WO2015160270A1

WO2015160270A1 - Targeted liposomal form of oligonucleotide-polyethyleneimine complex, its use and method of obtaining

Info

Publication number: WO2015160270A1
Application number: PCT/PL2015/050010
Authority: WO
Inventors: Justyna MEISSNER; Monika TOPORKIEWICZ; Kazimierz Kuliczkowski; Aleksander CZOGALLA; Aleksander SIKORSKI
Original assignee: Wrocławskie Centrum Badań Eit+ Sp. Z O.O.
Priority date: 2014-04-18
Filing date: 2015-04-18
Publication date: 2015-10-22
Also published as: PL407947A1

Abstract

The invention relates to the targeted, liposomal form of oligonucleotide-polyethyleneimine complex, characterized in that the oligonucleotide to polyethylenimine ratio ranges from 1:10 to 1:3, and the complex is encapsulated with a lipid bilayer enabling attachment of a targeting molecule recognizing the surface marker of pathological cells, as well as the method of obtaining the same and its application.

Description

Targeted liposomal form of oligonucleotide-polyethyleneimine complex, its use and method of obtaining

The invention relates to the targeted, liposomal form of oligonucleotide-polyethylenimine complex, its application and the method of obtaining the same. The invention finds its application particularly in treatment of cardiovascular system neoplasms. Research works were carried out at the University of Wroclaw, Faculty of Biotechnology, Department of Cytobiochemistry and at the Wroclaw Medical University in the Hematology, Blood Neoplasms and Bone Marrow Transplantation Clinic. Anitsense gene therapy, as one of the younger branches of medicine, is a promising strategy in the treatment of many neoplasm diseases, including chronic lymphocytic leukemia (CLL). It has been found that ca. 70% of diagnosed CLL are related with overexpression of BCL-2 gene. High level of Bcl-2 protein in the cell inhibits releasing cytochrome c to cytosol thus blocking the activation of caspases 9 and 3 playing the key role in inducing programmed death of a cell, called apoptosis. The process of apoptosis has a significant meaning in origins of many diseases, therefore a great hope is put in the possibility to silence the expression level of genes like BCL-2 by introducing genetic drugs into cells (antinsense oligonucleotides, siRNA). Also, the relationship between high level of BCL-2 gene expression and the low effectiveness of chemotherapy was found. A great progress has been made in the treatment of cardiovascular system neoplasms due to the combination of chemotherapy with bone marrow transplantation or application of specific, humanized antibodies targeted against antigens presented by neoplastic cells. Learning the biology of neoplastic cells enabled development of new treatment methods, specific for individual types of neoplasms. The major assumption in the targeted therapy is specific delivery of the drug to the neoplastic cells with concurrent lack of toxicity against healthy cells. Many of the so called targeted drugs are now under the clinical studies . The main task of such therapy is to block specific signaling pathways or specific transporters or enzymes in neoplastic cells, which, for example, leads to sensitization of the cell to a decreased dose of cytostatic drugs such as anthracycline or taxane derivatives. Currently, among others, angiogenesis inhibitors, monoclonal antibodies, signal transduction inhibitors, apoptosis inducing antisense oligonucleotides, and siRNA constructs silencing expression of specific anti-apoptotic genes are often used in research. The desired effect is the death of neoplastic cells with concurrent minimization of side effects on the patient's body. Having learned the structure of antigens specific to neoplastic cells enabled the development of monoclonal antibodies which can not only direct the cytostatic drug towards the desired site, but can also directly induce apoptosis. An example of the new generation drug, currently widely used in CLL therapy, is Rituximab^R (MabThera) being a first generation of targeted therapeutic monoclonal antibody against lymphocytic leukemia B lymphocytes exposing CD20 antigen. There are many known genetic drugs which received CHMP (The Committee for Medicinal Products for Human Use) approval, however, nucleic acids are very sensitive to degradation by endonucleases both in room temperature and in bodily fluids. The key task here is effective and specific delivery of therapeutic drugs to neoplastic cells and reaching efficient transfection in order to silence expression of genes taking part in the genesis of disease , genes determining survival of neoplastic cells, or genes responsible for their resistance to drugs. Due to the physical and chemical properties of nucleic acids (e.g. antisense oligonucleotides - asODN) and sensitivity to enzymatic hydrolysis, it is necessary to use a carrier which protects them from enzymatic degradation without changing their properties, effectively deliver into the target cells, leading to inhibition of transcription or translation of the specific gene. Liposome carriers are widely used in the research on delivering genetic drugs in therapy of, among others, lymphatic leukemia. Liposomes are the alternative to viral carriers which are characterized with many drawbacks eliminating them for the moment from therapeutic applications. From the present state of art, it is known that having positive charge complexes of cationic lipid-nucleic acid, the so-called "lipoplexes" proved to be very toxic for the organism and they were characterized with a very short half- life period in the bloodstream. Carrier-DNA complexes with the resultant positive charge in the bloodstream have obstructed access to the target tissues. This results from the aggregation of carriers in the cardiovascular system and non-specific interaction with the extracellular matrix, surface of many cells and plasma proteins, which may lead to the complement system activation, and elimination of the therapeutic drug. Currently, polyethylene glycol is commonly used as the component of the liposomal coat. Long chains of PEG covering such liposomes hinder them from opsonization, thus enabling longer time of circulation in the blood. Anionic liposomes are characterized with the long circulation time, but due to their resultant charge, the effectiveness of the bare genetic drug encapsulation into this kind of liposomal constructs is very low. From the international patent application No. WO 98/43095, a liposome composition is known, comprising antisense oligonucleotide c-Raf-1 . Liposomes with the resultant positive charge in the bloodstream exhibit limited access to target tissues. This results from the aggregation of complexes in the blood and non-specific interaction with the extracellular matrix components, surfaces of many cells and plasma proteins, which leads to the complement system activation, and elimination of the carriers and the drugs. Furthermore, the invention of patent application WO 98/43095 does not act selectively. US Patent No. US7666674 discloses sterically stabilized cationic liposomes (SSCL) encapsulating a K-type oligodeoxynucleotide (ODN) including a CpG motif. The solution referred to the above does not allow for encapsulation of nucleotide sequences of various types. Furthermore, the carriers from US7666674 patent has the positive resultant charge. Surface cationic charge induces reactions with negatively charged blood components resulting in the loss of colloidal stability of such liposomes. The technical problem that the present invention is facing is to propose an effective liposome carrier - genetic drug - specific antibody system which could be effectively used in neoplasm therapy. The main goal of the invention is to develop a liposome carrier for genetic drugs, characterized with selective action, low non-specific cytotoxicity, and high efficiency in delivering drugs to neoplastic cells, which, separately or in combination with commonly used cytostatics, will significantly improve the treatment effectiveness. In order for liposomes to be applied as pharmaceutical technology, they must retain stability during the many months long storage. Therefore, the purpose of the invention is also obtaining a carrier retaining specified physical and chemical parameters during long-term storage, which would protect the encapsulated nucleic acid from hydrolysis by enzymes present in biological fluids. Unexpectedly, the problems mentioned above have been solved by the present invention.

The first object of the invention is a targeted, liposomal form of oligonucleotide-polyethyleneimine complex, characterized in that the oligonucleotide to polyethyleneimine ratio ranges from 1 :10 to 1 :3., and the complex is encapsulated in a lipid bilayer enabling attachment of a molecule recognizing the specific surface marker expressed on pathologically modified cells. Equally preferably, the molecule recognizing the surface marker of pathological cells is selected from a group comprising: humanized antibodies, antibody derivatives, proteins, aptamers, small targeting molecules or designed ankyrin repeat proteins (DARPins) and attached to the liposomes. The second object of the present invention is a use of targeted, liposomal form of the oligonucleotide-polyethyleneimine complex as specified in first object of the invention for production of a drug intended for the human blood neoplastic diseases treatment.

The third object of the present invention is the method of obtaining the targeted, liposomal form of the oligonucleotide-polyethyleneimine complex, which includes the following steps: a) asODN is mixed with polyethyleneimine in 3:1 to 1 0:1 ratio. The PEI/DNA ratio means the amount of polyethyleneimine nitrogen (N) moles to the amount of phosphorus (P) moles in the nucleic acid. To calculate the PEI and DNA amounts, the following values were used : 1 μg PEI in the solution contains 23 nmoles of N whereas 1 μg pDNA contains 3 nmoles of P. b) the PEI and DNA mixture is complexed, preferably by incubation, for 30-60 min. at room temperature in a ratio of 3-10 mole of PEI (N) in miliQ water to 1 mole of DNA (P) also in a miliQ class water, supplementing the mixture with 0.1 volume of 10 times concentrated PBS, c) chloroform solutions of lipids constituting the casing are evaporated until obtaining dry lipid film, d) the residual organic solvents are removed, preferably in a vacuum evaporator, e) the dry lipid film of step d) is hydrated with the mixture, preferably PEI/asODN obtained in step b), more preferably for 40 minutes, f) the liposomes obtained in step e) are calibrated, preferably in a high pressure extruder using polycarbonate filters. Equally preferably, in step f) 400 nm, 200 nm, and 100 nm filters are used consecutively. More preferably, the filtration in step f) is repeated ten times.

The solution according to the present invention allows for encapsulation of any genetic drug inside the carrier, and by attaching any targeting molecules (here: antibodies), directing to the specific cells of the organism. The proposed solution enables obtaining liposomes characterized by stability of particle size, zeta potential and nucleic acid content during long-term storage, lack of sensitivity to the components of human blood and plasma, good protective properties against effects of nucleolytic enzymes, effectiveness of action against cells exposing the specific surface antigen, and lack of toxicity to the cells free of that antigen, and first of all, high effectiveness against the specific neoplasm in vivo, in animal tests. Furthermore, the present invention can be used in a combined therapy together with relatively low doses of cytostatics. The construction of the proposed is flexible enough to be easily used as carrier for any genetic drug, like for example asODN, siRNA, miRNA, or DNAzyme targeted against expression of any gene, as well as to attach any humanized antibody or any other protein agent, or e.g. aptamer directing to the cells characterized with a specific surface marker. In the disclosed invention, the stability of the carrier and DNA complexes in the bloodstream has been increased by creating a hydrophilic layer of polyethylene glycol on the external surface of the carriers. Furthermore, the disclosed invention allows for introduction of genetic drug (antisense oligonucleotides as-ODNs, siRNA, shRNA, miRNA, DNAzym etc.) of predetermined sequence into the cells. Exemplary embodiments of the invention have been presented in the drawings, where fig. 1 represents a diagram illustrating the carrier particle diameter stability during twelve months storage in the form of suspension or freeze-dried powder, fig. 2 represents a diagram illustrating the fluctuations in the particle zeta potential during twelve months storage in the form of suspension or freeze-dried powder, fig. 3 represents a diagram illustrating fluctuations in the genetic drug content in liposomes during twelve months long storage in the form of suspension and freeze-dried powder, fig. 4 represents a diagram illustrating the effect of human serum and plasma proteins upon the stability of the proposed liposomes, fig. 5 represents the result of electrophoretic analysis confirming the protective properties of the carrier, fig. 6 represents a diagram illustrating the hemolytic properties of liposomes towards human erythrocytes within 1 -4h period, fig. 7 represents confocal microscopic images of target and control cells mixed in equal quantities and incubated with immuno-liposomes, fig. 8 represents the result of Western blot analysis illustrating the Bcl-2 protein level in Daudi cells incubated for 24 and 72 hours with immuno-liposomes, fig. 9 presents the compared doses of immuno-liposomes corresponding to IC50 and IC90 for control (Jurkat T and L1210) and target cells, fig. 10 represents the apoptosis and necrosis level in Daudi cell line, fig. 1 1 represents the survival curves for Daudi cell line in the function of concentration of immuno-liposome with the addition of mitoxantrone and control preparations, fig. 12 represents the diagram illustrating the tumor volume growth dynamics in individual groups of NOD/SCID mice bearing human Daudi Burkitt's lymphoma tumor xenograft treated with immuno-liposome preparations

Example

In order to obtain 1 ml of the preparation in the form of liposomal suspension, 100 μg asODN was mixed with polyethyleneimine in 1 :5 ratio. The PEI/DNA ratio is understood as the amount of polyethyleneimine nitrogen (N) moles to the amount of phosphorus (P) moles in the nucleic acid. The PEI and DNA complexation was carried out for 30 minutes at room temperature with supplementing the mixture with 0.1 volume of ten times concentrated PBS. So, prepared solution of the complexes in water was used for hydration of dry lipid film (room temperature, 30 min). The next step was the encapsulation of asODN-PEI complexes with lipid mixture prepared as mentioned above casing. For this purpose, the chloroform solutions of lipids constituting the composition of the casing 4.3 mq/ml HEPC, 1 .5 mq/ml DOPE, 0.6 mg/ml DC-CHOL, 1 .2 mg/ml DSPE-PEG, 0.51 mg/ml DSPE-PEG-Mal (weight ratio 2.8:1 :0.4:0.8:0.34 which makes molar ratio: 59.67 mol% HEPC, 21 .73 mol% DOPE, 12.1 1 mol% DC-CHOL, 4.65 mol% DSPE-PEG, 1 .84 mol% DSPE-PEG-Mal) were evaporated until dry lipid film was obtained. Next, the residual organic solvent was removed in the vacuum evaporator and hydrated with 1 ml PEI/asODN complex solution for 40 minutes. The obtained liposomes were calibrated in a high pressure extruder with the application of polycarbonate filters (Nulceopore, WHATMAN) of the pore diameters of 400, 200 and 100 nm, consecutively; the suspension was passed ten times through each filter. The size of the resulting particles was measured via dynamic light scattering technique, using a ZetaSizer apparatus from Malvern Ltd., UK. In order to obtain immune-liposomes, the maleimide DSPE-PEG derivative (DSPE-PEG-Mal) was used. The maleimide group enables covalent attachment of protein thiol groups forming thio-ether bond characterized with high stability. Thiol groups in the attached antibodies were obtained in the result of thiolation of free amine groups with Traut's reagent for 4h at +4°C in PBS. The remaining Traufs reagent was removed by dialysis against PBS buffer.. The reaction of attaching antibodies to the liposomes containing DSPE-PEG-Mal in the liposomal coat was carried out for 24 hours at +4°C. The molar ratio of DSPE-PEG-Mal in liposomal bilayer to IgG was adjusted to 13:1 . Thereafter, unbound antibodies were removed by filtration of the reactant product via size-exclusion chromatography on a Sepharose 4B column equilibrated in PBS The contents of antibodies in the liposome fraction was determined via ELISA test (specifically) or BCA method (protein content). Description of animal model experiment: The experiment was performed on mice within the ethical approval of First Local Committee for Experiments with Laboratory Animals Wroclaw, Poland permit No. 04/2013. The 7- 8-week-old male NOD/SCID mice (weighing 20-25g) were obtained from the Children's Clinic Hospital of the Jagiellonian University in Krakow and maintained under standard pathogen-free (SPF) conditions. The mice were injected subcutaneously (s.c.) in the left flank of each animal 5x10⁶ Daudi cells in 0.2 ml medium/mouse. Medium contained Matrigel (BD, Immunogen) and Hanks liquid (General Chemistry Laboratory, Institute of Immunology and Experimental Therapy) in a ratio of 1 :1 . . When the tumor volume reached 50-70 mm³, on the fourth day after tumor inoculation, the animals were divided randomly into groups of 8 individuals. The mice were administered the preparation of immunoliposomes intravenously (i.v.) into the tail vein of the animal three times at weekly intervals. MTO was administered intraperitoneally (i.p.) in a dose 0.3 mg/kg body mass. . Table 1 illustrates the drug and control preparations dosage administered to CD20 Daudi Burkitt's lymphoma tumor bearing NOD/SCID mice . Abbreviations: MTO-mitoxantrone in 0.3 mg/kg body weight dose, CCLII asODN - immuno-liposomes according to the present invention targeted by antibody, containing anti-Bcl-2 asODN, CCLIIscODN - immuno-liposomes according to the present invention targeted with antibody, but containing the "scrambled" sequence of oligonucleotide, CCLIIasODN + MTO - immuno-liposomes according to the present invention, targeted with antibody, containing anti-Bcl-2 asODN in combined therapy with a small predetermined dose of mitoxantrone (0.3 mg/kg body weight), administered twice, intraperitoneally. The targeted by antibodies liposomal carrier of encapsulated asODN anti-Bcl-2 proposed herein, effectively prevents the growth of neoplastic tumors developing from the inoculated CD20+ human cells. In the course of the experiment, complete remission of the tumor growth was observed in mice groups treated with CCLII immuno-liposomes containing anti-Bcl-2 asODN according to the present invention alone and in combination with low concentration of mitoxantrone. The effectiveness of the treatment is presented in the graph illustrating the dependency of tumor volume on time of treatment for each preparation administered (Fig. 12). The assumed therapeutic dose necessary to reach the neoplasm healing effect in 100% of mice treated with Rituximab® antibodies is 250 μg per mice (10 mg/kg body weight). The dose of attached to CCLII liposomes that was administered to the model organisms was 16.2 μg per mice (0.65 mg/kg body weight). Therefore, the therapeutic effect of antibodies attached to the liposomal carriers according to the present invention was excluded, which was also confirmed in control experiments.

Tab. 1 Doses of Drugs and Control Preparation Applied to NOD/SCID mice

Medicine groups CCL Dose ODN Dose IgG Dose MTO Dose PBS

fag/kg] [mg/kg] [ml/kg]

MTO - - - 0.36

CCLII asODN 64 720 650 - 8

CCLII scODN 64 720 650 - 8

CCLII asODN + MTO 64 720 650 0.36 8 In order to examine the long-term stability, the liposomal preparations were stored in the form of suspension at the temperature of +4° and in freeze-dried form at -20° C. During the twelve months of storage, the preparations were analyzed for particle size and zeta potential with dynamic light scattering technique using the Zetasizer Nano system. Also, the decrease of DNA content inside the liposomes was tested. For freeze-drying, the liposomal preparation was frozen at -80 ° with sucrose as the cryoprotedant in the weight ratio of 13:1 (sucrose to lipids), then it was dehydrated in Savant Modulo Yod type freeze- drier (Thermo NY, USA) at the temperature of -50°Cfor 12h in reduced pressure conditions (0.370 mbar) and were stored at the temperature of -20°C for 12 months. Liposomes according to the present invention retain their stability in terms of the particle size and zeta potential during the twelve months long storage period both in suspension and freeze-dried form, which was presented in fig. 1 and fig. 2. They also retain high, over eighty percent, content of encapsulated genetic drug (Fig. 3). The stability of CCLII carriers was also confirmed in in vitro tests in the presence of human serum. For this purpose, the liposomes encapsulated calcein (fluorescent marker) were incubated with human serum or plasma for 8 hours. The calcein leakage even after 8h did not exceed 20% as compared to the control, which proves the high stability of liposomes being the subject of the invention (Fig. 4). Further, CCLII liposomes after incubation with serum were checked for protective properties towards the encapsulated nucleic acid. The liposomal carrier CCLII, being the object of the present invention, effectively protects the encapsulated drug against the effects of serum enzymes. The preparations stored for a year in the suspension or in freeze-dried form were incubated with commercially available DNAse I (Sigma). In both cases, the liposomes efficiently protected the nucleic acidagainst enzymatic degradation (Fig. 5). Moreover the obtained preparation does not show hemolytic activity towards human erythrocytes. Obtained hemolysis values of up to 7% after 1 hour of incubation was lower then 10% after 2h which is essentially within the experimental error of this assay (Fig. 6). The interaction of liposomes labeled with afluorescent dye, DiD with the cell membrane of various cell lines was monitored by confocal microscopy. The target and control cells mixed in equal number were incubated with the CCLII immuno-liposomes, and were discriminated by their size (Fig. 7A) or morphology (Fig. 7B). As a result of contact with DiD-labeled immune-liposomes, the cell membrane emitted fluorescence at λ = 633nm. These observations clearly indicate the difference in directing immuno-liposomes towards the cells bearing the CD20 marker on their surface. The liposomal preparation acts selectively, due to the attached antibodies.

The in vitro analysis of the efficacy ofCCLII carrier in antisense oligonucleotides ("anti-Bcl-2) delivery to various leukemia cells was monitored by analysis of downregulation level of protein of interest. After the incubation with immuno-liposomes, the level of Bcl-2 protein in the cells exposing CD20 marker on their surface was examined viaWestern Blot technique (chemiluminescence detection). After 48 hours of incubation with CCLII liposomes, a significant reduction of Bcl-2 protein expression in CD20+ cells was observed (Fig. 8). The analysis of cytotoxicity against target cells was carried out using the MTT test and/or the Alamar Blue test. It has been demonstrated that liposomes targeted by antibodies are specific towards human B cells. CCLII immuno-liposomes demonstrate high specificity towards Daudi and Raji cells exposing CD20 antigen on their surface, and are characterized by low IC10 and IC50 values. Those values for the control cells not exposing CD20 were twice as high, as can be observed in fig. 9. Moreover, the apoptosis level in target cells was examined after 48 hours incubation with CCLII immunoliposomes. The negative control consisted of cells untreated with liposomes. For that purpose, after 48 hours of incubation, the cells were stained with fluorescein-labeled annexin V and with propidium iodide (PI). The apoptosis and necrosis levels were analyzed using the flow cytometry. After 48h culture with immunoliposomes major population of cells (approximately 80%) was Annexin V and PI positive, indicating that therapeutic activity of proposed here liposomes containing asODNs against BCL2 is based on apoptosis induction. In control cells incubated with liposomes and which encapsulated scODNs approximately 87% of population were Annexin V and PI negative, indicating that they were viable and not undergoing apoptosisThe obtained results, presented in fig. 10 and in table 2, indicate that the object of the present invention induces apoptosis in Daudi cells by silencing the expression of the BCL-2 gene. The reduction of Bcl-2 protein level in the cells enables releasing the cytochrome c from the mitochondria, which in turn activates caspases 9 and 3 consecutively, eventually resulting in apoptosis.

Tab. 2. The apoptosis and necrosis level in Daudi cells.

The cytotoxic activity of CCLII immuno-liposomes in combination with the effects of cytostatic - mitoxantrone (MTO) was tested against Daudi cell line expressing CD20 antigen. High effectiveness of the combined system of CCLII liposomal preparation targeted with antibodies with the cytostatic was demonstrated.

Claims

Targeted liposomal form of oligonucleotide-polyethyleneimine complex, characterized in that the oligonucleotide to polyethyleneimine ratio ranges from 1 :10 to 1 :3, and the complex is encapsulated with a lipid bilayer enabling attachment of a molecule recognizing the surface marker on pathological cells.

The form of the complex of claim 1 , characterized in that the molecule which exhibits high affinity toward specific tumor markers can be selected from a group comprising: humanized antibodies, antibody derivatives, proteins, aptamers, small targeting molecules or designed ankyrin repeat proteins (DARPins) and attached to the liposomes.

A use of the targeted, liposomal form of the oligonucleotide-polyethyleneimine specified in claims 1 and 2 for production of a drug intended for the human blood neoplastic diseases treatment.

The method of obtaining the targeted, liposomal form of the oligonucleotide-polyethyleneimine complex, which include the following steps: a) asODN is mixed with polyethyleneimine in 3:1 to 10:1 ratio, b) the PEI and DNA mixture is complexed, preferably by incubation, for 30-60 min. at room temperature in ratio 3-10 moleof PEI (N) in miliQ water to 1 mole of DNA (P) also in a miliQ class water, supplementing the mixture with 0.1 volume of 10 times concentrated PBS c) chloroform solutions of lipids constituting the casing are evaporated until obtaining dry lipid film, d) the residual organic solvents are removed, preferably in a vacuum evaporator, e) dry lipid film of step d) is hydrated with the mixture, preferably PEI/asODN obtained in step b), more preferably for 40 minutes, f) liposomes obtained in step e) are calibrated, preferably in a high pressure extruder using polycarbonate filters.

The method of claim 4, characterized in that in step f), 400 nm, 200 nm, and 100 nm filters are used consecutively.

The method of claim 4 or 5, characterized in that the filtration in step f) is repeated ten times.