.
- the present invention relates to means and methods to overcome the mentioned difficulties. More specifically, the present invention provides a pre-determined substance delivery system in which liposome encapsulated substances are triggered to be released by a rather small, preferably non-toxic inorganic compound. While previous drug delivery systems relied on triggering stimuli, such as irradiation or physiological changes in the environment of the targeted tissue, the present invention makes use of a class of compounds that have been surprisingly found to specifically interact with liposomes so as to cause them to set their content free.
- the present invention is directed to the use of boron cluster compounds to trigger the release of agents encapsulated in lipid carriers, allowing to trigger the release even at sites, which are hardly (or even not at all) accessible to external triggers.
- the present invention further relates to a composition
- a composition comprising as a first component a boron cluster and as a second component a liposome according to the present invention.
- This composition may include liposomes encapsulating an agent and the components can be administered or applied concurrently or sequentially.
- the present invention relates to methods for releasing encapsulated agents from lipid carriers, such as liposomes by exposure to boron cluster compounds for identifying and obtaining a therapeutically active agent, for synthesizing a therapeutically active agent in a sufficient amount, for obtaining genetically altered cells, and for assaying liposomes or their encapsulated agents by exposure to a boron cluster compound.
- the present invention relates to the use of a boron cluster compound or components thereof, a liposome and an agent for the use in the above-mentioned methods.
- the present invention relates to the use of liposomes which are designed to release their encapsulated agent upon a trigger by boron cluster compounds.
- the present invention provides the possibility not only to treat primary tumors but rather also to treat metastases, one major problem in cancer therapy.
- the present invention relates in some aspects to the treatment of cancer, it is not at all limited to this disease, as will be clear from the following.
- Fig. 1 illustrates the aggregation and structural transformation of DPPC-liposomes after exposure to mercaptoundecahydro-closo-dodecaborat (BSH).
- BSH mercaptoundecahydro-closo-dodecaborat
- Fig. 2 illustrates the release of liposome-encapsulated calcein at 37°C. It is shown that the fluorescence of the fluorescent dye calcein is only detectable when encapsulated in the DPPC/DOTAP-liposomes.
- the x-axis refers to time and the y-axis refers to fluorescence intensity. After exposure to BSH and Triton, respectively, fluorescence decreases, since it is effectively abolished by the surrounding Co 2+ -ions.
- the determined calcein released during the 600 s was 87 % of the total amount.
- Fig. 3 illustrates the results of a cell survival experiment, measured by the WST assay. Survival of V79 cells incubated with BSH in the absence (open symbols) and presence (closed symbols) of doxorubicin-containing DPPC liposomes was measured. The curve reflects cell survival, ranging from 0% to 100% (y-axis) plotted against the concentration of BSH, ranging from 0 mM to 20 mM in the incubation medium after incubation for 24 h.
- Fig. 4 illustrates the release at 37°C of carboxyfluorescein encapsulated in DPPC-DSPE- PEG 2 ooo-liposomes. It is shown that the fluorescence of carboxyfluorescein is only detectable after exposure to BSH and Triton, respectively. Fluorescence increases, since the quenching of fluorescence when encapsulated in the liposome is abolished.
- the x-axis refers to time and the y-axis refers to fluorescence intensity.
- Liposome generally refers to any lipid carrier which is suitable for the purposes of the present invention.
- lipid carriers as used herein can comprise a variety of lipid aggregates which may all be used for the purpose of the present invention. They comprise for example micelles, lipid microspheres, lipid nanospheres, lipid vesicles also referred to as liposomes, such as ULV (unilamellar vesicles), MLV (multilamellar vesicles), MVL (multivesicular liposomes), sandwich liposomes (comprising an agent internalized between two bilamellar liposomes), giant liposomes (having diameters of 10 to 60 ⁇ m), anionic or cationic liposomes, liposome beads (aggregated or globulized liposomes with multiple encapsulations of agents), reconstituted liposomes (liposomes having incorporated protein components of membranes), hexasomes, cubosomes, and any membrane bilayers including an aqueous compartment and forming a permeability barrier between the encapsul
- Lipid complexes have been used for a myriad of drug therapies, and these delivery systems have shown promising results in for example gene therapy, as for gene therapy to be successful efficient and safe transfer of genes or biologically active reagent to a target cell is required.
- Lipid carrier refers to entity made from lipids or mixtures containing lipids, comprising an outer lipid layer surrounding a volume not containing lipids.
- the phase can consist of an ordered micellar structure of a single layer of lipid molecules, a two-dimensional arrangement of uni-, bi- or multi-layer membranes, or three-dimensional arrangements consisting of uni-, bi- or multi-layer membranes.
- the present invention therefore includes within its scope pharmaceutical compositions comprising at least one lipid carrier formulated for use in human or veterinary medicine.
- lipid carrier may be used interchangeably with the term “lipid vesicle”, and also comprises lipid aggregates.
- any artificial or naturally occurring vehicle of a monolayer, bilayer or multibilayer of lipids enclosing an open or closed volume is subsumed under the term "lipid carrier".
- the lipid carriers used in the present invention may comprise one type of lipid as wells as a mixture of different lipid molecules.
- cubosome as a sub-class of lipid carriers, as it is used herein, refers to dispersed particles of bicontinuous cubic liquid crystalline phase, self-assembled monostructured particles that can be formed in aqueous, lipid, and surfactant systems.
- One of the most common surfactants used to make cubosomes is the monoglyceride glycerol monoolein.
- the expression “bicontinuous” refers to the division of two continuous but non-intersecting aqueous regions by a lipid bilayer that is contorted into a space-filling structure.
- hexasome describes a further microstructure, typically hexagonally arranged, containing channels of aqueous material surrounded by a single layer of lipid molecules.
- liposome refers to microscopic lipid bilayer vesicles that enclose a compartment.
- Liposomal vesicles can contain a single bilayer (unilamellar vesicle) or multiple bilayers (multilamellar vesicle). These vesicles can encapsulate water- soluble agents in their aqueous cavities, or carry lipid soluble agents within the membrane itself.
- Liposomes have already been used for encapsulating a variety of agents, for example therapeutic agents such as cytotoxic drugs, and carrying them to biological target sites. Encapsulation of for example pharmaceuticals in liposomes can reduce drug side-effects, improve pharmacokinetics of delivery to a target site, and improve the therapeutic index of a drug.
- any of the aforementioned lipid carriers can be used for the purpose of the present invention, it is exemplarily made use of liposomes which is not to be understood as limitation of the scope of the present invention, since the subject matter and teaching of the present invention can be easily applied and transferred, respectively, to the aforementioned lipid carriers without undue burden for those skilled in the art.
- biocompatible is intended to describe compounds that are not toxic to cells. Compounds are “biocompatible” if their addition to cells in vitro does not lead to cell death and does not induce inflammation or other such adverse effects in vivo.
- Biodegradable compounds are those that, when introduced into cells, are broken down by the cellular machinery into components that the cells can either reuse or dispose of without significant toxic effect on the cells.
- trigger refers to a mechanism or agent, giving an impulse, activating or inducing activation to a system or object. Within the present invention the term “trigger” is also to be understood in the sense of "mediator”.
- lipid is any chemical compound with a hydrophobic portion, such as oils, fats and fatlike substances, either naturally occurring or synthesized, and that characteristically are soluble in apolar solvents but only sparingly soluble in aqueous solvents.
- lipids comprise also fatty acids, neutral fats (such as triacylglycerol), other fatty acid esters and soaps, long chain (or fatty) alcohols and waxes; sphingoids and other long-chain bases, glycolipids, phospholipids and sphingolipids, carotenes, polyphenols, glycopolyphenols, sterols (and related compounds), terpenes, and other isoprenoids.
- neutral fats such as triacylglycerol
- other fatty acid esters and soaps long chain (or fatty) alcohols and waxes
- sphingoids and other long-chain bases glycolipids, phospholipids and sphingolipids, carotenes, polyphenols, glycopolyphenol
- the lipids used in the present invention may be extracted and purified from a natural source or may be prepared synthetically in a laboratory. In a preferred embodiment, the lipids are commercially available.
- nanospheres refers to spherical structures having a size in the range of nanometers.
- microspheres refers to spherical structures having a size in the range of micrometers.
- micelle refers to aggregates of colloidal dimension, of oriented molecules of amphiphatic substances. Micelles are made from amphiphilic components, and in aqueous solutions the individual molecules of the micelle are oriented with their polar groups pointing towards the aqueous medium and their hydrophobic moiety directed into the center of the micelle. At low water content or in apolar solvents micelles can exist with inverted orientation of the molecules, called "inverse micelles”.
- surfactant refers to any agent, which preferentially absorbs to an interface between two immiscible phases, such as the interface between water and an organic polymer solution, a water/air interface or organic solvent/air interface.
- Surfactants generally possess a hydrophilic moiety and a lipophilic moiety, such that, upon absorbing to microparticles, they tend to present moieties to the external environment that do not attract similarly-coated particles, thus reducing particle agglomeration.
- Surfactants may also promote absorption of a therapeutic or diagnostic agent and increase bioavailability of the agent.
- Surfactants may also be used in the preparation of a pharmaceutical composition of the present invention.
- multivesicular liposomes refers to liposomes having a size of 1-20 ⁇ m, containing multiple non-concentric chambers within each liposome particle, resembling a "foam-like" matrix.
- multilamellar liposomes or multilamellar vesicles refers to liposomes containing multiple, often close to concentric chambers within each liposome particle, resembling the "layers of an onion". Multilamellar liposomes characteristically have mean diameters in the range of micrometers, usually from 0.5 to 25 ⁇ m.
- unilamellar liposomes or unilamellar vesicles refers to liposomes that enclose a single internal aqueous compartment and usually having a mean diameter range from about 20 to 500 run.
- Unilamellar liposomes include small unilamellar vesicles (SUV), and large unilamellar vesicles (LUV).
- the liposomes used in the present invention are unilamellar liposomes.
- amphipathic lipid refers to a molecule that has a hydrophilic "head” group and a hydrophobic "tail” group and has membrane-forming capability.
- the amphipathic lipids can be neutral, zwitterionic, anionic, or cationic lipids.
- zwitterionic lipid refers to an amphipathic lipid having a net charge of zero at pH 7.4, and includes for example phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, just to name a few.
- anionic lipid refers to an amphipathic lipid having a net negative charge at pH 7.4, and includes for example phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, phosphatidic acids, just to name a few.
- cationic lipid refers to an amphipathic lipid having a net positive charge at pH 7.4, and includes for example diacyl trimethylammoniumpropane and ethyl phosphatidylcholine.
- administering refers to any mode of application to a cell, tissue, organ, or whole organism, which results in the physical contact of the composition with an anatomical site.
- therapeutic index refers to a medication as a comparison of the amount that causes the therapeutic effect to the amount that causes toxic effects. Quantitatively, it is the ratio of the dose required to produce the toxic effect and the therapeutic dose. A commonly used measure of therapeutic index is the lethal dose of a drug for 50% of the population (LD 50 ) divided by the effective dose for 50% of the population (ED 50 ).
- LD 50 lethal dose of a drug for 50% of the population
- ED 50 effective dose for 50% of the population
- Small molecule refers to organic and inorganic compounds, whether naturally-occurring or artificially designed (e.g., via chemical synthesis), that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Typically, small molecules have a molecular weight of less than about 1500 g/mol.
- lipid carrier e.g. liposome may be loaded with proteinaceous material, i.e. proteins, peptides such as antibacterial peptides and derivatives thereof or nucleic acid based molecules, see also infra.
- Multilamellar and unilamellar liposomes can be made by several relatively simple methods, as for example a number of techniques for producing ULV and MLV is described in (for example U.S. patent numbers 4,522,803; 4,310,506; 4,235,871 ; 4,224,179; 4,078,052; 4,394,372; 4,308,166; 4,485,054; and 4,508,703).
- Giant liposomes can be constructed as described for example in US patent application 2004/0062797. Loading drugs into liposomes
- a liposome drug complex with the hydrophobic drug primarily in the membrane.
- Water soluble drugs can be sequestered in the liposome cavity by submitting liposomes to several cycles of freezing and thawing in an aqueous solution containing the drug.
- charged amphipathic drugs can be loaded into preformed liposomes using transmembrane pH gradients, as described in Bally et al, Biochem. Biophys. Acta 812 (1985), 66-76.
- the present invention generally relates to compound triggered delivery systems. More specifically, the present invention pertains to the use of inorganic compounds for triggering the release of an agent present in a liposome. In particular, the triggered release of agents encapsulated in lipid carriers by a boron cluster compound is disclosed. Accordingly, for the purpose of the present invention and as used in the claims the term "liposome" includes any suitable lipid carrier which is suitable to be triggered for release of an entrapped agent such as a drug.
- the present invention is based on the observation that an inorganic compound, i.e. the boron cluster compound disodium mercapto-undecahydro-dodecaborate (BSH) strongly interacts with a lipid bilayer. This interaction surprisingly leads to changes in the liposomes' structure. Prior to exposure to BSH, the liposomes exhibit a spherical structure, whereas after addition of BSH areas of thickened membranes as well as multilayer structures are observable, as well as aggregation of the liposomes, membrane fusion, liposome collapse and release of the liposomes' content; see Example 2 and Figure 1 a, b.
- BSH disodium mercapto-undecahydro-dodecaborate
- BSH is a boron cluster compound known from and used in the boron neutron capture therapy (BNCT), which is a specified form of neutron capture therapy (NCT).
- BNCT is a binary radiotherapy used for treatment of patients with malignant glioma.
- NCT neutron capture therapy
- BNCT a 10 B- containing compound is administered to the patient, in whom it accumulates preferentially into the neoplastic tissue. The tumor is then irradiated with low-energy neutrons produced by a nuclear reactor.
- boron- 10 absorbs neutrons and its subsequent decay emits energetic particles (charged particles) which can kill nearby tumor cells.
- the energetic and cytotoxic alpha particles and lithium-7 particles travel only about one cell diameter in tissue, preferably one may specify the cell type to be destroyed by placing the charged particle precursors only on or within the tumor cells.
- sufficient 10 B must be localized in a tumor to generate the required density of particles. Large numbers of boron containing compounds have been tested for their ability to satisfy the above criteria.
- the current boron carriers in clinical use are a boronated aromatic amino acid derivative, L-4-dihydroxyboryl phenylalanine, 4-borono- L-phenylalanine, L-BPA, and disodium mercaptoundecahydro-closo-dodecaborate (Na 2 10 Bi 2 HuSH, sodium borocaptate, sodium mercaptododecaborate, BSH); see for example Kageji et al, Biochim. Biophys. Acta 1391 (1998), 377-383; Alberts, Gabel and Moss, Boron Neutron Capture Therapy: Towards Clinical Trials of Glioma Treatment; Plenum Press, 1992.
- boron cluster compound as used in accordance with the present invention and in the appended claims naturally includes the use of suitable pro-drugs from which the active boron cluster compound is liberated.
- suitable pro-drugs from which the active boron cluster compound is liberated.
- One example and the preferred boron cluster compound to be used in accordance with the present invention has been described by Lechtenberg and Gabel for BSH in J. Organometallic Chemistry 690 (2005), 2780-2782.
- a glucuronidated derivative of BSH has been synthesized from which BSH could be liberated by tumor-inherently overexpressed glucuronidase.
- the extracellular conversion of a non- active pro-drug of the boron cluster compound to a lipid carrier release-triggering compound can be achieved by using a secreted form of the normally lysosomal human beta- glucuronidase to establish an extracellular effector system that converts an inactivated glucuronidated derivative of the boron cluster compound to the trigger-effective drug.
- a secreted form of the normally lysosomal human beta- glucuronidase to establish an extracellular effector system that converts an inactivated glucuronidated derivative of the boron cluster compound to the trigger-effective drug.
- any other suitable pro-drug of a boron cluster compound may be used within the scope of the present invention as long as the subject boron cluster compound liberated thereof exerts its desired effect, i.e. triggering the release of the liposome content.
- BSH exhibits surprising properties, rendering it useful as a trigger for the release of liposome content.
- this effect is thought to be due to the nature of BSH, being a twofold negatively charged anion, and therefore predestinated to interact with positively charged groups, i.e. cations, such as the positively charged part of the polar head group of the phospholipids in the bilayer.
- cluster compounds featuring another atom instead of or in addition to boron having similar chemical properties such as those listed in the same main group of the periodic table of the elements as boron or other equivalent compounds, in particular those which are capable of forming clusters may be also useful for the purpose of the present invention. Therefore, the present invention also encompasses the use of cluster compounds or compositions comprising said compounds, capable of effecting liposomal carriers in the way described, leading to changes in the membrane structure and finally to the release of the content.
- the boron cluster contains a closo-dodecaborate cluster.
- the boron cluster compound is disodium mercapto-undecahydro-dodecaborate (BSH).
- the present invention for the first time describes a substance release system which does not need the prerequisites described in the prior art as well as no further activation upon administration.
- the liposomes triggered for release do not need to be designed in a certain way to be sensitive to the trigger of the present invention.
- a special liposome design to be triggered for release by the boron cluster compound of the present invention is not required, it is not excluded from the scope of the present invention, since there may exist cases in which a special design of the liposomes can have a beneficial effect, such as accelerated or targeted release of the content.
- the present invention for the first time provides the use of a boron cluster compound for the preparation of a composition for triggering the release of an agent present in a liposome.
- boron cluster compounds such as BSH are known to be substantially non-toxic in humans, at least at the concentrations required in accordance with the present invention to trigger the release of the liposome encapsulated substance.
- boron cluster compounds as triggers for release according to the present invention exhibits major advantages compared to the triggering methods of the prior art, such as an easy way of administration. Since it is not toxic in the doses tested by the present inventors, it can be injected into the blood flow, i.e. be administered systemically, and therefore enables the treatment of hardly accessible cells, organs or tissues (or those which are not accessible at all), such as metastases in the field of cancer. Thus, no targeting of the boron cluster compound is required, rendering administration easy and avoiding any possibly undesired influence on the trigger (and therefore on the trigger capacity) upon targeting.
- the boron cluster compound and the composition comprising the boron cluster compound, respectively can - if desired - be targeted to special cells, tissues or organs, if it is intended by the application strategy used.
- the strategy comprises for example either the sequential administration of liposomes and boron cluster compound (or the composition comprising the boron cluster compound), wherein the boron cluster compound (or the composition comprising the boron cluster compound) for example is administered first, it may be targeted to the desired target cell, tissue, or organ, such as a tumor. After reaching the desired target, it "waits" for "arriving" liposomes which are administered later.
- the liposomes may also be targeted either to the tumor or the boron cluster compound and the corresponding composition, respectively, or the liposomes are not targeted and reach the desired area by systemic distribution.
- both components liposomes and boron cluster compound
- both components may be administered concurrently and may be targeted for the purpose of accelerated delivery to the desired cell, tissue, or organ.
- the invention relates to a composition, wherein the composition is a diagnostic or pharmaceutically active composition and more preferably wherein said composition is designed to be administered systemically.
- the composition is a diagnostic or pharmaceutically active composition and more preferably wherein said composition is designed to be administered systemically.
- liposomes generally depend on pH and ionic strength. They characteristically show low permeability to ionic and polar substances, but at certain temperatures can undergo a gel-liquid crystalline phase transition dependent upon the physical properties of the lipids used in their manufacture which markedly alters their permeability.
- the phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less ordered structure, known as the liquid crystalline phase.
- Various types of lipids differing in chain length, saturation, and head group have been used in liposomal drug formulations for many years, including the unilamellar, multilamellar and multivesicular liposomes, described in the definitions; see supra.
- phospholipids by themselves are sufficient for the formation of liposomes, some of the properties of the latter can be improved upon by the incorporation of other lipid soluble compounds into the liposomal structure.
- the stability of the phospholipid bilayers can be altered by the inclusion of for example a sterol, and the incorporation of for example a charged amphiphile can not only render the liposomal surface positively or negatively charged, it can also increase the distance, and hence aqueous volume, between the bilayers. It is obvious that it is no undue burden for those skilled in the art to select the appropriate liposome, i.e. the liposome having the appropriate properties for the purposes they are intended to be used for.
- boron cluster compound and as mentioned supra, of the present invention, without intending to be bound by theory, also other boron cluster compounds can be used which are capable of achieving the desired effect, i.e. interacting with liposome's membrane and thereby releasing the encapsulated content.
- This interaction may in one embodiment lead to aggregation of the liposomes, but is not restricted to this effect, since any other effect is assumable and included within the scope of the invention which leads to leakage or other release of the liposomes content upon addition of the trigger, such as the boron cluster compound.
- the release of the liposomes content can be triggered in any type of lipid vesicle, such as ULV (unilamellar vesicle), MLV (multilamellar vesicle), MVL (multivesicular liposomes), sandwich liposomes, micelles (some of which are further characterized in the definitions; see supra), and any membrane bilayers including an aqueous cavity.
- ULV unilamellar vesicle
- MLV multilamellar vesicle
- MVL multivesicular liposomes
- sandwich liposomes micelles (some of which are further characterized in the definitions; see supra)
- micelles some of which are further characterized in the definitions; see supra
- any membrane bilayers including an aqueous cavity.
- liposomes Since either as multilamellar or unilamellar vesicles, liposomes have proven valuable as vehicles for drug delivery in animals and in humans, a variety of agents can be trapped in the aqueous core of the liposome, while hydrophobic substances can be dissolved in the liposome bilayer membrane.
- the liposome structure can be readily injected and can form the basis for both sustained release and drug delivery to specific cell types, or parts of the body.
- Multilamellar vesicles primarily because they are relatively large, are usually rapidly taken up by the reticuloendothelial system (the liver and spleen).
- the invention typically utilizes vesicles which remain in the circulatory system for hours, such as pegylated liposomes (stealth liposomes) and preferably unilamellar vesicles having a diameter of less than 250 run, and more preferably about 100 nm.
- vesicles which remain in the circulatory system for hours, such as pegylated liposomes (stealth liposomes) and preferably unilamellar vesicles having a diameter of less than 250 run, and more preferably about 100 nm.
- the liposome of the present invention can comprise any combination of lipids, preferably it comprises a mixture of lipids, more preferably it comprises amphipathic lipids, most preferably dipalmitoylphosphatidylcholine (DPPC), whereas in another embodiment of the present invention the liposome comprises positively charged lipids, preferably N-[I -(2,3- Dioleoyloxy)]-N,N,N-trimethylammonium propan (DOTAP) methylsulfate. Methylsulfate can be exchanged for other anions.
- DPPC dipalmitoylphosphatidylcholine
- DOTAP dipalmitoylphosphatidylcholine
- Methylsulfate can be exchanged for other anions.
- the liposome of the invention comprises dipalmitoylphosphatidylcholine (DPPC) and l,2-disteaoryl-sn-glycero-3-phosphatidyl- ethanolamine-N[methoxypoly(ethylene glycol)] (DSPE-PEG).
- DPPC dipalmitoylphosphatidylcholine
- DSPE-PEG l,2-disteaoryl-sn-glycero-3-phosphatidyl- ethanolamine-N[methoxypoly(ethylene glycol)]
- This class of liposomes has been shown to be not capable of aggregating and more importantly, to have increased circulation times in blood. For some applications it may be desirable to use this kind of liposomes. Accordingly, the liposomes to be used in accordance with the compositions, methods and uses of the present invention may be chosen in view of the intended application.
- long-circulation and short-circulation liposomes may be used.
- the liposomes and/or the boron cluster compound can be targeted, i.e. actively directed to a desired cell, tissue, or organ.
- the liposomes and/or the boron cluster compound used according to the invention may be modified to comprise targeting agents, wherein "targeting” includes for example decorating the outside of the liposome with one or more ligands specific for a particular target site.
- targeting agents include for example decorating the outside of the liposome with one or more ligands specific for a particular target site.
- a variety of targeting agents that direct pharmaceutical compositions to particular cells as well as means of coupling targeting molecules to liposomes are known in the art; see, for example, Cotten et al, Methods Enzym. 217 (1993), 618; U.S. patent number 5,258,499.
- the targeting agents may be included throughout the particle or may be only on the surface.
- the targeting agent may be used to target specific cells or tissues or may be used to promote endocytosis or phagocytosis of the liposome.
- targeting agents include, but are not limited to natural and synthetic peptides, hormones, antibodies (as for example described in patent application US 2002/0035084), fragments of antibodies, low-density lipoproteins (LDLs), transferrin, proteins, asialycoproteins, gpl20 envelope protein of the human immunodeficiency virus (HIV), carbohydrates residues, substrates for enzymes highly expressed on the tumors surface, natural and synthetic ligands such as receptor ligands or other receptor-binding molecules, receptor-binding proteins, specific cell surface receptors, glycoproteins, lipids, small molecules, etc.
- the targeting agent may be included in the bilayer. If the targeting agent is only on the surface, the targeting agent may be associated with liposomes (i.e., by covalent, hydrophobic, hydrogen bonding, van der Waals, or other interactions) using standard chemical techniques.
- the targeting molecule comprises an antibody, receptor binding protein or peptide or a carbohydrate residue.
- any suitable liposome or equivalent complex capable of encapsulating a given substance can be used in accordance with the present invention.
- reference is also made to the pertinent scientific and patent literature concerning the provision of appropriate liposomes for example international application WO2000/051565 which describes a method to prepare liposome-encapsulated bioactive agents such as nucleic acids; international application WO2001/037807 describing liposomes including a fusogenic liposome, a linking moiety and a targeting moiety, wherein the fusogenic liposome is a lipid bilayer encapsulating contents.
- the liposome containing pharmaceutical composition can be administered systemically and does not necessarily need to be targeted, although targeting is also included within the scope of the present invention.
- agents with which they can be “loaded” encompass a wide variety.
- the agents to be delivered and released by the trigger of the present invention may be investigative, toxic, therapeutically active, diagnostic, or prophylactic agents.
- the agent is a pharmaceutically active agent.
- Suitable therapeutically active agents include for example but are not limited to inorganic and organic chemical entities, biologically active substances, such as human genes, proteins, enzymes, and the like, as well as genetically engineered copies of the same, nutrients, vitamins, minerals, other plant and animal substances, a clinically used drug that has been approved by the FDA, such as an antibiotic, anti-viral agent, anesthetic, steroidal agent, anti- inflammatory agent, anti-neoplastic agent, antigen, vaccine, antibody, decongestant, antihypertensive, sedative, birth control agent, progestational agent, anti-cholinergic, analgesic, anti-depressant, anti-psychotic, p-adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, non-steroidal anti-inflammatory agent, nutritional agent, etc., or a mixture of pharmaceutically active agents, like for example, two or more antibiotics may be combined in the same microparticle, or two or more anti-neoplastic agents may be combined in the
- antifungals include further topically applied antifungals, antiarthritics, corticosteroids, whitening agents, antioxidants, polyphenols, nitrus oxide, moisturizers, anabolics, analgesics, anesthetics, antiasthmatics, antibacterial, antihistaminics, antiparasitics, vasodilators, vasoconstrictors, anti-tumor, i.e., seborrheic keratosis to malignant tumors such as basal cell carcinoma, anti seborrheic, anti-vertigo such as compazine, anti insects, or toxins, such as botox (nerve paralysis), hormones such as estrogen, nicotine, amino acids, lipids, herbs, metabolite supplements, and other therapeutic or medicinal compositions.
- antifungals include further topically applied antifungals, antiarthritics, corticosteroids, whitening agents, antioxidants, polyphenols, nitrus oxide, moisturizers, anabolics,
- a chemotherapeutic agent may be the therapeutically active agent and be encapsulated within a liposome, thereby sequestering its toxic effects from non-targeted tissues.
- the agent is a therapeutically active agent, preferably an anti tumor agent, although the precise compounds to be used will occur to those skilled in the art according to the problem to be solved.
- Suitable diagnostic agents include for example but are not limited to agents that can be radio labelled, fluorescently labelled, enzymatically labelled and/or include magnetic compounds, gases; commercially available imaging agents used in positron emissions tomography (PET), computer assisted tomography (CAT) and x-ray imaging, such as iodine-based materials, single photon emission computerized tomography, fluoroscopy, and magnetic resonance imaging (MRI), such as gadolinium chelates, ultrasound, and computed tomography (CT).
- PET positron emissions tomography
- CAT computer assisted tomography
- MRI magnetic resonance imaging
- gadolinium chelates such as gadolinium chelates, ultrasound, and computed tomography (CT).
- the invention relates to a composition, wherein the agent is a diagnostic agent, preferably a dye, more preferably a fluorescent dye and most preferably carboxyfluorescein or calcein.
- Suitable prophylactic agents include for example but are not limited to vaccines, which may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, and cell extracts. Vaccines may also include polynucleotides which encode antigenic protein or peptides. Prophylactic agents may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, Freund's adjuvant, etc.
- Prophylactic agents include antigens of such bacterial organisms as Streptococccus pnuemoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridiumbotulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonasaeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholera, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum
- antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof. More than one antigen may be combined in a particular liposomal carrier, or a pharmaceutical composition may include liposomes each containing different antigens or combinations of antigens.
- the present invention additionally relates to a composition
- a composition comprising as a first component an inorganic compound, i.e.
- boron cluster compound and as a second component a liposome as described hereinbefore, preferably wherein said liposome comprises a therapeutically active or a diagnostic agent as defined above and in a further embodiment the above compositions are designed to be administered or applied concurrently or sequentially.
- the present invention relates in a further embodiment to a method of releasing an agent from a liposome under predetermined conditions comprising the step of exposing the liposome to a boron cluster compound such as to release the agent.
- boron cluster compounds as described hereinbefore for triggered release of a liposome-encapsulated agent and the composition, comprising the boron cluster compound, respectively can of course also be used for further investigations, such as in vitro assays, like for example cell culture experiments, to trigger release of an liposome encapsulated agent, said agent intended to be tested concerning its (for example therapeutic) effect on the cells and cell culture, respectively after release.
- the present invention further provides a method of identifying and obtaining a therapeutically active agent comprising
- cell culture such as suspension cultures, callus-cultures, as well as tissue cultures or cultivated organs, as well as any type of cells, such as endodermal cells, ectodermal cells, mesodermal cells, stem cells, tumor cells, such as cancer cells etc., just to name a few.
- cell line V-79 derived from the lung tissue of a Chinese hamster is used; see also example 4.
- This cell line as well as other cell lines which are suitable for the screening methods of the present invention can be ordered from official depositary institutions, for example the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Culture (DSMZ).
- ATCC American Type Culture Collection
- DSMZ German Collection of Microorganisms and Cell Culture
- the method further comprises synthesizing the therapeutically active agent determined in step (c) in a therapeutically effective amount.
- a therapeutically effective amount means the total amount of the therapeutically active agent, also called drug or pro-drug that is sufficient to show a meaningful patient benefit, i.e. treatment, healing, prevention or amelioration of for example damaged tissue, or an increase in the rate of treatment, healing, prevention or amelioration of such conditions.
- therapeutically effective amount includes the total amount of the drug or pro-drug that is sufficient to elicit a physiological response in a non-human animal test.
- the appropriate concentration of the therapeutic agent might be dependent on the particular agent.
- the therapeutically effective dose has to be compared with the toxic concentrations; the clearance rate as well as the metabolic products play a role as to the solubility and the formulation.
- Therapeutic efficacy and toxicity of compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50 % of the population) and LD50 (the dose lethal to 50 % of the population).
- the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
- the liposomes and/or the boron cluster compounds may be formulated and administered systemically, topically, orally, pulmonary or otherwise locally.
- suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intra-lesional, or intraocular injections, just to name a few.
- the present invention provides in a further embodiment a method of obtaining a genetically altered cell comprising any one of the above described methods.
- the method of the present invention may be used to deliver substances capable of inducing mutations into the genome of a given cell thereby generating spontaneous cell lines that exhibit new properties.
- recombinant DNA constructs may be delivered in order to transform a given target cell and to come up with a pre-determined genetically engineered cell.
- the recombinant DNA construct preferably comprises a selectable marker gene and thus may be encapsulated preferably with a selective agent into the liposome. Accordingly, the present invention also provides transgene delivery systems which allow both in time, i.e. transformation and selection for transformants. Means and methods for preparing recombinant DNA constructs for this purpose including selective agents are well known to the person skilled in the art and are described in the literature including those cited below, for example Sambrook et ⁇ /., (1989).
- the present invention further relates to a method of assaying liposomes or agents encapsulated by liposomes, comprising subjecting the liposome to a boron cluster compound. This includes for example assaying the liposomes with respect to: stability, size, biodegradability, capability of encapsulating the desired agent, leakage, shelf-life, etc.
- assaying parameters concerning the encapsulated agent may be: effectivity after release, intactness after release, tolerance, shelf-life within the liposome or lipid vesicle, biodegradability, uptake by the cell, optionally undesired chemical modifications upon uptake in or release from the vesicle used, such as a liposome, rate of release, amount of release, just to name a few.
- kits for using in the methods described above comprising a liposome, optionally containing an agent, a boron cluster compound, and optionally means for detection. It may further comprise the liposome structures ready for the user to add the biological reagent of interest, as well as one or more specific biologically-active reagents for addition to the liposome structure.
- Another kit of the present invention may comprise a set of lipid carriers, such as liposomes each containing a specific, biologically-active reagent, which when administered together or sequentially, are particularly suited for the treatment of a particular disease or condition.
- Such kits would typically comprise a compartmentalized carrier suitable to hold in close confinement at least one container and the compounds of the kit may be sterile, where appropriate.
- the kit may further include a transfer means, such as pipets, for transferring the suspended cells.
- the kit's carrier could further comprise reagents useful for performing said methods and may also contain means for detection such as labeled enzyme substrates or the like. Instructions can be provided to detail the use of the components of the kit, such as written instructions, video presentations, or instructions in a format that can be opened on a computer (e.g. a diskette or CD-ROM disk).
- a computer e.g. a diskette or CD-ROM disk.
- gene therapy approaches for treating cancer which can employ the triggered release by the boron cluster compound of the present invention including for example but not being limited to: antisense or ribozyme therapy (to block synthesis of proteins encoded by deleterious genes), chemoprotection (to add proteins to normal cells to protect them from chemotherapies), immunotherapy (to enhance the body's immune defenses against cancer), pro-drug or suicide gene therapy (to render cancer cells highly sensitive to selected drugs), tumor suppressor genes (to replace a lost or damaged cancer-blocking gene), antibody genes (to interfere with the activity of cancer-related proteins in tumor cells) and oncogene down-regulation (to shut off genes that favor uncontrolled growth and spread of tumor cells) (Blaese, Scientific American 6 (1997), 111), the present invention also concerns other diseases, such as diabetes, atherosclerosis, chemotherapy- induced multi-drug resistance, and generally, immunological, neurological (Ho and Sapolsky, Scientific American 6 (1997), 116) and viral diseases (Friedmann
- liposomes as carriers for agents, such as therapeutic agents could indeed be a useful tool in drug delivery, but to date is hampered by the unsatisfying release of the encapsulated agents.
- the triggers for release used in the prior art either rely on properties of the diseased tissue such as different pH value or presence/overexpression of specific enzymes, both of which are difficult to manipulate, or require knowledge about the location of the tumors or the cells, tissues, or organs, which are intended to be treated, as well as some of the triggers require a number of prerequisites, such as accessibility of the desired target, knowledge of a special type of tumor (i.e.
- the present invention provides means and methods for triggering the release of for example therapeutic agents in or at least in the vicinity of metastases, which is one major advantage compared to the prior art lacking this feature, the present invention may for instance be used as a tool for the detection of alteration, aberrations, disorders, cellular abnormalities, and the like at sites which are not accessible to prior triggers of release, and therefore, may also enable the diagnosis of for example metastases which are maybe not detectable or locatable by common means.
- a diagnostic agent such as a labelled tumor-marker
- the present invention provides new perspectives to directly affect metastases, such as killing the cells of the secondary tumor, as they are now -using the means and methods provided by the present invention- accessible for cytotoxic agents, which can be delivered and released without doing damage to non-diseased cells.
- compositions, methods and uses in accordance with the present invention can be generally applied to the diagnosis and therapy of any disease or condition for which a rather targeted approach is needed or advantageous.
- a non-limited list of diseases and disorders which may be diagnosed or treated in accordance with the present invention is set forth below.
- disorders to be treated in accordance with the method of the present invention which include, but are not limited to, an immune system disorder such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis,
- an immune system disorder
- the present invention generally relates to the use of any compound, i.e. inorganic compound with a special emphasis on boron cluster compounds such as BSH or components thereof, liposomes, substances, i.e. therapeutically active or diagnostic agents for use in the above described embodiments.
- the present invention relates to the use of a liposome of a pharmaceutical or diagnostic composition, encapsulating an agent, wherein the liposome is designed for being triggered to release the agent by a boron cluster compound.
- the triggering effect is not restricted to the release of substances, since those skilled in the art may recognize that the opening of the liposome's membrane can also be exploited for other purposes than releasing agents, such as maybe the uptake of substances, if a subsequent "closing" of the liposomes is achieved. It may further be intended to render this triggering effect (opening of the membrane) reversible.
- agents could be encapsulated in liposomes and released by the boron cluster compound of the present invention, to monitor the influence of encapsulating and releasing of such an agent, i.e. whether the released agent is identical to the one which was encapsulated, thereby investigating, whether the drugs reach their target having their original chemical identity or if they are altered. This would also be an important feature, since in terms of therapeutically active drugs, they need to reach the target in their therapeutically active form; otherwise their delivery towards diseased sites is useless.
- the public database "Medline” may be utilized, which is hosted by the National Center for Biotechnology Information and/or the National Library of Medicine at the National Institutes of Health. Further databases and web addresses, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL) are known to the person skilled in the art and can also be obtained using internet search engines.
- EBI European Bioinformatics Institute
- EBL European Molecular Biology Laboratory
- Example 1 Preparation of liposomes containing carboxyfluorescein or calcein
- liposomes were prepared according to standard procedures. Briefly, to obtain a lipid film, an appropriate amount of lipid and optionally further compounds can be weighed out and subsequently be dissolved in appropriate volumes of chloroform and methanol. The mixture of solvents can afterwards be removed by use of a rotary evaporator under vacuum and the thusly generated lipid layer can be further dried by use of an oil pump.
- Hydration of the lipid layer is achievable by adding corresponding volumes of a 100 mM carboxyfluorescein-solution or a 0.1 mM calcein-solution.
- the whole mixture can be heated in a water bath to a temperature being preferably at least 10 °C higher than the liposome's phase transition point. It is known to those skilled in the art that this cycle of "heating and vortexing" is repeated until the lipid film is completely dissolved generating a lipid emulsion.
- freeze-thaw cycle This procedure is often followed by a freeze-thaw cycle, which is optionally run 5 times, i.e. transferring the sample into a cryo-tube and freezing it in liquid nitrogen for two to three minutes, and thawing the samples for about 60 seconds in a water bath. Any subsequent step is carried out 10 0 C higher than the liposome's phase transition point.
- Thawing the sample is usually followed by vortexing, a procedure after which the sample comprises multilamellar and unilamellar liposomes exhibiting a wide size range. To exclusively obtain unilamellar liposomes of a defined size, this mixture is extruded, a technique which is known to those in the art.
- this mixture can be enriched for unilamellar liposomes.
- the sample containing the differently sized liposomes is usually pressed through said membrane using two syringes at each side of the extruder, a procedure in the course of witch the liposomes are forced to pass through the membrane, thereby stripping off the multiple lipid layers resulting in unilamellar liposomes.
- the liposomes being flexible enough and capable of passing the pores of the membrane, they usually are heated up to their phase transition temperature, a fact known to those of skill. Therefore, the extruder will be put into a water bath and heated up to the corresponding temperature. Liposomes can be pressed through the membrane for several times (up to about 20 times).
- the obtained unilamellar liposomes (having a size of about 100 nm) are separated from the not encapsulated, i.e. free carboxyfluorescein by gel filtration, using for example a Sephadex G-25 column. Separation of liposomes and free dye can be followed by visual inspection. Where calcein is used as fluorescent dye, it can be removed by using G50 Sephadex spin- columns, loading the liposome samples onto the G50 column and spinning for example for two minutes at 1000 rpm.
- Lipid concentration is determined by Stewart assay, a colorimetric method commonly used to determine phospholipid (phosphatidylcholine) concentrations. Usually for the determination of choline, DPPC is used as a standard. Stewart assay is suited for determination of phosphatidylcholine content of samples in the presence of inorganic phosphate. The test exploits the tendency of lipids to form complexes with ammonium iron(III)thiocyanate. Since complex formation depends on the head group of the phospholipids, this test is not suitable for analysis of samples, containing an unknown mixture of different phospholipids. Since concerning the present invention, only liposomes of known compositions have been measured, Stewart test was the appropriate method.
- Such a test can be carried out using an aqueous solution of 0.1 M iron chloride and 0.4 M ammonium thiocyanate, subsequent mixing with the test liposome solution and chloroform, followed by short vortexing and centrifugation for 10 minutes at 2700 rpm (for example using a Heraeus Biofuge 15 R, Rotor HSA 4100).
- Optical density of the lower chloroform phase will be measured using a photometer at 485 nm compared to the blank and concentration of the test samples will be calculated by comparison with the calibration curve obtained for the standard samples of known concentrations.
- DPPC dipalmitoylphosphytidylcholine
- DOTAP dipalmitoylphosphytidylcholine
- DPPC-DSPE-PEG 2OO o-IiPOSOIrIeS containing dipalmitoylphosphytidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylenglyco ⁇ ooo in a molar ratio of 98:2 were prepared as described above, using the corresponding components. Extrusion was carried out in the presence of carboxyfluorescein (concentration 10OmM) and liposomes were freed from excessive carboxyfluorescein by gel filtration.
- Example 2 Determination of structural changes of liposomes upon addition of BSH using cryo-Transmission Electron Microscopy (TEM) Liposomes (lipid concentration 10 mM) were incubated at 45°C in 0.1 M Tris buffer pH 7.4. The samples were prepared for cryo-transmission electron microscopy 36 hours after mixing. From independent experiments it is known that the major changes occur within a few minutes. For liposomes prepared from other lipids with lower phase transition temperatures, the same effect is achieved with lower temperatures. After addition of mercaptoundecahydro- closo-dodecaborat (BSH) the liposomes, having a spherical shape prior to BSH addition (see Fig.
- BSH mercaptoundecahydro- closo-dodecaborat
- Example 3 Monitoring calcein release from DPPC/DOTAP liposomes after exposure to BSH using fluorometry
- Liposomes from DPPC containing 5 mol% DOTAP were prepared by extrusion in the presence of 100 ⁇ M calcein and freed from excessive calcein by gel filtration.
- BSH final concentration 16 mM
- Triton X -100 at a final concentration of 0.1 % was added to completely destroy the liposomes.
- WST-I test is a non radioactive method to test and quantify cells for proliferation and viability. It is based on measuring the activity of the mitochondrial succinate-tetrazolium reductase system of cells by measuring the metabolization of the WST-I reagent (which is slightly red) to formazane (which is deep dark red). The change in colour which can be measured, using enzyme-linked immunosorbent assay (ELISA)-reader at a wavelength of 420 nm to 480 run and is a measure for the amount of metabolically active mitochondria of cells. As described by Ishiyama et al, Biol. Pharm. Bull.
- the WST-I test can either be used to test for cell proliferation in response to stimulation with for example growth factors or cytokines, or for detection of cell death by addition of fore example apoptosis reducing agents or chemotherapeutics.
- V-79 cells were cultivated in 96-well plates (8000 cells per well).
- doxorubicin-filled liposomes were added to the cultivation medium (total doxorubicin concentration 0.9 ⁇ M), followed by BSH at a concentration ranging from 0 mM to 20 mM.
- BSH total doxorubicin concentration 0.9 ⁇ M
- the medium was removed after 24 hours, and the viability was assayed with the WST assay.
- WST-I reagent can be added in an amount corresponding to 10% of total reaction volume and plates can be further incubated.
- absorption is measured in an ELISA-reader at a wavelength of 420 nm to 480 nm, see supra.
- ODsampie optical density of treated cells OD 1OO r 0 : optical density of untreated cells (controls)
- DPPC-DSPE-PEG 2OO o-IiPOSOnIeS containing dipalmitoylphosphytidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylenglyco I 20 O O in a molar ratio of 98:2 were prepared as described hereinbefore, see supra.
- the above described experiments performed in accordance with the present invention demonstrate that substances encapsulated in custom designed liposomes can be specifically triggered to the release with an inorganic compound, i.e. the boron cluster compound, BSH. Moreover, it could be shown that this novel delivery system can be used effectively to target and kill the predetermined cells. Accordingly, the delivery system presented in accordance with the present invention can be used in many ways including diagnostic and therapeutic uses but will also be a valuable tool in basic research.
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Abstract
A compound triggered release system for liposome-encapsulated agents is provided. Furthermore, diagnostic and therapeutic uses of this novel delivery system are described.
Description
Triggered release of liposome contents
Field of the invention
The present invention relates to drug delivery systems using compositions of lipid carrier, such as liposomes or liposomal formulations or compositions. In particular, the present invention relates to compounds and methods for triggering the release of agents encapsulated by lipid carriers under pre-determined conditions. Such pre-determined conditions may be the release at a pre-determined site or the release in the presence of a pre-determined material, i.e. by the choice of the trigger, or both. More particular, it relates to the release of an agent for therapeutic, diagnostic, or investigative purposes. In particular, the present invention relates to the use of boron cluster compounds in drug discovery, to preparation of pharmaceutical compositions containing the boron cluster compound and to methods for drug release using said boron cluster compound.
Background of the invention
A common manner of administering agents, such as drugs and dyes to the body is via injection into the bloodstream or a localized area in the body. When injected into the bloodstream, the injected material is carried throughout the blood system and therefore the entire body, known as systemic delivery. The drawbacks to this delivery system are that the concentration of the injected material is extremely diluted and the material acts on most tissues in the body and may be toxic to some of them. In addition, the location and duration of exposure of these injected materials cannot be controlled.
Since optimal treatment of a variety of diseases, requires both maintenance of a drug level for a prolonged period of time and drugs being precisely administered to the desired cell, tissue, or organ, "vehicles" have been designed allowing the exact delivery or transport to the desired site of activity. Ideal delivery "vehicles" should be bio-absorbable, non-toxic, non- immunogenic, stable during storage and, after administration, able to access target cells. Additionally, especially the triggered release of the "vehicle"-encapsulated agent in a controlled fashion at the site of action to enable the for example therapeutic agent to locally exert its effect is one important feature. Since lipid particles have been shown to be efficient
"vehicles" for many in vitro and in vivo applications, this can be realized by encapsulating drugs in lipid vesicles, such as liposomes.
Beside the difficulty to direct the "liposome" containing compositions or formulations to a specific target side (i.e. challenged cell tissues or organs), the other major difficulty is to trigger the release of the liposome's content at the site of activity. Several methods of controlled drug release delivery have been employed, for example the release by exposing the liposome composition to a number of different triggers, such as acidic environment (pH- value), to irradiation, to heat, as well as to enzymes or enzyme activating agents, all indeed resulting more or less in the destruction or rearrangement of the liposome membrane, thereby releasing the encapsulated agent, but exhibiting severe drawbacks, as will be described infra.
Especially in the field of disease therapy, more particularly cancer therapy, tumor specific drug delivery has become of increasing interest, since the use of chemotherapeutics is often limited due to their severe side-effects. As conventional drug delivery systems have shown low efficiency, alternative drug delivery principles were needed, and therefore liposome- based drug delivery has been introduced on the market, and are still being developed as an alternative way of administering therapeutically active agents.
Thus, one major requirement, among others, for a successful treatment using a liposome- based strategy is that the cells, tissues, or organs challenged with cancer can be exposed to a high concentration of a therapeutically active agent without doing irreversible harm to the host, i.e. without affecting the surrounding healthy cells, tissues, or organs.
Since cancer is still a life-threatening disease which causes more than ten million deaths every year worldwide the number of which is still growing. Local treatment is usually effected by surgery and radiotherapy. For chemotherapy, there is to date still no general appropriate treatment available which is well tolerable and effective at the same time, since the clinical use of most conventional chemotherapeutics is often limited either due to inadequate delivery of the required drug concentrations to the tumor target tissue or due to severe and harmful toxic effects on healthy cell tissues or organs. Therefore, liposomes were suggested as a sort of micro carrier-technology, useful as drug carriers in cancer chemotherapy, the use of which requires first, prolonged blood circulation of the liposomes, second, a sufficient accumulation
at the sites of challenged cells, tissues, or organs, i.e. for example tumors, and third, the controlled release of the drug and uptake of the same by the tumor.
In this context, earlier described limitations on the application of liposomal vesicles in patients due to the relatively fragile nature of liposomes and the very fast blood clearance by the reticuloendothelial system (RES), respectively have been more or less overcome by use of for example pegylated liposomes, i.e. stealth liposomes. Stealth liposomes contain a "stealth component", such as monosialoganglioside, or polyethyleneglycol-conjugated lipids such as polyethyleneglycol-distearoylphosphatidylethanolamine or -ceramide both having a strong influence on the clearance profile. This is one basic requirement to provide controlled and increased concentration of active agents at or near the desired cells, tissues, or organs.
Concerning the second prerequisite, i.e. the accumulation of the liposomes at the sites of effected cells, tissues, or organs, it was found that liposomes significantly accumulate in tumors due to a leaky vasculature of the tumors and the lack of an effective lymphatic drainage system.
However, the third demand for successful drug delivery by liposomal carriers, i.e. the release of the encapsulated drug still remains one major problem. Several strategies have been suggested to trigger the release, each bearing severe drawbacks. Many drugs are released fast enough to give a therapeutic concentration of the drug, but this occurs indiscriminately also during circulation of the liposome. Other liposomes are so stable that they do not release their contents at the site of action. Since this problem, i.e. the release of the encapsulated agent is not yet satisfactorily solved, the site-specific delivery of the liposomes to the desired cells, tissues, or organs for example by targeting the liposomes to the sites of activity, which was discussed as a big improvement for accelerated and selective drug delivery, is of less value, as without either retaining the encapsulated drug until it reaches the tumor, or releasing the encapsulated agents once the liposomes have reached the tumor site, the liposomes are more or less useless.
As mentioned before several strategies have been proposed to accomplish site-specific triggered drug release in tumor tissue for example triggering by acid (i.e. pH value), radiation (such as laser or microwave irradiation) heat (i.e. temperature), light (such as UV light),
photo oxidation, or enzyme-mediated triggering. Each of the methods suggested to date, for triggered release exhibit severe drawbacks, a detailed discussion of which is presented later.
Briefly, one of the major drawbacks of the prior triggering methods is that they all require either the targeting of the trigger (towards the liposomal carrier or desired cell, tissue, or organ) or predetermined (and therefore specially modified) properties of the liposomes used, which can then be exploited by an external stimulus, having the disadvantage that the reach of most of the external triggers is poor and therefore their effect is locally restricted. Thus, they cannot trigger drug release from liposomes at sites which are either not yet known or only hardly (if at all) accessible to the external stimulus. Alternatively, when internal triggering mechanisms such as low pH values or specific enzymatic setup of the target tissue are used, these cannot be manipulated. For that reason, treatment of metastases (as an example in the field of cancer for a tumor which is inaccessible, and/or the location or even existence of which is unknown) is not possible with external triggers, and cannot be controlled when using internal triggers.
Since most of the cancer patients do not die from the primary tumor but rather of metastases, which are often located at sites rendering them inoperable or which more often are not yet detected or localized, an approach is needed which is independent of the aforementioned requirements, e.g. knowledge of location and accessibility of the tumor or metastasis. Therefore, there is a need in the art for improved methods for the release of encapsulated agents.
As it is known that liposomes can accumulate at the sites of tumors due to the tumor's leaky vasculature and the lack of an effective lymphatic drainage system, it is one major object of the present invention to provide a trigger for release of encapsulated agents and a composition containing the trigger, respectively, which is independent of the aforementioned requirements, and allows the release of the encapsulated agents even at sites which are not accessible to the triggers of the prior art, i.e. at or at least in the vicinity of hardly (if at all) accessible cells, tissues, or organs, such as metastases. This would be an important step forward in the field of cancer treatment, since metastases are those tumors where most of the patients suffering from cancer die from.
The present invention solves the problems of unsatisfactorily (since insufficient and therefore ineffective) release of liposomal carrier-encapsulated substances, and permits said substances to be released at or at least in the vicinity of the specific and desired target sites by the administration of a boron cluster compound, triggering the release of the liposome- encapsulated substance, allowing the substance to exert its desired effect substantially only on the targeted cells, tissues, or organs.
Summary of the invention
Lipid carriers, such as liposomes offer considerable promise as vehicles for delivery of agents for use in a variety of applications including biochemical and immunological assays, diagnosis, and also pharmaceutical delivery systems. Unfortunately their application is undermined by the difficulty associated with selectively releasing their contents at a specific time or location.
The present invention relates to means and methods to overcome the mentioned difficulties. More specifically, the present invention provides a pre-determined substance delivery system in which liposome encapsulated substances are triggered to be released by a rather small, preferably non-toxic inorganic compound. While previous drug delivery systems relied on triggering stimuli, such as irradiation or physiological changes in the environment of the targeted tissue, the present invention makes use of a class of compounds that have been surprisingly found to specifically interact with liposomes so as to cause them to set their content free.
In particular, the present invention is directed to the use of boron cluster compounds to trigger the release of agents encapsulated in lipid carriers, allowing to trigger the release even at sites, which are hardly (or even not at all) accessible to external triggers.
The present invention further relates to a composition comprising as a first component a boron cluster and as a second component a liposome according to the present invention. This composition may include liposomes encapsulating an agent and the components can be administered or applied concurrently or sequentially.
In a further aspect, the present invention relates to methods for releasing encapsulated agents from lipid carriers, such as liposomes by exposure to boron cluster compounds for identifying
and obtaining a therapeutically active agent, for synthesizing a therapeutically active agent in a sufficient amount, for obtaining genetically altered cells, and for assaying liposomes or their encapsulated agents by exposure to a boron cluster compound.
Furthermore, the present invention relates to the use of a boron cluster compound or components thereof, a liposome and an agent for the use in the above-mentioned methods.
Moreover, the present invention relates to the use of liposomes which are designed to release their encapsulated agent upon a trigger by boron cluster compounds.
The present invention provides the possibility not only to treat primary tumors but rather also to treat metastases, one major problem in cancer therapy. Although the present invention relates in some aspects to the treatment of cancer, it is not at all limited to this disease, as will be clear from the following.
Brief description of the drawings
Fig. 1: illustrates the aggregation and structural transformation of DPPC-liposomes after exposure to mercaptoundecahydro-closo-dodecaborat (BSH). Liposomes after incubation at a lipid-concentration of 10 mM at 45 °C in 0.1 M Tris buffer pH 7.4 and preparation for cryo-transmission electron microscopy 36 h after mixing are shown, demonstrating the change of liposomal shape from a spherical form (A), prior to BSH addition, to a more flattened structure (B) after BSH addition. Additionally multilayer structures and thickened membranes are observed.
Fig. 2: illustrates the release of liposome-encapsulated calcein at 37°C. It is shown that the fluorescence of the fluorescent dye calcein is only detectable when encapsulated in the DPPC/DOTAP-liposomes. The x-axis refers to time and the y-axis refers to fluorescence intensity. After exposure to BSH and Triton, respectively, fluorescence decreases, since it is effectively abolished by the surrounding Co2+-ions. The determined calcein released during the 600 s was 87 % of the total amount.
Fig. 3: illustrates the results of a cell survival experiment, measured by the WST assay. Survival of V79 cells incubated with BSH in the absence (open symbols) and presence
(closed symbols) of doxorubicin-containing DPPC liposomes was measured. The curve reflects cell survival, ranging from 0% to 100% (y-axis) plotted against the concentration of BSH, ranging from 0 mM to 20 mM in the incubation medium after incubation for 24 h.
Fig. 4: illustrates the release at 37°C of carboxyfluorescein encapsulated in DPPC-DSPE- PEG2ooo-liposomes. It is shown that the fluorescence of carboxyfluorescein is only detectable after exposure to BSH and Triton, respectively. Fluorescence increases, since the quenching of fluorescence when encapsulated in the liposome is abolished. The x-axis refers to time and the y-axis refers to fluorescence intensity. BSH is added after t = 120 s in final concentrations ranging from O mM to 32 mM. At t = 240 s Triton is added to the liposomes.
Definitions "Liposome", as the term is used herein, generally refers to any lipid carrier which is suitable for the purposes of the present invention.
In general, lipid carriers as used herein can comprise a variety of lipid aggregates which may all be used for the purpose of the present invention. They comprise for example micelles, lipid microspheres, lipid nanospheres, lipid vesicles also referred to as liposomes, such as ULV (unilamellar vesicles), MLV (multilamellar vesicles), MVL (multivesicular liposomes), sandwich liposomes (comprising an agent internalized between two bilamellar liposomes), giant liposomes (having diameters of 10 to 60 μm), anionic or cationic liposomes, liposome beads (aggregated or globulized liposomes with multiple encapsulations of agents), reconstituted liposomes (liposomes having incorporated protein components of membranes), hexasomes, cubosomes, and any membrane bilayers including an aqueous compartment and forming a permeability barrier between the encapsulated volume and the exterior solution. The minimum requirement is to form at least an aqueous compartment. Lipid complexes have been used for a myriad of drug therapies, and these delivery systems have shown promising results in for example gene therapy, as for gene therapy to be successful efficient and safe transfer of genes or biologically active reagent to a target cell is required.
"Lipid carrier", as the term is used herein, refers to entity made from lipids or mixtures containing lipids, comprising an outer lipid layer surrounding a volume not containing lipids.
The phase can consist of an ordered micellar structure of a single layer of lipid molecules, a two-dimensional arrangement of uni-, bi- or multi-layer membranes, or three-dimensional arrangements consisting of uni-, bi- or multi-layer membranes. The present invention therefore includes within its scope pharmaceutical compositions comprising at least one lipid carrier formulated for use in human or veterinary medicine. The term "lipid carrier" may be used interchangeably with the term "lipid vesicle", and also comprises lipid aggregates. In principal, according to the present invention, any artificial or naturally occurring vehicle of a monolayer, bilayer or multibilayer of lipids enclosing an open or closed volume is subsumed under the term "lipid carrier". The lipid carriers used in the present invention may comprise one type of lipid as wells as a mixture of different lipid molecules.
The term "cubosome" as a sub-class of lipid carriers, as it is used herein, refers to dispersed particles of bicontinuous cubic liquid crystalline phase, self-assembled monostructured particles that can be formed in aqueous, lipid, and surfactant systems. One of the most common surfactants used to make cubosomes is the monoglyceride glycerol monoolein. The expression "bicontinuous" refers to the division of two continuous but non-intersecting aqueous regions by a lipid bilayer that is contorted into a space-filling structure.
The term "hexasome", as referred to herein, describes a further microstructure, typically hexagonally arranged, containing channels of aqueous material surrounded by a single layer of lipid molecules.
As commonly understood, the term "liposome" refers to microscopic lipid bilayer vesicles that enclose a compartment. Liposomal vesicles can contain a single bilayer (unilamellar vesicle) or multiple bilayers (multilamellar vesicle). These vesicles can encapsulate water- soluble agents in their aqueous cavities, or carry lipid soluble agents within the membrane itself. Liposomes have already been used for encapsulating a variety of agents, for example therapeutic agents such as cytotoxic drugs, and carrying them to biological target sites. Encapsulation of for example pharmaceuticals in liposomes can reduce drug side-effects, improve pharmacokinetics of delivery to a target site, and improve the therapeutic index of a drug.
Although any of the aforementioned lipid carriers can be used for the purpose of the present invention, it is exemplarily made use of liposomes which is not to be understood as limitation of the scope of the present invention, since the subject matter and teaching of the present
invention can be easily applied and transferred, respectively, to the aforementioned lipid carriers without undue burden for those skilled in the art.
Methods for preparing these liposomes are by now well known in the art and any such methods (some of which are described below) can be employed in the context of the present invention.
For the purposes of this description, the term "biocompatible" is intended to describe compounds that are not toxic to cells. Compounds are "biocompatible" if their addition to cells in vitro does not lead to cell death and does not induce inflammation or other such adverse effects in vivo.
"Biodegradable" compounds, as used herein, are those that, when introduced into cells, are broken down by the cellular machinery into components that the cells can either reuse or dispose of without significant toxic effect on the cells.
The term "trigger", as used herein, refers to a mechanism or agent, giving an impulse, activating or inducing activation to a system or object. Within the present invention the term "trigger" is also to be understood in the sense of "mediator".
The term "lipid", as defined herein, is any chemical compound with a hydrophobic portion, such as oils, fats and fatlike substances, either naturally occurring or synthesized, and that characteristically are soluble in apolar solvents but only sparingly soluble in aqueous solvents. According to the invention lipids comprise also fatty acids, neutral fats (such as triacylglycerol), other fatty acid esters and soaps, long chain (or fatty) alcohols and waxes; sphingoids and other long-chain bases, glycolipids, phospholipids and sphingolipids, carotenes, polyphenols, glycopolyphenols, sterols (and related compounds), terpenes, and other isoprenoids. For the sake of clarity, some of the abbreviations used in the present invention are explained in the following: DPPC dipalmitoylphosphatidylcholine DSPE distearoylphosphatidylethanolamine PEG polyethyleneglycol
DOTAP N-[l-(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propane methylsulfate. Methylsulfate can be exchanged for other anions.
The lipids used in the present invention may be extracted and purified from a natural source or may be prepared synthetically in a laboratory. In a preferred embodiment, the lipids are commercially available.
The term "nanospheres", as used herein, refers to spherical structures having a size in the range of nanometers.
The term "microspheres", as used herein, refers to spherical structures having a size in the range of micrometers.
The term "micelle", as used herein refers to aggregates of colloidal dimension, of oriented molecules of amphiphatic substances. Micelles are made from amphiphilic components, and in aqueous solutions the individual molecules of the micelle are oriented with their polar groups pointing towards the aqueous medium and their hydrophobic moiety directed into the center of the micelle. At low water content or in apolar solvents micelles can exist with inverted orientation of the molecules, called "inverse micelles".
The term "surfactant", as used herein, refers to any agent, which preferentially absorbs to an interface between two immiscible phases, such as the interface between water and an organic polymer solution, a water/air interface or organic solvent/air interface. Surfactants generally possess a hydrophilic moiety and a lipophilic moiety, such that, upon absorbing to microparticles, they tend to present moieties to the external environment that do not attract similarly-coated particles, thus reducing particle agglomeration. Surfactants may also promote absorption of a therapeutic or diagnostic agent and increase bioavailability of the agent. Surfactants may also be used in the preparation of a pharmaceutical composition of the present invention.
The term "multivesicular liposomes (MVL)", as used herein, refers to liposomes having a size of 1-20 μm, containing multiple non-concentric chambers within each liposome particle, resembling a "foam-like" matrix.
The term "multilamellar liposomes or multilamellar vesicles (MLV)", as used herein, refers to liposomes containing multiple, often close to concentric chambers within each liposome
particle, resembling the "layers of an onion". Multilamellar liposomes characteristically have mean diameters in the range of micrometers, usually from 0.5 to 25 μm.
The term "unilamellar liposomes or unilamellar vesicles (ULV)", as used herein, refers to liposomes that enclose a single internal aqueous compartment and usually having a mean diameter range from about 20 to 500 run. Unilamellar liposomes include small unilamellar vesicles (SUV), and large unilamellar vesicles (LUV). Preferably, the liposomes used in the present invention are unilamellar liposomes.
The term "amphipathic lipid" refers to a molecule that has a hydrophilic "head" group and a hydrophobic "tail" group and has membrane-forming capability. The amphipathic lipids can be neutral, zwitterionic, anionic, or cationic lipids.
The term "zwitterionic lipid" refers to an amphipathic lipid having a net charge of zero at pH 7.4, and includes for example phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, just to name a few.
The term "anionic lipid" refers to an amphipathic lipid having a net negative charge at pH 7.4, and includes for example phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, phosphatidic acids, just to name a few.
The term "cationic lipid" refers to an amphipathic lipid having a net positive charge at pH 7.4, and includes for example diacyl trimethylammoniumpropane and ethyl phosphatidylcholine.
The term "administering", as used herein, refers to any mode of application to a cell, tissue, organ, or whole organism, which results in the physical contact of the composition with an anatomical site.
The term "therapeutic index", as used herein, refers to a medication as a comparison of the amount that causes the therapeutic effect to the amount that causes toxic effects. Quantitatively, it is the ratio of the dose required to produce the toxic effect and the therapeutic dose. A commonly used measure of therapeutic index is the lethal dose of a drug for 50% of the population (LD50) divided by the effective dose for 50% of the population (ED50).
"Small molecule", as used herein, refers to organic and inorganic compounds, whether naturally-occurring or artificially designed (e.g., via chemical synthesis), that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Typically, small molecules have a molecular weight of less than about 1500 g/mol. Known naturally- occurring small molecules include, but are not limited to, penicillin, erythromycin, paclitaxel, cyclosporin, and rapamycin. Known synthetic small molecules include, but are not limited to, ampicillin, methicillin, sulfamethoxazole, and sulfonamides. Alternatively, or in addition, the lipid carrier, e.g. liposome may be loaded with proteinaceous material, i.e. proteins, peptides such as antibacterial peptides and derivatives thereof or nucleic acid based molecules, see also infra.
General techniques Preparation of liposomes
Methods for producing liposomes are, by now, well known in the art and any of such methods can be employed in the context of the present invention; see, e.g., U.S. patent numbers
4,753,788 and 4,935,171 ; Szoka, et al, Proc. Natl. Acad. Sci. USA, 75 (1978), 4194-4198;
U.S. patent number 4,882,165; Deamer and Uster, "Liposome Preparation: Methods and
Mechanisms," in Liposomes, Marcel Dekker, Inc., New York (1983); Kim et al, Biochim.
Biophys. Acta 728 (1983), 339-348; Liu et al, Biochim. Biophys. Acta 1104 (1992), 95-101 ; Lee et al., Biochim. Biophys. Acta., 1103 (1992), 185-197). Many liposome formulations using many different lipid components have been used in various in vitro-, cell culture-, and animal experiments. Parameters have been identified that determine liposomal properties and are reported in the literature, for example, by Wang and Huang, Biochem. 28 (1989), 9508-
9514; Thierry and Dritschilo, Nuc. Acids Res. 20 (1992), 5691-5698, Gregoriadis, Drug Carriers in Biology and Medicine, Chapter 14, pages 288-341, Academic Press, 1979, describes liposomes on pp. 288-297.
Multilamellar and unilamellar liposomes can be made by several relatively simple methods, as for example a number of techniques for producing ULV and MLV is described in (for example U.S. patent numbers 4,522,803; 4,310,506; 4,235,871 ; 4,224,179; 4,078,052; 4,394,372; 4,308,166; 4,485,054; and 4,508,703).
Giant liposomes can be constructed as described for example in US patent application 2004/0062797.
Loading drugs into liposomes
Several methods by which drugs are loaded into liposomes are described in Ostro and Cullis, Am. J. Hosp. Pharm. 456 (1989), 1576-1587; and by Juliano, "Interactions of Proteins and Drugs with Liposomes," in Liposomes, Ibid. Most drugs are loaded at the time the liposome is formed by cosolubilizing the drug with the starting materials. The site of the liposome (cavity or membrane) into which the drug is located depends on the properties of the drug. A hydrophobic drug such as amphotericin B, for example, is cosolubilized with lipid in an organic solvent; see Lopez-Bernstein, J. Infect. Dis. 147 (1983), 939-945. Subsequent removal of the solvent and subsequent hydration of the liposome yields a liposome drug complex with the hydrophobic drug primarily in the membrane. Water soluble drugs can be sequestered in the liposome cavity by submitting liposomes to several cycles of freezing and thawing in an aqueous solution containing the drug. Finally, charged amphipathic drugs can be loaded into preformed liposomes using transmembrane pH gradients, as described in Bally et al, Biochem. Biophys. Acta 812 (1985), 66-76.
Detailed description of the invention
The present invention generally relates to compound triggered delivery systems. More specifically, the present invention pertains to the use of inorganic compounds for triggering the release of an agent present in a liposome. In particular, the triggered release of agents encapsulated in lipid carriers by a boron cluster compound is disclosed. Accordingly, for the purpose of the present invention and as used in the claims the term "liposome" includes any suitable lipid carrier which is suitable to be triggered for release of an entrapped agent such as a drug.
The present invention is based on the observation that an inorganic compound, i.e. the boron cluster compound disodium mercapto-undecahydro-dodecaborate (BSH) strongly interacts with a lipid bilayer. This interaction surprisingly leads to changes in the liposomes' structure. Prior to exposure to BSH, the liposomes exhibit a spherical structure, whereas after addition of BSH areas of thickened membranes as well as multilayer structures are observable, as well as aggregation of the liposomes, membrane fusion, liposome collapse and release of the liposomes' content; see Example 2 and Figure 1 a, b.
BSH is a boron cluster compound known from and used in the boron neutron capture therapy (BNCT), which is a specified form of neutron capture therapy (NCT). BNCT is a binary radiotherapy used for treatment of patients with malignant glioma. In BNCT a 10B- containing compound is administered to the patient, in whom it accumulates preferentially into the neoplastic tissue. The tumor is then irradiated with low-energy neutrons produced by a nuclear reactor. In the ensuing neutron capture reaction boron- 10 absorbs neutrons and its subsequent decay emits energetic particles (charged particles) which can kill nearby tumor cells. Since the energetic and cytotoxic alpha particles and lithium-7 particles travel only about one cell diameter in tissue, preferably one may specify the cell type to be destroyed by placing the charged particle precursors only on or within the tumor cells. In order for this therapy to be effective, sufficient 10B must be localized in a tumor to generate the required density of particles. Large numbers of boron containing compounds have been tested for their ability to satisfy the above criteria. The current boron carriers in clinical use are a boronated aromatic amino acid derivative, L-4-dihydroxyboryl phenylalanine, 4-borono- L-phenylalanine, L-BPA, and disodium mercaptoundecahydro-closo-dodecaborate (Na2 10Bi2HuSH, sodium borocaptate, sodium mercaptododecaborate, BSH); see for example Kageji et al, Biochim. Biophys. Acta 1391 (1998), 377-383; Alberts, Gabel and Moss, Boron Neutron Capture Therapy: Towards Clinical Trials of Glioma Treatment; Plenum Press, 1992. However, numerous potential boron compounds have been synthesised and many compounds have been tested preclinically (Hawthorne, Angew. Chem. 105 (1993) 997-1033; Morin, Tetrahedon 50 (1994), 12521-12569; Wyzlic et al., Int. J. Radiat. Oncol. Biol. Phys. 28 (1994), 1203-1213; Lesnikowski and Schinazi, Pol. J. Chem. 69 (1995), 827-840; Gabel, B. Cancer. Radiother. 83 (1996), 186-190; Mehta and Lu, Pharmaceut. Res. 13 (1996), 344-351 ; Sjόberg et al., J. Neuro-oncol. 33 (1997), 41-52; Soloway et al., Chem. Rev. 98 (1998), 1515-1562; Hawthorne and Lee, J. Neuro-oncol. 63 (2003), 33-45).
Furthermore, it is to be understood that the term "boron cluster compound" as used in accordance with the present invention and in the appended claims naturally includes the use of suitable pro-drugs from which the active boron cluster compound is liberated. One example and the preferred boron cluster compound to be used in accordance with the present invention has been described by Lechtenberg and Gabel for BSH in J. Organometallic Chemistry 690 (2005), 2780-2782. Here a glucuronidated derivative of BSH has been synthesized from which BSH could be liberated by tumor-inherently overexpressed glucuronidase. In this context, the person skilled in the art will recognize that the extracellular conversion of a non-
active pro-drug of the boron cluster compound to a lipid carrier release-triggering compound can be achieved by using a secreted form of the normally lysosomal human beta- glucuronidase to establish an extracellular effector system that converts an inactivated glucuronidated derivative of the boron cluster compound to the trigger-effective drug. A similar approach though within a different context, has been described in Weyel et al., Gene Ther. 7 (2000), 224-231.
Of course, any other suitable pro-drug of a boron cluster compound may be used within the scope of the present invention as long as the subject boron cluster compound liberated thereof exerts its desired effect, i.e. triggering the release of the liposome content.
Within the present invention, one critical feature in the discovery of BSH's considerable effect on the shape and structures of liposomes, respectively, is the finding that BSH not only induces rearrangement of the liposome structure, but even more important, leads by way of altering the structure to the release of the liposome content. Therefore, BSH exhibits surprising properties, rendering it useful as a trigger for the release of liposome content. Without intending to be bound by theory, in accordance with the present invention, this effect is thought to be due to the nature of BSH, being a twofold negatively charged anion, and therefore predestinated to interact with positively charged groups, i.e. cations, such as the positively charged part of the polar head group of the phospholipids in the bilayer.
Since this may be one possible way of inducing distortion to the membrane, those skilled in the art will recognize that other negatively charged cluster compounds such as twofold negatively charged boron cluster compounds like Bi2H12 2", B10H1O 2" may exert a comparable effect. Moreover, it is envisaged that other cluster compounds exert a similar effect, such as ammonioundecahydro-c/ojO-dodecaborate (comprising a NH3 +-group) or hydroxyundecahydro-closo-dodecaborate (replacing the SH group with an OH group). Additionally, cluster compounds featuring another atom instead of or in addition to boron having similar chemical properties such as those listed in the same main group of the periodic table of the elements as boron or other equivalent compounds, in particular those which are capable of forming clusters may be also useful for the purpose of the present invention. Therefore, the present invention also encompasses the use of cluster compounds or compositions comprising said compounds, capable of effecting liposomal carriers in the way described, leading to changes in the membrane structure and finally to the release of the
content. Preferably, the boron cluster contains a closo-dodecaborate cluster. In a particularly preferred embodiment of the present invention the boron cluster compound is disodium mercapto-undecahydro-dodecaborate (BSH).
One major surprising observation in accordance with the present invention compared to the currently available state of the art is that the release of the content, is inducible simply by addition of BSH, without any further stimulation, i.e. without any further kind of "activation", either of BSH or the liposome, and also without the liposomes being specifically designed to render them BSH-sensitive; see Examples 3 and 5 and Figures 2 and 4. In case of liposomes not containing PEG, aggregation is observed.
Hence, the present invention for the first time describes a substance release system which does not need the prerequisites described in the prior art as well as no further activation upon administration. Moreover, the liposomes triggered for release do not need to be designed in a certain way to be sensitive to the trigger of the present invention. Although a special liposome design to be triggered for release by the boron cluster compound of the present invention is not required, it is not excluded from the scope of the present invention, since there may exist cases in which a special design of the liposomes can have a beneficial effect, such as accelerated or targeted release of the content.
Thus, the present invention for the first time provides the use of a boron cluster compound for the preparation of a composition for triggering the release of an agent present in a liposome.
One particular advantage of the substance release system of the present invention, especially in view of therapeutic applications is that boron cluster compounds such as BSH are known to be substantially non-toxic in humans, at least at the concentrations required in accordance with the present invention to trigger the release of the liposome encapsulated substance.
This provides an enormous advantage, since an accumulation of liposomes, which is often found for example at sites of tumors due to reduced lymphatic drainage, can be triggered with the boron cluster compound or any composition comprising such a compound to release their encapsulated content.
A precisely triggered release of agents in or near by the desired target, including also tumors and metastases, respectively, will allow a precise administration of for example anti-cancer therapeutics and therefore be a big step towards an improved and more effective anti-cancer therapy.
Hence, the use of boron cluster compounds as triggers for release according to the present invention exhibits major advantages compared to the triggering methods of the prior art, such as an easy way of administration. Since it is not toxic in the doses tested by the present inventors, it can be injected into the blood flow, i.e. be administered systemically, and therefore enables the treatment of hardly accessible cells, organs or tissues (or those which are not accessible at all), such as metastases in the field of cancer. Thus, no targeting of the boron cluster compound is required, rendering administration easy and avoiding any possibly undesired influence on the trigger (and therefore on the trigger capacity) upon targeting.
Nevertheless, in one alternative embodiment of the present invention, the boron cluster compound and the composition comprising the boron cluster compound, respectively, can - if desired - be targeted to special cells, tissues or organs, if it is intended by the application strategy used. If the strategy comprises for example either the sequential administration of liposomes and boron cluster compound (or the composition comprising the boron cluster compound), wherein the boron cluster compound (or the composition comprising the boron cluster compound) for example is administered first, it may be targeted to the desired target cell, tissue, or organ, such as a tumor. After reaching the desired target, it "waits" for "arriving" liposomes which are administered later. In this case the liposomes may also be targeted either to the tumor or the boron cluster compound and the corresponding composition, respectively, or the liposomes are not targeted and reach the desired area by systemic distribution. Alternatively, both components (liposomes and boron cluster compound) may be administered concurrently and may be targeted for the purpose of accelerated delivery to the desired cell, tissue, or organ.
Thus, in a further embodiment the invention relates to a composition, wherein the composition is a diagnostic or pharmaceutically active composition and more preferably wherein said composition is designed to be administered systemically.
Concerning the broad field in which liposomes are used as "carriers" and the number of different compositions of liposomes to be used, differing for example in the composition, such as different amounts of anionic, cationic or neutral lipids, those skilled in the art will easily recognize that each variation in the composition can be connected to a certain property.
The physical characteristics of liposomes generally depend on pH and ionic strength. They characteristically show low permeability to ionic and polar substances, but at certain temperatures can undergo a gel-liquid crystalline phase transition dependent upon the physical properties of the lipids used in their manufacture which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less ordered structure, known as the liquid crystalline phase. Various types of lipids differing in chain length, saturation, and head group have been used in liposomal drug formulations for many years, including the unilamellar, multilamellar and multivesicular liposomes, described in the definitions; see supra.
Although phospholipids by themselves are sufficient for the formation of liposomes, some of the properties of the latter can be improved upon by the incorporation of other lipid soluble compounds into the liposomal structure. Thus, the stability of the phospholipid bilayers (in terms of both rigidity and permeability) can be altered by the inclusion of for example a sterol, and the incorporation of for example a charged amphiphile can not only render the liposomal surface positively or negatively charged, it can also increase the distance, and hence aqueous volume, between the bilayers. It is obvious that it is no undue burden for those skilled in the art to select the appropriate liposome, i.e. the liposome having the appropriate properties for the purposes they are intended to be used for.
With respect to the boron cluster compound and as mentioned supra, of the present invention, without intending to be bound by theory, also other boron cluster compounds can be used which are capable of achieving the desired effect, i.e. interacting with liposome's membrane and thereby releasing the encapsulated content. This interaction may in one embodiment lead to aggregation of the liposomes, but is not restricted to this effect, since any other effect is assumable and included within the scope of the invention which leads to leakage or other release of the liposomes content upon addition of the trigger, such as the boron cluster compound.
In principal the release of the liposomes content can be triggered in any type of lipid vesicle, such as ULV (unilamellar vesicle), MLV (multilamellar vesicle), MVL (multivesicular liposomes), sandwich liposomes, micelles (some of which are further characterized in the definitions; see supra), and any membrane bilayers including an aqueous cavity.
Since either as multilamellar or unilamellar vesicles, liposomes have proven valuable as vehicles for drug delivery in animals and in humans, a variety of agents can be trapped in the aqueous core of the liposome, while hydrophobic substances can be dissolved in the liposome bilayer membrane. The liposome structure can be readily injected and can form the basis for both sustained release and drug delivery to specific cell types, or parts of the body. Multilamellar vesicles, primarily because they are relatively large, are usually rapidly taken up by the reticuloendothelial system (the liver and spleen). The invention typically utilizes vesicles which remain in the circulatory system for hours, such as pegylated liposomes (stealth liposomes) and preferably unilamellar vesicles having a diameter of less than 250 run, and more preferably about 100 nm.
In principal, the liposome of the present invention can comprise any combination of lipids, preferably it comprises a mixture of lipids, more preferably it comprises amphipathic lipids, most preferably dipalmitoylphosphatidylcholine (DPPC), whereas in another embodiment of the present invention the liposome comprises positively charged lipids, preferably N-[I -(2,3- Dioleoyloxy)]-N,N,N-trimethylammonium propan (DOTAP) methylsulfate. Methylsulfate can be exchanged for other anions.
In a further preferred embodiment the liposome of the invention comprises dipalmitoylphosphatidylcholine (DPPC) and l,2-disteaoryl-sn-glycero-3-phosphatidyl- ethanolamine-N[methoxypoly(ethylene glycol)] (DSPE-PEG). This class of liposomes has been shown to be not capable of aggregating and more importantly, to have increased circulation times in blood. For some applications it may be desirable to use this kind of liposomes. Accordingly, the liposomes to be used in accordance with the compositions, methods and uses of the present invention may be chosen in view of the intended application.
Thus, long-circulation and short-circulation liposomes, respectively, or mixtures thereof may be used.
As already described supra, there exists an alternative way of administering the liposomes besides the above mentioned accumulation of systematically administered liposomes at for example sites of leaky vascularization and the lack of an effective lymphatic drainage system like in tumors. In an alternative embodiment, the liposomes and/or the boron cluster compound can be targeted, i.e. actively directed to a desired cell, tissue, or organ.
Thus, the liposomes and/or the boron cluster compound used according to the invention may be modified to comprise targeting agents, wherein "targeting" includes for example decorating the outside of the liposome with one or more ligands specific for a particular target site. A variety of targeting agents that direct pharmaceutical compositions to particular cells as well as means of coupling targeting molecules to liposomes are known in the art; see, for example, Cotten et al, Methods Enzym. 217 (1993), 618; U.S. patent number 5,258,499. The targeting agents may be included throughout the particle or may be only on the surface.
The targeting agent may be used to target specific cells or tissues or may be used to promote endocytosis or phagocytosis of the liposome. Examples of targeting agents include, but are not limited to natural and synthetic peptides, hormones, antibodies (as for example described in patent application US 2002/0035084), fragments of antibodies, low-density lipoproteins (LDLs), transferrin, proteins, asialycoproteins, gpl20 envelope protein of the human immunodeficiency virus (HIV), carbohydrates residues, substrates for enzymes highly expressed on the tumors surface, natural and synthetic ligands such as receptor ligands or other receptor-binding molecules, receptor-binding proteins, specific cell surface receptors, glycoproteins, lipids, small molecules, etc. If the targeting agent is included throughout the liposome, the targeting agent may be included in the bilayer. If the targeting agent is only on the surface, the targeting agent may be associated with liposomes (i.e., by covalent, hydrophobic, hydrogen bonding, van der Waals, or other interactions) using standard chemical techniques. In a preferred embodiment of the invention the targeting molecule comprises an antibody, receptor binding protein or peptide or a carbohydrate residue.
Although targeting seems to have several advantages, there are also some drawbacks, the major of which is sterical hindrance of ligands which may occur, for example, when strong binding ligands are bound to the liposome, which in turn is intended to accumulate for example at a tumor, the ligands may obstruct the way for more liposomes to accumulate; see Barenholz, Curr. Opin. Colloid Interface Sci. 6 (2001), 66-77. In addition,
"immunoliposomes" (liposomes carrying an antibody as targeting molecule) show enhanced liposome clearance (Harding et ah, Biochim. Biophys. Acta 1327 (1997), 181-192; Koning et al., Liposome Res. 12 (2002), 107-119).
In principal any suitable liposome or equivalent complex capable of encapsulating a given substance can be used in accordance with the present invention. In this respect, reference is also made to the pertinent scientific and patent literature concerning the provision of appropriate liposomes, for example international application WO2000/051565 which describes a method to prepare liposome-encapsulated bioactive agents such as nucleic acids; international application WO2001/037807 describing liposomes including a fusogenic liposome, a linking moiety and a targeting moiety, wherein the fusogenic liposome is a lipid bilayer encapsulating contents. Furthermore, international application WO2001/074333 describes a liposome having a lipid bilayer, where the lipid bilayer includes either the D or L steroisomer of an ether lipid or a non-equal mixture of both. Naturally, all the liposomes and methods of preparation thereof are envisaged to be used in accordance with the present invention. The disclosure content of the mentioned international applications, in particular with respect to the description of the there disclosed liposomes is specifically incorporated herein by reference.
In this context, it is one major advantage of the present invention that the liposome containing pharmaceutical composition can be administered systemically and does not necessarily need to be targeted, although targeting is also included within the scope of the present invention.
Since it is known that liposomes can be used as "carriers", agents with which they can be "loaded" encompass a wide variety. In this context, the agents to be delivered and released by the trigger of the present invention may be investigative, toxic, therapeutically active, diagnostic, or prophylactic agents. In a preferred embodiment, the agent is a pharmaceutically active agent.
Suitable therapeutically active agents, include for example but are not limited to inorganic and organic chemical entities, biologically active substances, such as human genes, proteins, enzymes, and the like, as well as genetically engineered copies of the same, nutrients, vitamins, minerals, other plant and animal substances, a clinically used drug that has been approved by the FDA, such as an antibiotic, anti-viral agent, anesthetic, steroidal agent, anti-
inflammatory agent, anti-neoplastic agent, antigen, vaccine, antibody, decongestant, antihypertensive, sedative, birth control agent, progestational agent, anti-cholinergic, analgesic, anti-depressant, anti-psychotic, p-adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, non-steroidal anti-inflammatory agent, nutritional agent, etc., or a mixture of pharmaceutically active agents, like for example, two or more antibiotics may be combined in the same microparticle, or two or more anti-neoplastic agents may be combined in the same microparticle; or alternatively, an antibiotic may be combined with an inhibitor of the enzyme commonly produced by bacteria to inactivate the antibiotic (e.g., penicillin and clavulanic acid). They include further topically applied antifungals, antiarthritics, corticosteroids, whitening agents, antioxidants, polyphenols, nitrus oxide, moisturizers, anabolics, analgesics, anesthetics, antiasthmatics, antibacterial, antihistaminics, antiparasitics, vasodilators, vasoconstrictors, anti-tumor, i.e., seborrheic keratosis to malignant tumors such as basal cell carcinoma, anti seborrheic, anti-vertigo such as compazine, anti insects, or toxins, such as botox (nerve paralysis), hormones such as estrogen, nicotine, amino acids, lipids, herbs, metabolite supplements, and other therapeutic or medicinal compositions.
Alternatively, a chemotherapeutic agent may be the therapeutically active agent and be encapsulated within a liposome, thereby sequestering its toxic effects from non-targeted tissues. Thus, in a preferred embodiment of the invention, the agent is a therapeutically active agent, preferably an anti tumor agent, although the precise compounds to be used will occur to those skilled in the art according to the problem to be solved.
Suitable diagnostic agents include for example but are not limited to agents that can be radio labelled, fluorescently labelled, enzymatically labelled and/or include magnetic compounds, gases; commercially available imaging agents used in positron emissions tomography (PET), computer assisted tomography (CAT) and x-ray imaging, such as iodine-based materials, single photon emission computerized tomography, fluoroscopy, and magnetic resonance imaging (MRI), such as gadolinium chelates, ultrasound, and computed tomography (CT).
In a preferred embodiment the invention relates to a composition, wherein the agent is a diagnostic agent, preferably a dye, more preferably a fluorescent dye and most preferably carboxyfluorescein or calcein.
Suitable prophylactic agents include for example but are not limited to vaccines, which may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, and cell extracts. Vaccines may also include polynucleotides which encode antigenic protein or peptides. Prophylactic agents may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, Freund's adjuvant, etc. Prophylactic agents include antigens of such bacterial organisms as Streptococccus pnuemoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridiumbotulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonasaeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholera, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter jejuni, and the like; antigens of such viruses as smallpox, influenza A and B, respiratory syncytial virus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackie viruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and the like; antigens of fungal, protozoan, and parasitic organisms such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydialpsittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and the like. These antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof. More than one antigen may be combined in a particular liposomal carrier, or a pharmaceutical composition may include liposomes each containing different antigens or combinations of antigens.
Even stem cells, autologous or embryonic endothelial cells (ECs), and/or endothelial progenitor cells (EPCs) may also be encapsulated in liposomes, as described in for example U.S. application US2004/0062797 Al. In fact, the minimum requirement is that the nature of the agent must be such that it is protectable by a liquid structure.
The present invention additionally relates to a composition comprising as a first component an inorganic compound, i.e. a boron cluster compound and as a second component a liposome as described hereinbefore, preferably wherein said liposome comprises a therapeutically active or a diagnostic agent as defined above and in a further embodiment the above compositions are designed to be administered or applied concurrently or sequentially.
As it is one major object of the present invention to provide a successful trigger for the release of a liposome-encapsulated agent, since even a successful targeting of the liposome and the boron cluster compound, respectively is of less value if the content is not released at the desired site where it should be effective, the present invention relates in a further embodiment to a method of releasing an agent from a liposome under predetermined conditions comprising the step of exposing the liposome to a boron cluster compound such as to release the agent.
The use of the boron cluster compounds as described hereinbefore for triggered release of a liposome-encapsulated agent and the composition, comprising the boron cluster compound, respectively, can of course also be used for further investigations, such as in vitro assays, like for example cell culture experiments, to trigger release of an liposome encapsulated agent, said agent intended to be tested concerning its (for example therapeutic) effect on the cells and cell culture, respectively after release.
Therefore, the present invention further provides a method of identifying and obtaining a therapeutically active agent comprising
(a) incubating a cell culture with a liposome containing the test compound
(b) exposing the cell culture to a boron cluster compound; and (c) determining a detectable response of a cell in the cell culture compared to a control, thereby identifying the therapeutically active agent.
Those skilled in the art will recognize that any kind of cell culture is suitable, such as suspension cultures, callus-cultures, as well as tissue cultures or cultivated organs, as well as any type of cells, such as endodermal cells, ectodermal cells, mesodermal cells, stem cells, tumor cells, such as cancer cells etc., just to name a few. In a preferred embodiment, cell line V-79, derived from the lung tissue of a Chinese hamster is used; see also example 4. This cell line as well as other cell lines which are suitable for the screening methods of the present invention can be ordered from official depositary institutions, for example the American Type
Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Culture (DSMZ).
Of course, it is included by the present invention that once a therapeutically active agent is identified, by the above described method, the method further comprises synthesizing the therapeutically active agent determined in step (c) in a therapeutically effective amount. As used herein, the term "a therapeutically effective amount" means the total amount of the therapeutically active agent, also called drug or pro-drug that is sufficient to show a meaningful patient benefit, i.e. treatment, healing, prevention or amelioration of for example damaged tissue, or an increase in the rate of treatment, healing, prevention or amelioration of such conditions. In addition or alternatively, in particular with respect to pre-clinical testing of the drug the term "therapeutically effective amount" includes the total amount of the drug or pro-drug that is sufficient to elicit a physiological response in a non-human animal test.
The appropriate concentration of the therapeutic agent might be dependent on the particular agent. The therapeutically effective dose has to be compared with the toxic concentrations; the clearance rate as well as the metabolic products play a role as to the solubility and the formulation. Therapeutic efficacy and toxicity of compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50 % of the population) and LD50 (the dose lethal to 50 % of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
Depending on the specific conditions being treated, the liposomes and/or the boron cluster compounds (or compositions containing the boron cluster compound) may be formulated and administered systemically, topically, orally, pulmonary or otherwise locally. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, supra. Other suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intra-lesional, or intraocular injections, just to name a few. Further routes of administration will be known to those skilled in the art and can be readily used to administer the liposome and the boron cluster compound of the present invention.
As screening for genetically altered cells has become of increased interest, the present invention provides in a further embodiment a method of obtaining a genetically altered cell comprising any one of the above described methods. For example, the method of the present invention may be used to deliver substances capable of inducing mutations into the genome of a given cell thereby generating spontaneous cell lines that exhibit new properties. Alternatively, recombinant DNA constructs may be delivered in order to transform a given target cell and to come up with a pre-determined genetically engineered cell. In this context, the person skilled in the art will immediately recognize that in this embodiment the recombinant DNA construct preferably comprises a selectable marker gene and thus may be encapsulated preferably with a selective agent into the liposome. Accordingly, the present invention also provides transgene delivery systems which allow both in time, i.e. transformation and selection for transformants. Means and methods for preparing recombinant DNA constructs for this purpose including selective agents are well known to the person skilled in the art and are described in the literature including those cited below, for example Sambrook et α/., (1989).
As there is always a need for optimizing, assaying, controlling, or monitoring pharmaceutical compositions, this also applies to the components of various liposomal pharmaceutical compositions encapsulating an agent. Therefore, the present invention further relates to a method of assaying liposomes or agents encapsulated by liposomes, comprising subjecting the liposome to a boron cluster compound. This includes for example assaying the liposomes with respect to: stability, size, biodegradability, capability of encapsulating the desired agent, leakage, shelf-life, etc. Similarly assaying parameters concerning the encapsulated agent may be: effectivity after release, intactness after release, tolerance, shelf-life within the liposome or lipid vesicle, biodegradability, uptake by the cell, optionally undesired chemical modifications upon uptake in or release from the vesicle used, such as a liposome, rate of release, amount of release, just to name a few.
The present invention also provides one or more kits for using in the methods described above, said kit comprising a liposome, optionally containing an agent, a boron cluster compound, and optionally means for detection. It may further comprise the liposome structures ready for the user to add the biological reagent of interest, as well as one or more specific biologically-active reagents for addition to the liposome structure. Another kit of the present invention may comprise a set of lipid carriers, such as liposomes each containing a
specific, biologically-active reagent, which when administered together or sequentially, are particularly suited for the treatment of a particular disease or condition. Such kits would typically comprise a compartmentalized carrier suitable to hold in close confinement at least one container and the compounds of the kit may be sterile, where appropriate. The kit may further include a transfer means, such as pipets, for transferring the suspended cells.
The kit's carrier could further comprise reagents useful for performing said methods and may also contain means for detection such as labeled enzyme substrates or the like. Instructions can be provided to detail the use of the components of the kit, such as written instructions, video presentations, or instructions in a format that can be opened on a computer (e.g. a diskette or CD-ROM disk).
Although there are almost non-limiting examples of gene therapy approaches for treating cancer which can employ the triggered release by the boron cluster compound of the present invention including for example but not being limited to: antisense or ribozyme therapy (to block synthesis of proteins encoded by deleterious genes), chemoprotection (to add proteins to normal cells to protect them from chemotherapies), immunotherapy (to enhance the body's immune defenses against cancer), pro-drug or suicide gene therapy (to render cancer cells highly sensitive to selected drugs), tumor suppressor genes (to replace a lost or damaged cancer-blocking gene), antibody genes (to interfere with the activity of cancer-related proteins in tumor cells) and oncogene down-regulation (to shut off genes that favor uncontrolled growth and spread of tumor cells) (Blaese, Scientific American 6 (1997), 111), the present invention also concerns other diseases, such as diabetes, atherosclerosis, chemotherapy- induced multi-drug resistance, and generally, immunological, neurological (Ho and Sapolsky, Scientific American 6 (1997), 116) and viral diseases (Friedmann, Scientific American 6 (1997), 96), as well as correcting the ion transport defect in cystic fibrosis patients by inserting the human CFTR (cystic fibrosis transmembrane conductance regulator) gene.
In fact, the current use of liposomes as carriers for agents, such as therapeutic agents could indeed be a useful tool in drug delivery, but to date is hampered by the unsatisfying release of the encapsulated agents. The triggers for release used in the prior art either rely on properties of the diseased tissue such as different pH value or presence/overexpression of specific enzymes, both of which are difficult to manipulate, or require knowledge about the location of the tumors or the cells, tissues, or organs, which are intended to be treated, as well as some of the triggers require a number of prerequisites, such as accessibility of the desired target,
knowledge of a special type of tumor (i.e. the enzymes which are present on the tumor's surface), design of specific trigger-sensitive liposomes, to trigger the release of encapsulated agents. For those reasons, there is a strong need in the art for a triggered release provided by the trigger of the present invention and the methods and uses, respectively, which are independent from these factors, therefore being highly effective and solving the prior problems.
Moreover, since the present invention provides means and methods for triggering the release of for example therapeutic agents in or at least in the vicinity of metastases, which is one major advantage compared to the prior art lacking this feature, the present invention may for instance be used as a tool for the detection of alteration, aberrations, disorders, cellular abnormalities, and the like at sites which are not accessible to prior triggers of release, and therefore, may also enable the diagnosis of for example metastases which are maybe not detectable or locatable by common means. This can be achieved by for example preparing liposomes encapsulating a diagnostic agent, such as a labelled tumor-marker, subsequently administering said liposomes either targeted or via the systemic way to the desired site, such as a secondary tumor (metastasis), then triggering the release of the diagnostic compound from the liposomes using the boron cluster compound (or a composition comprising the boron cluster compound) of the present invention, and finally detecting the labelled cells.
Furthermore, the present invention provides new perspectives to directly affect metastases, such as killing the cells of the secondary tumor, as they are now -using the means and methods provided by the present invention- accessible for cytotoxic agents, which can be delivered and released without doing damage to non-diseased cells.
Having described the preferred embodiments of the present invention with respect to its application in tumor therapy and diagnostic, the person skilled in the art will acknowledge that the present invention is not limited thereto. Indeed, because of the generality of liposome mediated delivery of substances, the compositions, methods and uses in accordance with the present invention can be generally applied to the diagnosis and therapy of any disease or condition for which a rather targeted approach is needed or advantageous. A non-limited list of diseases and disorders which may be diagnosed or treated in accordance with the present invention is set forth below.
The following listing reflects examples of disorders to be treated in accordance with the method of the present invention which include, but are not limited to, an immune system disorder such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjgren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, trauma, and hematopoietic cancer including lymphoma, leukemia, and myeloma; a reproductive disorder such as a disorder of prolactin production, infertility, including tubal disease, ovulatory defects, and endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune disorders, an ectopic pregnancy, and teratogenesis, cancer of the breast, fibrocystic breast disease, and galactorrhea, a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia; a nervous system disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; a cell signaling disorder including endocrine disorders such as disorders of the hypothalamus and pituitary resulting from lesions such as primary brain tumors, adenomas, infarction associated with pregnancy, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immunological disorders, and complications due to head trauma; disorders associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma; disorders associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection; disorders associated with hyperparathyroidism including Conn disease (chronic hypercalemia); pancreatic disorders such as Type I or Type II diabetes mellitus and associated complications; disorders associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis; disorders associated with gonadal steroid hormones such as: in women, abnormal prolactin production, infertility, endometriosis, perturbations of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in postmenopausal women, osteoporosis; and, in men, Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, hypergonadal disorders associated with Leydig cell tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 α-reductase, and gynecomastia; and a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
Hence, it will be appreciated that the present invention generally relates to the use of any compound, i.e. inorganic compound with a special emphasis on boron cluster compounds such as BSH or components thereof, liposomes, substances, i.e. therapeutically active or diagnostic agents for use in the above described embodiments.
In one further embodiment the present invention relates to the use of a liposome of a pharmaceutical or diagnostic composition, encapsulating an agent, wherein the liposome is designed for being triggered to release the agent by a boron cluster compound.
Additionally, the triggering effect, achieved by using the means and methods of the present invention, is not restricted to the release of substances, since those skilled in the art may recognize that the opening of the liposome's membrane can also be exploited for other purposes than releasing agents, such as maybe the uptake of substances, if a subsequent "closing" of the liposomes is achieved. It may further be intended to render this triggering effect (opening of the membrane) reversible.
Even if the purpose would be restricted to the release of agents, it is not necessarily restricted to release agents to enable them to exert a certain effect. Alternatively agents, could be encapsulated in liposomes and released by the boron cluster compound of the present invention, to monitor the influence of encapsulating and releasing of such an agent, i.e. whether the released agent is identical to the one which was encapsulated, thereby investigating, whether the drugs reach their target having their original chemical identity or if they are altered. This would also be an important feature, since in terms of therapeutically active drugs, they need to reach the target in their therapeutically active form; otherwise their delivery towards diseased sites is useless.
It will be apparent that the boron cluster compounds, compositions, methods, and uses as substantially described herein or illustrated in the description and the examples, are also subject of the present invention and claimed herewith. In this respect, it is also understood that the embodiments as described in any one of the examples, can be independently used and combined with any one of the embodiments described hereinbefore and claimed in the appended claims set.
These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the materials, methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries and databases, using for example electronic devices. For example, the public database "Medline" may be utilized, which is hosted by the National Center for Biotechnology Information and/or the National Library of Medicine at the National Institutes of Health. Further databases and web addresses, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL) are known to the person skilled in the art and can also be obtained using internet search engines. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.
The above disclosure generally describes the present invention. Several documents are cited throughout the text of this specification. Full bibliographic citations may be found at the end of the specification immediately preceding the claims. The contents of all cited references
(including literature references, issued patents, published patent applications as cited throughout this application and manufacturer's specifications, instructions, etc) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.
EXAMPLES
The examples which follow further illustrate the invention, but should not be construed to limit the scope of the invention in any way. Detailed descriptions of conventional methods, such as those employed herein can be found in the cited literature; see also "The Merck Manual of Diagnosis and Therapy" Seventeenth Ed. Ed by Beers and Berkow (Merck & Co., Inc. 2003).
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Methods in molecular genetics and genetic engineering are described generally in the current editions of Molecular Cloning: A Laboratory Manual, (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1984); Transcription And Translation (Hames and Higgins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (Ausubel et al., eds.); and Recombinant DNA Methodology (Wu, ed., Academic Press). Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, VoIs. 154 and 155 (Wu et al., eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir and Blackwell, eds., 1986). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, and Clontech. General techniques in cell culture and media collection are outlined in Large Scale Mammalian Cell Culture (Hu et al, Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); and Suspension Culture of Mammalian Cells (Birch et al.,
Bioprocess Technol. 19 (1990), 251); Extracting information from cDNA arrays, Herzel et al., CHAOS 11, (2001), 98-107.
Example 1: Preparation of liposomes containing carboxyfluorescein or calcein As mentioned in the description hereinbefore, liposomes were prepared according to standard procedures. Briefly, to obtain a lipid film, an appropriate amount of lipid and optionally further compounds can be weighed out and subsequently be dissolved in appropriate volumes of chloroform and methanol. The mixture of solvents can afterwards be removed by use of a rotary evaporator under vacuum and the thusly generated lipid layer can be further dried by use of an oil pump.
Hydration of the lipid layer is achievable by adding corresponding volumes of a 100 mM carboxyfluorescein-solution or a 0.1 mM calcein-solution. The whole mixture can be heated in a water bath to a temperature being preferably at least 10 °C higher than the liposome's phase transition point. It is known to those skilled in the art that this cycle of "heating and vortexing" is repeated until the lipid film is completely dissolved generating a lipid emulsion.
This procedure is often followed by a freeze-thaw cycle, which is optionally run 5 times, i.e. transferring the sample into a cryo-tube and freezing it in liquid nitrogen for two to three minutes, and thawing the samples for about 60 seconds in a water bath. Any subsequent step is carried out 10 0C higher than the liposome's phase transition point. Thawing the sample is usually followed by vortexing, a procedure after which the sample comprises multilamellar and unilamellar liposomes exhibiting a wide size range. To exclusively obtain unilamellar liposomes of a defined size, this mixture is extruded, a technique which is known to those in the art. Using for example a commonly available standard extruder (LiposoFast, Avestin Inc.) having an inner polycarbonate membrane with a pore diameter of 100 nm, this mixture can be enriched for unilamellar liposomes. The sample containing the differently sized liposomes is usually pressed through said membrane using two syringes at each side of the extruder, a procedure in the course of witch the liposomes are forced to pass through the membrane, thereby stripping off the multiple lipid layers resulting in unilamellar liposomes.
To ensure the liposomes being flexible enough and capable of passing the pores of the membrane, they usually are heated up to their phase transition temperature, a fact known to those of skill. Therefore, the extruder will be put into a water bath and heated up to the
corresponding temperature. Liposomes can be pressed through the membrane for several times (up to about 20 times).
The obtained unilamellar liposomes (having a size of about 100 nm) are separated from the not encapsulated, i.e. free carboxyfluorescein by gel filtration, using for example a Sephadex G-25 column. Separation of liposomes and free dye can be followed by visual inspection. Where calcein is used as fluorescent dye, it can be removed by using G50 Sephadex spin- columns, loading the liposome samples onto the G50 column and spinning for example for two minutes at 1000 rpm.
Lipid concentration is determined by Stewart assay, a colorimetric method commonly used to determine phospholipid (phosphatidylcholine) concentrations. Usually for the determination of choline, DPPC is used as a standard. Stewart assay is suited for determination of phosphatidylcholine content of samples in the presence of inorganic phosphate. The test exploits the tendency of lipids to form complexes with ammonium iron(III)thiocyanate. Since complex formation depends on the head group of the phospholipids, this test is not suitable for analysis of samples, containing an unknown mixture of different phospholipids. Since concerning the present invention, only liposomes of known compositions have been measured, Stewart test was the appropriate method. Such a test can be carried out using an aqueous solution of 0.1 M iron chloride and 0.4 M ammonium thiocyanate, subsequent mixing with the test liposome solution and chloroform, followed by short vortexing and centrifugation for 10 minutes at 2700 rpm (for example using a Heraeus Biofuge 15 R, Rotor HSA 4100). Optical density of the lower chloroform phase will be measured using a photometer at 485 nm compared to the blank and concentration of the test samples will be calculated by comparison with the calibration curve obtained for the standard samples of known concentrations.
Preparation of DPPC+5%DOTAP-liposomes
Liposomes of dipalmitoylphosphytidylcholine (DPPC) containing 5 mol% N-[l-(2,3- Dioleoyloxy)]-N,N,N-trimethylammonium propane methylsulfate. (DOTAP) were prepared as described above, using the corresponding components. Extrusion was carried out in the presence of 100 μM calcein and liposomes were freed from excessive calcein by gel filtration.
Preparation of DPPC-DSPE-PEG?n0n-liposomes
DPPC-DSPE-PEG2OOo-IiPOSOIrIeS containing dipalmitoylphosphytidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylenglyco^ooo in a molar ratio of 98:2 were prepared as described above, using the corresponding components. Extrusion was carried out in the presence of carboxyfluorescein (concentration 10OmM) and liposomes were freed from excessive carboxyfluorescein by gel filtration.
Example 2: Determination of structural changes of liposomes upon addition of BSH using cryo-Transmission Electron Microscopy (TEM) Liposomes (lipid concentration 10 mM) were incubated at 45°C in 0.1 M Tris buffer pH 7.4. The samples were prepared for cryo-transmission electron microscopy 36 hours after mixing. From independent experiments it is known that the major changes occur within a few minutes. For liposomes prepared from other lipids with lower phase transition temperatures, the same effect is achieved with lower temperatures. After addition of mercaptoundecahydro- closo-dodecaborat (BSH) the liposomes, having a spherical shape prior to BSH addition (see Fig. Ia) start to aggregate, thereby changing their shape from isolated round-shaped liposomes into a more flattened structure (see Fig. Ib). Moreover multilayer structures and thickened membranes are observed, indicating release of the whole content of the liposomes.
Example 3: Monitoring calcein release from DPPC/DOTAP liposomes after exposure to BSH using fluorometry
Liposomes from DPPC containing 5 mol% DOTAP were prepared by extrusion in the presence of 100 μM calcein and freed from excessive calcein by gel filtration. At 37°C, Co2+ ions, capable of abolishing very effectively the fluorescence of calcein even in small concentrations were added to the Tris-buffered liposome-solution (concentration 25 μM) to a final concentration of 100 μM at t = 100 s. At t = 200 s BSH (final concentration 16 mM) was added and at t = 500 s, Triton X -100 at a final concentration of 0.1 % was added to completely destroy the liposomes. Fluorescence was measured over a time scale ranging from t = 0 s to t = 600 s using a Perkin-Elmer LS 50 B spectrofluorometer. The decrease of fluorescence is a measure for the leakage, since the fluorescence of calcein released from the liposome is abolished by the surrounding Co2+ (100 μM). After addition of either BSH or Triton release of the liposom's content can be observed; see Fig. 2.
Example 4: Determination of viability of V-79 cells after incubation with doxorubicin- filled liposomes and exposure to different concentrations of BSH
WST-I test is a non radioactive method to test and quantify cells for proliferation and viability. It is based on measuring the activity of the mitochondrial succinate-tetrazolium reductase system of cells by measuring the metabolization of the WST-I reagent (which is slightly red) to formazane (which is deep dark red). The change in colour which can be measured, using enzyme-linked immunosorbent assay (ELISA)-reader at a wavelength of 420 nm to 480 run and is a measure for the amount of metabolically active mitochondria of cells. As described by Ishiyama et al, Biol. Pharm. Bull. 19 (1996), 1518-1520, the WST-I test can either be used to test for cell proliferation in response to stimulation with for example growth factors or cytokines, or for detection of cell death by addition of fore example apoptosis reducing agents or chemotherapeutics.
According to the present invention, V-79 cells were cultivated in 96-well plates (8000 cells per well). At 37°C, doxorubicin-filled liposomes were added to the cultivation medium (total doxorubicin concentration 0.9 μM), followed by BSH at a concentration ranging from 0 mM to 20 mM. The medium was removed after 24 hours, and the viability was assayed with the WST assay. According to known protocols, WST-I reagent can be added in an amount corresponding to 10% of total reaction volume and plates can be further incubated. Usually, absorption is measured in an ELISA-reader at a wavelength of 420 nm to 480 nm, see supra. It is obvious that in some cases a control, i.e. reference value is needed. Therefore, usually cells are taken which have only be incubated with the WST-I reagent, i.e. which should show maximum metabolic activity, being fixed as 100%. Cell viability can be calculated using the following equation: Cell viability [%] = ODsample / OD,00% x 100
ODsampie :optical density of treated cells OD1OOr0 : optical density of untreated cells (controls)
The results obtained by the WST-I test are shown in Fig. 3
Example 5: Monitoring carboxyfluorescein release from DPPC-DSPE- Polyethylenglycol (PEG)2ooo liposomes after addition of BSH at various concentrations using fluorometry
DPPC-DSPE-PEG2OOo-IiPOSOnIeS containing dipalmitoylphosphytidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylenglyco I20OO in a molar ratio of 98:2 were prepared as described hereinbefore, see supra. Carboxyfluorescein was encapsulated in liposomes in self-quenching concentrations. Liposomes were incubated in buffer in a fluorescence cuvette, and BSH was added at t = 120 s at final concentrations ranging from 0 mM to 32 mM. Fluorescence of released carboxyfluorescein was monitored using a Perkin- Elmer LS 50 B spectrofluorometer. After a total of 240 seconds, Triton X-100 (final concentration 0.1%) was added to completely release carboxyfluorescein. It was observable that the addition of increasing concentrations of BSH led to an increased release of carboxyfluorescein; see Fig. 4.
In summary, the above described experiments performed in accordance with the present invention demonstrate that substances encapsulated in custom designed liposomes can be specifically triggered to the release with an inorganic compound, i.e. the boron cluster compound, BSH. Moreover, it could be shown that this novel delivery system can be used effectively to target and kill the predetermined cells. Accordingly, the delivery system presented in accordance with the present invention can be used in many ways including diagnostic and therapeutic uses but will also be a valuable tool in basic research.
Claims
1. Use of a boron cluster compound for the preparation of a composition for triggering the release of an agent present in a liposome.
2. The use of claim 1, wherein the boron cluster compound is di-sodium mercapto- undecahydro-dodecaborate (BSH).
3. The use of claim 1 or 2, wherein the liposome is not capable of aggregating.
4. The use of any one of claims 1 to 3, wherein said liposome comprises a mixture of lipids.
5. The use of any one of claims 1 to 4, wherein the liposome comprises neutral lipids.
6. The use of claim 5, wherein the neutral lipid is dipalmitoylphosphatidylcholine (DPPC).
7. The use of any one of claims 4 to 6, wherein the liposome comprises positively charged lipids.
8. The use of claim 7, wherein the positively charged lipid is N-[l-(2,3- dioleoyloxy)propyl]-N,ΛfN-trimethylarnmonium methyl sulphate (DOTAP).
9. The use of any one of claims 1 to 8, wherein the liposome comprises dipalmitoylphosphatidylcholine (DPPC), 1,2- Dipalmitoyl-sn-glycero-3- phosphoethanolamine (DSPE) and polyethylenglycol (PEG).
10. The use of any one of claims 1 to 9, wherein the liposome and/or boron cluster compound is targeted to a desired cell, tissue or organ.
11. The use of any one of claims 1 to 10, wherein the liposome and/or boron cluster compound comprises a targeting molecule capable of specific binding to a predetermined target.
12. The use of claim 11, wherein the targeting molecule comprises an antibody, receptor- binding protein, or substrate.
13. The use of any one of claims 1 to 12 wherein the agent is a therapeutically active agent.
14. The use of claim 13, wherein the therapeutically active agent is an anti-tumor agent.
15. The use of any one of claims 1 to 14, wherein the composition is a pharmaceutical composition.
16. The use of claim 15, wherein said pharmaceutical composition is designed to be administered systemically.
17. The use of any one of claims 1 to 12, wherein the agent is a diagnostic agent.
18. The use of claim 17, wherein the diagnostic agent is a dye.
19. The use of claim 18, wherein the dye is carboxyfluoresceine.
20. The use of any one of claims 1 to 14 or 17 to 19, wherein the composition is a diagnostic composition.
21. A composition comprising as a first component a boron cluster compound and as a second component a liposome as defined in any one of claims 1 to 12.
22. The composition of claim 21, wherein said liposome comprises a therapeutically active or a diagnostic agent as defined in any one of claims 13 to 19.
23. The composition of claim 21 or 22, wherein said components are designed to be administered or applied concurrently or sequentially.
24. A method of releasing an agent from a liposome under predetermined conditions comprising the step of exposing the liposome to a boron cluster compound such as to release the agent.
25. A method of identifying and obtaining a therapeutically active agent comprising (a) incubating a cell culture with a liposome containing a test compound; (b) exposing the cell culture to a boron cluster compound; and
(c) determining a detectable response of a cell in the cell culture compared to a control, thereby identifying the therapeutically active agent.
26. The method of claim 25 further comprising synthesizing the therapeutically active agent comprising determined in step (c) in a therapeutically effective amount.
27. A method of obtaining a genetically altered cell comprising the method of claim 24 or
25.
28. A method of assaying liposomes or agents encapsulated by liposomes comprising subjecting the liposome to a boron cluster compound.
29. A kit for use in the method of any one of claims 24 to 28 comprising a liposome, optionally containing an agent, a boron cluster compound, and optionally means for detection.
30. Use of a boron cluster compound or components thereof, a liposome, a therapeutically active or diagnostic agent for use in the method of any one of claims 24 to 28.
31. Use of a liposome for the preparation of a pharmaceutical or diagnostic composition, encapsulating an agent, wherein the liposome is designed for being triggered to release the agent by a boron cluster compound.
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