Bisphosphonate-Modified Liposomes Containing Nanoparticles

Abstract

The invention relates to liposomes containing nanoparticles, wherein the nanoparticles are selected from magnetic, zparamagnetic, superparamagnetic and/or fluorescent and/or functionalized nanoparticles, and the liposomal sleeve contains lipid-derivatized bisphosphonic acid. The liposomes are suitable for preparing a solution for the diagnosis of pathological tissue degeneration or conversion processes on the bone and in the bone marrow, in particular for the treatment and diagnosis of bone tumors and bone metastases and disorders in the bone marrow (proliferative diseases of the blood-producing and lymphoreticular system).

Claims

1.-14. (canceled)

15. A liposome comprising: nanoparticles, the nanoparticles selected from magnetic, paramagnetic, superparamagnetic and/or fluorescent and/or functionalized nanoparticles; a liposomal envelope comprising lipid-derivatized bisphosphonic acid.

16. The liposome according to claim 15, wherein the lipid-derivatized bisphosphonic acid is selected from compounds having the general formula I ##STR00003## wherein R.sup.1 is H, OH, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-alkoxy, C.sub.1-C.sub.6-hydroxyalkyl, C.sub.1-C.sub.6-aminoalkyl, C.sub.1-C.sub.6-halogenalkyl, X is a direct bond, an alkylene group with 1 to 20 carbon atoms, (CH.sub.2).sub.m—(OCR.sup.3HCH.sub.2).sub.n—(O).sub.o—, in which R.sup.3 means H or CH.sub.3 and m is 0 or a number from 1 to 6, n is a number from 1 to 10, in particular 1 to 6, and o is 0 or 1, wherein m, n, and o are not 0 at the same time, —(CR.sup.4HCH.sub.2O).sub.p—, R.sup.4 means H or CH.sub.3, p is a number from 1 to 10, in particular 1 to 6, (CH.sub.2).sub.q—(OCR.sup.5HCH.sub.2).sub.r—(O).sub.s—(CH.sub.2).sub.t—, in which R.sup.5 means H or CH.sub.3, and q is 0 or a number from 1 bis 6, r is a number from 1 to 10, in particular 1 to 6, and s is 0 or 1, und t is a number from 1 to 6, R.sup.2 is a substituent with the formula (II) ##STR00004## or a fatty acid chain with 8 to 22 carbon atoms, wherein the substituents with the formula II and the fatty acid chain can comprise substituents such as halogen, in particular F, and physiologically acceptable derivates thereof, including salts and trimethylsilyl derivates.

17. The liposome as claimed in claim 16, wherein the liposomal envelope further contains phospholipids and/or a uronic acid derivative.

18. The liposome as claimed in claim 15, wherein the nanoparticles are selected from the group consisting of iron oxides, pure iron with an oxide layer, ferrofluids, QDs, gadolinium, silicate particles coated with paramagnetic or fluorescent substances, gold particles coated with paramagnetic or fluorescent substances, carbon particles coated with paramagnetic or fluorescent substances, and mixtures thereof.

19. The liposome as claimed in claim 18, wherein the nanoparticles comprise a protective envelope or a functionalized coating.

20. The liposome as claimed in claim 15, further comprising one or more therapeutically and/or diagnostically active substances.

21. The liposome as claimed in claim 20, wherein the one or more therapeutically and/or diagnostically active substances are present within the liposomal envelope or are bonded to the liposomal envelope.

22. The liposome as claimed in claim 20, wherein the one or more therapeutically active substances are selected from the group consisting of antiproliferative substances, antimigratory substances, antiangiogenic substances, antithrombotic substances, anti-inflammatory substances, antiphlogistic substances, cytostatic substances, cytotoxic substances, immunotherapeutic substances, anticoagulative substances, antibacterial substances, antiviral substances, antimycotic substances, and vaccines.

23. The liposome as claimed in claim 20, wherein the one or more diagnostically active substances are selected from the group consisting of contrast agents for imaging methods, radial nucleotides, and tumor markers.

24. A solution containing liposomes according to claim 15 for producing a therapeutic agent, a diagnostic agent or a combined theranostic system for diagnosis and/or treatment of pathological tissue degradation or conversion processes on the bone or in the bone marrow, including treatment and diagnosis of bone tumors and bone metastases and disorders in the bone marrow.

25. A method of producing a solution for localization and diagnosis of bone conversion processes, the method comprising using a liposome according to claim 15 in the solution.

26. A method of producing a solution for focal treatment of bone tumors and bone metastases, own and foreign metastases in bone tissue, and diseases of the blood-producing and lymphoproliferative system by thermal ablation, the method comprising using a liposome according to claim in the solution.

27. A method for therapeutic thermal ablation of tumors and metastases, the method comprising using the solution according to claim 24.

28. A diagnostic method for recognizing and marking tumor lesions at or in the bone, the method comprising using the solution according to claim 24.

Description

[0029] In a preferred embodiment, the liposomal envelope contains a compound with the general formula I, phospholipids, and/or a uronic acid derivative. The afore described bisphosphonates are characterized by a high affinity to the bone and are suitable as expedient for the active ingredient as well as for the transport of diagnostics, for example, supermagnetic particles, radioactive particles etc. The nanoparticles employed according to the invention comprise the above described bisphosphonic acids as envelope. In a preferred embodiment of the present invention, the envelope represents a liposomal encapsulation. Liposomes comprise colloid, vesicular structures on the basis of (phosphorus) lipid double bilayers. Because of these structural properties, they can incorporate hydrophilic as well as hydrophobic molecules. They are decomposable and substantially non-toxic because they are comprised of natural biomolecules.

[0030] The liposomes according to the invention comprise preferably a particle size of 50 nm to 200 μm (0.05 μm to 200 μm), in particular of 100 nm-250 nm. Such a particle size enables a stable transport of the liposomes to the site of action in the bone tissue.

[0031] According to the invention, the liposomes contain nanoparticles that are selected from magnetic and/or fluorescent nanoparticles. Suitable nanoparticles should have such a size that they can be encapsulated by liposomes and can comprise a size of 5 to 450 nm. A common particle size of the nanoparticles amounts to 5-20 nm. Preferably, the nanoparticles are selected from nanoparticles of iron oxides, pure iron with an oxide layer, ferrofluids, QDs, gadolinium, silicate, gold or carbon particles coated with magnetic or fluorescent substances, and any mixtures thereof.

[0032] The magnetic particles that are contained in the nanoparticles employed according to the invention are magnetic particles known from the prior art. They are comprised of a magnetic material, preferably a ferromagnetic, anti-ferromagnetic, ferrimagnetic, anti-ferrimagnetic or superparamagnetic material, further preferred of iron oxides, in particular superparamagnetic iron oxides or of pure iron which is provided with an oxide layer. The scope of the present invention encompasses also paramagnetically coated QDs (quantum dots), QDs which contain an Fe core or silicate-coated nanoparticles with a magnetic core as well as fluorescent and also radioactive substances or solutions, such as solutions of Tc-99, C-14, and stable isotopes, such as C-13, F-19, which can be used in MRT in bisphosphonate liposomes. The aforementioned particles can be heat-activated by means of electrical alternating fields.

[0033] In a possible embodiment, the liposomes according to the invention are heated by a magnetic alternating field. Heating of the tissue containing the nanoparticles to more than 50° C. is possible. Such high temperatures can be reached because up to 800 pg and more of iron in the form of the nanoparticles can be taken up per tumor cell.

[0034] Preferably, the nanoparticles are comprised of iron oxides and in particular of magnetite (Fe.sub.3O.sub.4), maghemite (γ-Fe.sub.2O.sub.3), or mixtures of these two oxides. In general, the preferred nanoparticles can be represented by the formula FeOx wherein x is a number from 1 to 2. The nanoparticles comprise preferably a diameter of less than 500 nm. Preferably, the nanoparticles have an average diameter of 15 nm or lie preferably in the size range of 1 to 100 nm and particularly preferred in the range of 10 to 20 nm.

[0035] In addition to the magnetic materials of the formula FeOx wherein X is a number in the range from 1.0 to 2.0, according to the invention also materials of the general formula MFe.sub.2O.sub.4 with M═Co, Ni, Mn, Zn, Cd, Ba, Gd or other ferrites can be used. Moreover, also silica-carbon or polymer particles are suitable in which the magnetic materials such as, for example, the herein mentioned magnetic materials, are incorporated and/or bonded.

[0036] Preferably, these particles are comprised of magnetic iron oxides or of pure iron with an oxide layer. These magnetic particles can be, for example, produced according to the method disclosed in DE 4428851.

[0037] In a further possible embodiment, the liposomes contain fluorescent nanoparticles, such as, for example, particles of silica or calcium phosphate doped with a dye or surface-modified semiconductor particles, such as those of binary compounds such as lead sulfide, lead selenide, cadmium selenide, cadmium sulfide or cadmium telluride or of ternary compounds such as cadmium selenide sulfide, zinc selenide, which in the biological research as imaging agents or in clinics as local markers or reference points for detection of NP by means of X-ray, carbon nanoparticles (Indian ink) and gold nanoparticles. Particularly suitable particles are also so-called QDs (quantum dots) which are superior to known fluorescent dyes due to their intensive fluorescence and photostability. QDs on the basis of zinc selenide are particularly preferred because of their fluorescent properties, good functional properties, and minimal toxicity. The excitation of the fluorescent nanoparticles is realized in conventional manner known to a person of skill in the art, such as photo excitation by means of suitable light sources.

[0038] In a further embodiment, the nanoparticles are selected from carbon nanoparticles which can also be coated and/or functionalized and can be imparted with semiconductor properties by the functionalization. The carbon nanoparticles have the advantage that they are less toxic in comparison to iron-containing QDs.

[0039] The nanoparticles and in particular the QDs can be bonded to proteins, oligonucleotides, smaller molecules etc. in order to bind them immediately to the target at the bone.

[0040] In a possible embodiment, the nanoparticles comprise a protective envelope or functionalized coating.

[0041] This protective envelope or coating can also comprise a functionalization of the surface. The functionalization of the surface comprises free amino groups, hydroxide groups, carboxyl groups or carbonyl groups to which an active ingredient or a functional linker can be bonded by means of an imine bond, amine bond, ester bond, amide bond or ketal bond. By means of this linker, also a therapeutically active or diagnostic substance—e.g. a receptor-binding antibody—can be bonded covalently, ionically, by complexing, lipophilically or by hydrogen bonds. The production of particles with a protective envelope and optionally a functionalization can be realized according to methods as they are disclosed in WO 2006108405.

[0042] In addition to the magnetic nanoparticles, the liposomes according to the invention can also contain therapeutically active and/or diagnostically active substances. These substances are transported by means of the liposomes directly to the site of action and can be released thereat. The release is realized usually spontaneously when the liposomal particle has reached the site of action/target, decomposes or the thermal ablation is carried out.

[0043] In a further embodiment, the optionally contained therapeutically active substances are not directly bonded to the magnetic particles but can be present within the envelope which contains the lipid-derivatized phosphonic acid. In case of a liposomal encapsulated form, the therapeutic as well as diagnostic active ingredient as well as the magnetic particles are liposomally encapsulated. The active ingredient and the magnetic particles can be present together encapsulated in a liposome but also in separate liposomes.

[0044] These substances can be present within the liposomal envelope or can be bonded to the envelope. For example, they can be located at the surface of the nanoparticles and/or present at the nanoparticles without being bonded thereto. At the surface, the substances can be bonded by binding locations, for example. Bonding to the surface can be covalent such as by a functional group arranged at the surface, any other bonding such as an ionic bond or other interactions. In a preferred embodiment, the diagnostically or therapeutically active substances are bonded to the lipid-derivatized bisphosphonic acid, for example, by a covalent bond, an ionic bond and/or by van der Waals interaction.

[0045] The therapeutically active substances can be selected from chemical or biological therapeutically active substances such as antiproliferative, antimigratory, antiangiogenic, antithrombotic, anti-inflammatory, antiphlogistic, cytostatic, cytotoxic, immunotherapeutic, anticoagulative, antibacterial, antiviral and/or antimycotic substances as well as vaccines. Particular preferred are antiproliferative, antimigratory, antiangiogenic, cytostatic and/or cytotoxic substances as well as nucleic acids, amino acids, peptides, proteins, carbohydrates, lipids, glycoproteins, glycans or lipoproteins with antiproliferative, antimigratory, antiangiogenic, antithrombotic, anti-inflammatory, antiphlogistic, cytostatic, cytotoxic, anticoagulative antibacterial, antiviral and/or antimycotic properties.

[0046] As cytotoxic and/or cytostatic components, for example, alkylation agents, antibiotics with cytostatic properties, anti-metabolites, microtubuli inhibitors and topoisomerase inhibitors, platinum-containing compounds, and other cytostatic agents, such as, for example, asparaginase, tretinoin, alkaloids, podophyllotoxins, taxanes, and Miltefosin®, hormones, immunomodulators, monoclonal antibodies, signal transducing agents (signal transduction molecules), and cytokines can be used.

[0047] As diagnostic substances, all conventional diagnostic agents which are used in clinical day-to-day operation and specialized centers can be used which are suitable in radiological methods such as CT, X-ray, MRT, NMR, and in the nuclear medical, such as isotope scintigraphy/Gamm camera, positron emission tomography (PET) and as radiopharmaceuticals. These substances also include therapeutically and/or diagnostically active substances such as contrast agents for imaging methods, radionucleotides, antibodies, and tumor markers.

[0048] Tumor markers are biochemical substances which, for some cancer types, are produced by the tumor cells, are expressed/exist on their cell surface, and are released into the blood. Accordingly, they can be diagnostically detected with sensitive methods on the tumor cells or in the blood of the patient.

[0049] Tumor markers are often built of sugars and proteins (so-called glycoproteins) such as e.g. the carcinoembryonic antigen (for short CEA), a marker for colon cancer. In addition to glycoproteins, hormones, and enzymes, genetic diagnostics are increasingly used. When a tumor shows certain genes (gene expression), this can be an indication of the special tumor cell type of which a primary tumor or its metastases are comprised.

[0050] Correspondingly, one can diagnose, localize and characterize the tumor, and then therapeutically attack it, e.g. operate or destroy in a targeted fashion, e.g. by thermal ablation or, newly, with antibodies as examples of an on-target therapy (“targeted therapy”). In this context, it is very important that as many as possible of the tumor cells are completely removed. This is often more likely the case for an on-targeted attack on the molecular level.

[0051] However, not all medications even for intravenous application reach their site of action in a sufficient quantity. Therefore, they must be e.g. locally injected or enriched in the tissue, but injection is hardly possible in case of bone.

[0052] For different cancer diseases, there are different markers. The known tumor markers include for example:

[0053] and other markers are continuously newly established.

TABLE-US-00001 Tumor Type Marker Breast cancer CA15-3, CEA, CA 125, HER2-new Ovarian cancer CA 125, beta-HCG, AFP Lung cancer NSE, CYFRA 21-1, SCC Stomach cancer CEA, CA-72-4, CA 19-9 Colon cancer CEA, EGFR Pancreatic cancer Cd 44 Prostate cancer PSA, PSMA, CG-1 Bone cancer RANKL

[0054] In a particularly preferred embodiment, the liposomes contain tumor markers.

[0055] Various cancer types, such as e.g. breast cancer or prostate cancer, metastasize into the bone. For example, a liposome according to the invention can contain a HER2 antibody-coated nanoparticle, such as e.g. an Fe nanoparticle, and can be injected intravenously as diagnostic agent and, for example, detected by means of MRT.

[0056] When as a result of the liposome transport to the bone after spontaneous release in the MRT a positive enrichment of the antibody-laden nanoparticles at the bone is found, these HER2 antibody nanoparticles can be used for therapy of the bone metastasis for a local thermal ablation, but the free antibodies (without Fe particles) can also be transported, liposomally encapsulated, in a targeted fashion to the bone metastases. This would be a treatment in the form of a theranostic system.

[0057] The production of the liposomes according to the invention can be carried out with methods known in the prior art. A possible method is, for example, the lipid film extrusion method. The production of liposomes is, for example, disclosed in the dissertation of Verena Hengst (Department of Pharmacy of the Philipps University of Marburg, 2007). Further production methods can be found at: https://de.wikipedia.org/wiki/Liposomenerzeugung.

[0058] The liposomes according to the invention are present in a conventional liposomal formulation, for example, in form of a liposomal dispersion. This liposomal formulation can be administered as such, or it can be further processed to a solution (application solution) as is generally known in the art.

[0059] As further components, the liposomes and liposome formulations according to the invention can contain expedients known in the prior art for the production of liposomes, such as solvents, rheological agents (dextrans, heparin derivatives), antioxidants, esterase inhibitors, pH buffering substances. In particular pH buffering substances are suitable to influence the stability of the liposomes and their interaction with the target cells.

[0060] A further subject matter of the invention is a solution which contains the above-described liposomes and for production of a therapeutic agent, diagnostic agent or a combined theranostic system for diagnosis and/or treatment of pathological tissue degradation or conversion processes at the bone or in the bone marrow, in particular for treatment and diagnosis of bone tumors and bone metastases as well as disorders in the bone marrow (proliferative diseases of the blood-producing and lymphoreticular system).

[0061] For example, for diagnosis and/or treatment of bone tumors and bone metastases, own and foreign metastases, and of pathological bone tissue processes. In a possible embodiment, the solution is an infusion solution or an injection solution.

[0062] A further subject matter is the use of liposomally encapsulated nanoparticles for producing a solution for focal treatment and/or diagnosis of bone conversion processes, wherein the nanoparticles are selected from magnetic, paramagnetic, superparamagnetic or/and fluorescent and/or functionalized nanoparticles and the liposomal envelope contains lipid-derivatized bisphosphonic acid or bisphosphonic acid derivatives.

[0063] Yet another subject matter is the use of the afore described liposomally encapsulated nanoparticles for producing a solution for localization, diagnosis and/or therapy of bone conversion processes.

[0064] The solutions (application solutions) are aqueous solutions that comprise a pH value in the physiological range, preferably between 6.8 and 8.0. These solutions, for example, can comprise emulsifiers and stabilizers, buffer systems such as HEPES, and further components that do not impair the stability of the liposomes and enhance the absorption into the cell. For example, the liposomes can be stable but the absorption into the cells is disturbed when the medium of the liposome formulation is not neutral or is too acidic. Also, the charge of the liposome envelope has an influence on the absorption into the cell; it should be as neutral as possible or only weakly negative/acidic.

[0065] In a possible embodiment, the liposomes according to the invention are used for producing a diagnostic agent for recognizing, marking and/or agent for removal of tumor lesions (solid tumors and metastases) at or in the bone.

[0066] In particular, the liposomes according to the invention are suitable for thermal ablation of tumors and metastases and foreign metastases, in particular in the bone tissue.

[0067] The solutions, for example, infusion solutions but also injection solutions, are preferably a physiological saline solution that is suitable for interstitial or intra-tumoral application.