Patent Application
20140350323
The finding of appropriate therapeutic treatments of diseases such as bacterial or fungal infections, or cancer is still a major task in the field of medicine and pharmacology. Different approaches have been established over time to be utilized in the treatment of these diseases or to minimize their symptoms. In Europe nearly every third man suffers from cancer during the course of his life. Upon diagnosis of cancer, the survival rate within a term of five years is approximately 55%. In Germany, roughly 400,000 new cases of patients that suffer of cancer are accounted per year. The most frequent type of cancers among the human population is breast cancer, intestinal cancer and lung cancer. These types of cancer are main targets for different medical treatment approaches. Cancer treatments generally include resections of tumor tissue, chemotherapy with cytostatics and angiogenesis inhibitors. In addition, irradiation therapy is supplied in combination with the application of radio pharmaceuticals, X-rays, thermionic irradiation and neutron irradiation.
One problem associated with the application of irradiation can be seen in the high doses required in order to diminish cell growth of treated cancer cells. The high doses of irradiation however cause a number of severe side effects with unpleasant outcome for the patient. One major problem of such irradiation treatment is that not only the cancer tissue is affected by irradiation but also surrounding healthy tissue. It is therefore an aim in radiology to decrease the irradiation doses required for treatment of diseases. Irradiation enhancers were found to maximize the irradiation effects, and thereby minimizing the doses required for diminishing cell growth. Such enhancers are able to achieve a reduction of the irradiation doses utilized for the treatment of target cells.
Lanthanide compounds and their uses for MRT and other applications have been extensively discussed (Caravan P., Ellison J. J., McMurry T. J., Lauffer R. B. (1999) Chem. Rev. 99, 2293-2352 “Gadolinium(III) Cheleates as MRI Contrast Agents: Structure, Dynamics, and Applications”; Wiener E. C., Konda S., Shadron A., Brechbiel M., Gansow O. (1997) Invest. Radiol. 32, 748-54 “Targeting dendrimer-chelates to tumors and tumor cells expressing the high-affinity folate receptor”).
U.S. Pat. No. 6,770,020 B2 describes a method of using gadolinium-containing compounds as agents for neutron capture therapy to treat neoplastic cell growth. The subject is exposed to a gadolinium-containing compound for a time sufficient to allow the compound to accumulate in neoplastic cells. The subject is then exposed to a thermal and/or epithermal neutron flux, thereby Initiating a neutron capture reaction In the gadolinium atoms that results in specific death of neoplastic cells.
U.S. Pat. No. 5,888,997 describes irradiation sensitizers and the use of texaphyrins for irradiation sensitization and other conditions for which X-ray irradiation has proven to be therapeutically effective.
EP 012 92 298 B describes halogen compounds for use in a photo therapeutic treatment of a disease. The compounds are used for increasing the efficiency of a radiation therapy.
U.S. Pat. No. 6,040,432 describes metal complexes of DTPA derivatives suitable for use in diagnosis and therapy. Heavy elements were used in NMR/MRT diagnostic and as irradiation therapeutics.
T. Nawroth, et al., SRMS 4, Conference “Synchrotron Irradiation in Material Sciences”, Grenoble, Aug. 23-25, 2004, describes magnetic liposomes and trapping target hollow magnetic particles for biomedical applications. The method described is used for imaging, and neutron and photodynamic X-ray therapy of cancer.
WO 2009/121631 A2 describes polymer based nano particles which comprises one or more soluble lanthanide compounds such as Erbium-169, Samarium-153, Yttrium-90 embedded in a solid biodegradable polymer particle. Preferred biocompartible polymers are polyesters such as polyhydroxybutyric acid, polyhydroxyvaleric acid, polycaprolactone, polycyanoacrylate, polycarbonate, polylactide (PLA), poly (lactideco-glycolide), polylactic (also termed polylactide), polyglycolic, acid (also termed polyglycolide), apolylactic-polyglycolic acid.
U.S. Pat. No. 6,770,020 B2 describes another method of using gadolinium-containing compounds as agents in the treatment of neoplastic cell growth. The subject is exposed to a gadolinium-containing compound for a time sufficient to allow the compound to accumulate in neoplastic cells. The subject is then exposed to a termal and/or epitermal neutron flex, thereby initiating a neutron capture reaction in the gadolinium atoms that results in specific death of neoplastic cells. Although the systems and methods described above may show some effects in killing cancer cells, there is a need to increase the efficiency in the treatment of cancer using lanthanide compounds-containing particles.
Against this background, it is object of the present invention to provide an improved irradiation enhancer system which is based on the a selection of lanthanide compounds for diminishing the growth of target cells and which allows for reducing the dose of irradiation applied to the target cells in order to minimize the risks of side effects and to increase the efficiency of irradiation treatment.
This object is solved by a particulate system with the technical features of claim 1. The sub claims relate to preferred embodiments of the present invention.
The present invention provides polymer particles that comprises one or more lanthanide compounds embedded in a solid biodegradable polymer particle, wherein the lanthanide compounds of the polymer particles have a photon energy that is greater than 38 keV and a K absorption edge Z that is greater than 56.
The use of heavy metal lanthanide compounds with a photon energy >38 keV and a K absorption edge Z >56 in the particulate system of the invention results in an enhancement of radiation by increasing the radiation absorption diameter due to photo electrical absorption of electrons at the K layer. The radiation of the lanthanide particles of the invention will deeply enter the tissue, in particular cancer tissue, and thus will able to reach the localisation of the tumour. At the same time, severe burns of the surrounding non-target tissue will be avoided. Contrary to other methods, the methods according to the present invention do not result in a higher sensitisation of cells (chemical sensitizer effect) but apply radiation enhancement. In addition, radioactive or toxic effects are avoided by the methods of the invention.
Preferred heavy metal lanthanide compounds having >38 keV and a K absorption edge Z >56 are compounds that are stable isotopes or long-term isotopes with a half-life of more than 1010 years. Preferred lanthanide compounds have a K absorption edge Z between 57 and 83 and include lanthanide compounds ranging from lanthanide up to bismuth and are non-radioactive.
In one embodiment the polymer particles loaded with one or more lanthanide compounds are provided in freeze-dried form, preferably in the form of freeze-dried powder. Surprisingly, the particulate system in combination with freeze-drying results in an increased efficiency In killing cancer cells as compared to non-modified polymer particles, which are provided in aqueous suspension.
In another embodiment, the polymer particles of the invention are provided in modified, stabilised form in suspension. In order to obtain such stable polymer particles, the surface of the polymer are stabilized by detergents or stabilizers.
The inventors of the particulate system according to the present invention discovered that embedding lanthanide compounds having photon energy that is greater than 38 keV and K absorption edge Z that is greater than 56 into biodegradable polymer particles is highly efficient for killing cancer cells.
The particulate system according to the present invention preferably comprises one or more water soluble lanthanide compounds (including their salts) that are embedded in a solid biodegradable polymer particle for delivery to the target cells. Preferred polymers used for embedding the lanthanide compound or a mixture thereof are polycarbonic acids, polylactic acids, polyglycolic acids, polypeptides or combinations thereof.
The lanthanide compound is preferably selected from the group consisting of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, erbium, dysprosium, holmium, erbium, thulium, yterbium, lutetium, scandium, yttrium, hafnium iridium, platin, gold, bismuth and their salts. The use of lanthanide salts, preferably acetate salts, is preferred.
Lanthanide compounds that show a characteristic irradiation pattern upon excitation are suitably detectable by well known analysis methods in the art. In one embodiment, erbium acetate is a preferred lanthanide compound to be packed in a solid, freeze-dried biodegradable polymer particle. In an alternative embodiment gadolinium acetate is preferred. It is further possible to combine one or more lanthanide compounds or their salts with other types of irradiation enhancers (e.g. cis-platin, resveratrol, hydroxychalcone, roscovitine, amrubicine, amrubicinol) or even cytostatics such as doxorubicine or Paclitaxel in the freeze-dried polymer particles of the invention.
The polymer of the particulate system Is preferably selected from the group consisting of any optically active (D-/L-/DL-) forms of poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly (lactide-co-glycolide) copolymers (PLGA), polydioxanone (PDS), polyacrylates, polyketales, polycyanoacrylates, polyorthoesters, polyacetates, poly(ε caprolactone), polyphosphozene, polycarbonates, polypeptides, polyiminocarbonates, poly(β-hydroxyester).
Poly(glycolic acid) (PGA), poly(lactic acid) (PLA) and their copolymers are preferred biodegradable polymers of the particulate system according to the present invention. These polymers degrade in the body by hydrolysis of the ester backbone to non-harmful and non-toxic compounds. The degradation products are either excreted by the kidneys or eliminated as carbon dioxide and water through well-known biochemical pathways. The polymers PGA, PLA, PLGA and PDS as well as their copolymers can be used in all optically active forms or as part of a racemic mixture of their active L- and D-forms.
The life-time of polymers within the human or animal body and therefore their effectiveness can be controlled by selecting different appropriate end-groups. Preferably, the polymer utilized in the particulate system of the invention has either a free carboxylic acid end group, an ester terminated end group or an alkyl ester end group. Polymers kept with ester terminated and alkyl ester groups typically show longer degradation life times than the free carboxylic analogues. One preferred polymer of the invention is poly(D,L-lactide-co-glycolide) with a free carboxylic acid end group.
The packaging of lanthanides/lanthanide salt compounds in solid biodegradable polymer particles surprisingly results in a rather high enrichment of the irradiation enhancer within the particle and hence high delivery doses to the target cells. The freeze-dried biodegradable polymer particles are superior in their efficiency to non-modified particles in aqueous suspensions. This may be explained by the formation of solid bounds between the nano-particles in suspension. As a result, agglomerates are formed, which are poorer absorbed by the cells as compared to freeze-dried particles. The inventors further showed by electronmicroscopy that the formation of solid bounds between the particles is essentially completed upon storage of the samples over night. By contrast, only little or no solid bounds could be observed using freeze-dried particles. Therefore, the uptake of a lanthanide compound of the invention is a significantly increased using freeze-dried particles.
The efficiency of polymer particles in suspension can be increased, however, by modifying the surface of the particles using detergents or other stabilizers. Such treated particles also show increased efficiency.
The use of lanthanide compounds in form of their acetate salts is preferred since there appears to be an unexpected and surprising molecular interaction between chemical residues of the acetate salt and structures of the polymer. The polymer particles provide a high efficiency in the uptake of the lanthanide compounds by the target cells. The polymer particle of the invention is thus a suitable means to deliver the lanthanide compounds to the target cell (e.g. bacterial, fungal or cancer cell).
One major advantage of the invention is that the particulate system uses polymer particles that are biodegradable. The use of biodegradable polymers avoids an unwanted accumulation of polymer compounds within the treated tissue, in particular in the human or animal body. The biodegradable polymer used in the particulate system of the invention will be physiologically degraded after a certain time.
The particulate system of the invention, consisting of lanthanide compounds embedded in solid biodegradable polymers is preferably produced by solvent evaporation. A defined amount of polymer is diluted in dichloromethane. A lanthanide salt (e.g. erbium acetate) is diluted at high concentration in water resulting in an aqueous phase. The aqueous phase is emulsified within the oily phase of the polymer fraction by treatment with an ultrasonic stirrer on ice. The resulting O/W emulsion is transferred into a W/O/W emulsion by addition of approximately 2.5× vol of a 1% aqueous solution of polyvinyl alcohol with an average molecule mass of 72,000 g/mol. A subsequent ultrasonic treatment is followed.
The resulting emulsion is stirred slowly in 3× vol of water, preferably in a round-bottomed flask on a magnetic stirrer at low pressure (approximately 500 mbar) for several hours. Following incubation, the solvent dichloromethane is allowed to enter into the aqueous phase and subsequently into the gas phase. The evaporation of the solvent results in a hardening of the polymer particles and their separation. Upon evaporation of the solvent, the size of the produced particles can be controlled by dynamic light scattering (DLS).
The invention further relates to a method for diminishing cell growth, comprising the steps of exposing cells to a particulate system, comprising one or more water soluble lanthanide compounds as irradiation enhancer, wherein the lanthanide compounds are embedded in a biodegradable polymer particle as carrier, the polymer being selected from the group consisting of polycarbonic acids, polylactic acids, polyglycolic acids, polypeptides or combinations thereof, freeze-drying the particles loaded with one or more lanthanide compounds and exposing the cells that are treated with the particulate system to irradiation at a wave length that results in an excitation of the lanthanide compound(s).
The method can be used both for in-vitro and in-vivo treatments. The carrier systems and the methods according to the invention can be used both for therapeutic and diagnostic purposes.
Depending on the lanthanide used, the irradiation dose for treating the target cells is at least 4 Gy. A suitable pre-incubation time for the irradiation treatment of the target cells that were exposed to lanthanide loaded polymer particles is at least 24 h.
The invention further relates to a method for producing particles, comprising one or more water soluble lanthanide compounds that are embedded in a solid biodegradable polymer particle, the polymer being selected from the group consisting of polycarbonic acids, polylactic acids, polyglycolic acids, polypeptides or combinations thereof by incubating the polymer with the lanthanide compound in a suitable solvent solution, emulsifying the polymer/lanthanide mixture and applying solvent evaporation to hardening the particles.
The invention also relates to a pharmaceutical composition, comprising one or more water soluble lanthanide compounds that are embedded in a solid biodegradable polymer particle, the polymer being selected from the group consisting of polycarbonic acids, polylactic acids, polyglycolic acids, polypeptides or combinations thereof for use in the treatment of a disease.
In a preferred embodiment, the disease is a bacterial or fungal Infection or cancer. The systems and methods according to the invention can be both applied to prokaryotic and eukaryotic cells. Pathological diseases that are caused by a bacterial or fungal infection are based on pathogenic bacteria or fungi. Both bacterial and fungal cells can be exposed to the particulate system according to the invention. Cell growth is inhibited or reduced in these cells by delivering the lanthanide compounds by the polymer carrier to the respective target cells. The systems and methods according to the invention are also suitable for treatment of pathogenic eukaryotic cells, in particular cancer cells. Cancer tissue/cells can be efficiently treated with freeze-dried polymer particles that are loaded with lanthanide compounds according to the present invention, thereby causing a dose-dependent reduction of cell growth or cell death.
The manufacture and applicability of the particulate system according to the invention is more fully explained in the following. The experimental data that support and demonstrate the present invention are shown in the accompanying Figures. Lanthanide compounds used in these experiments have a photon energy >38 keV and a K absorption edge Z >56.
The pharmacological effect of the particulate system was investigated in cells of the lung carcinoma cell line A549. In order to determine a reduction of cell growth, A549 cells were seeded in 96 well plates and incubated for approximately 24 h before further treatments in order to reach nearly complete fixation of the cells to the bottom of the plates. The cells were then incubated together with polymer particles that were loaded with erbium for approximately 3 hours. Following exposing the cells with lanthanide particles, the cell culture plates were irradiated with different doses of irradiation. The proliferation of cells was determined by using a MU growth assay in order to determine survival of irradiated tumor cells. The MTT assay allows the analysis of proliferation and determination of survival of cancer cells following irradiation and is based on a reduction of yellow water soluble tetra sodium salt to a purple water insoluble formazane dye by living cells. The cell proliferation over a period of 5 to 6 days following irradiation was analyzed. The results are presented in
Survival was calculated by using a mathematical approach in which cell survival is calculated using the following formula:
Survival=2 ̂−(tdelay/tdoubling time)
T doubling time=time for cell doubling
T delay=time required to achieve a specific absorption value in the MTT test of the irradiated sample in comparison to the control.
In order to determine lanthanide-dependent absorption properties, A549 cells were incubated with different test samples and irradiated with variable irradiation doses. Irradiation with a monochromatic pattern above and below the k absorption profile of the lanthanide was applied.
According to
In
According to
| Number | Date | Country | Kind |
|---|---|---|---|
| 11007401.0 | Sep 2011 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2012/003825 | 9/12/2012 | WO | 00 | 8/18/2014 |
