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  • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• • A—HUMAN NECESSITIES
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  • A61P33/00—Antiparasitic agents
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  • A61P33/04—Amoebicides
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  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10323—Virus like particles [VLP]
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  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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Definitions

• a virus-like particle vector for delivery of pharmaceutical agents for delivery of pharmaceutical agents, a process for the manufacture thereof, its uses and a pharmaceutical composition.
• the present invention relates to the field of drug delivery. More particularly, the invention includes a virus-like particle (VLP) vector, a process for the manufacture thereof, use of the virus-like particle vector and a pharmaceutical composition, which contains the virus-like particle vector. More precisely, the invention relates to the virus-like particle vector, which constitutes an adenoviral dodecahedron with a therapeutic substance attached, wherein the vector is intended for delivering therapeutic agents into mammalian tissues, especially low molecular weight medical agents, in particular low molecular weight anti-cancer drugs into animal cancer tissues.
• VLP virus-like particle
• Adenoviruses are medium-sized non-enveloped DNA viruses, which infect humans and animals.
• Two adenoviral capsid proteins are responsible for virus penetration at the beginning of infection. These include: trimeric fibre protein, responsible for virus attachment to the host cell surface, and pentameric penton base protein, involved in virion internalisation. These two proteins form a non-covalent complex called penton, presented schematically below.
• dodecahedric nanoparticles comprising 12 pentons form spontaneously.
• dodecahedric virus-like particles which comprises 12 penton base proteins only can be formed via a similar procedure.
• Both types of dodecahedra (Dd) retain the functionality of their constituents and show an extraordinary ability of cell penetration (Fender et al., 1997; Fender et al., 2003; Vives et al., 2004).
• the dodecahedron recognises two types of receptors.
• the penton base protein that recognises ⁇ v integrins, it shows affinity to the ⁇ v integrins, whose levels are elevated in newly grown vessels, which supply blood to the cancer tissue.
• the dodecahedron has strong affinity to heparin sulphates (Vives et al., 2004), located at the surface of all epithelial cells.
• Bleomycin is a glycopeptide antibiotic used in the treatment of various types of cancers (Lazo and Sebti, 1999). The antibiotic acts by cleaving DNA in the cell nucleus, thus inhibiting cell division (Sausville et al., 1978; Carter et al., 1990). Bleomycin is exceptionally cytotoxic when located in the cell nucleus (Poddevin et al., 1991). However, the activity of the antibiotic is limited because, being hydrophilic, the particle has low penetration potential through cell membranes, has limited receptors on the plasma membrane and undergoes very quickly intracytoplasmic proteolysis (Mir et al., 1996; Lazo, 1996; Tounekti et al. 1993).
• Electroporation has been the method used heretofore to facilitate BLM penetration into the cancer tissue, which resultes in the increase of the anti-cancer effect of bleomycin (Gehl et al., 1998; Orlowski et al., 1988).
• bleomycin electrochemotherapy was carried out in 1991.
• Bleomycin was administered directly into the tumour or intravenously, concomitantly with electric shock.
• Such therapies make it possible to stop tumour growth, inducing various levels of necrotic changes within the cancer tissue.
• partial or general anaesthesia an additionally stressing procedure, was used during electroporation,
• a protein complex (adenoviral dodecahedron comprising the penton base protein and optionally containing the fibre protein) was disclosed.
• the adenoviral protein complex contains: (a) 12 pentons (P), each of, which contains at least one penton base (Pb) and one fibre protein (F), wherein the pentons are linked through the Pb so that they form a dodecahedric structure resistant to proteolysis with a molecular weight of 4.8-6.6 MD, or (b) 12 Pbs form a dodecahedron as above, the molecular weight, however, is 3.2 to 4 MD.
• P pentons
• F fibre protein
• an adenoviral dodecahedron was claimed, being a protein complex, a composition containing such a complex and use thereof.
• a native or recombinant adenoviral protein complex is used in the treatment and prevention of human and animal diseases.
• the complex disclosed contains 12 pentons, wherein each contains at least one fibre protein and penton base protein, without any additional adenoviral elements, and the said fibre protein originates from one or more adenoviral serotypes, and the penton base proteins are linked with one another and form a stable dodecahedric structure resistant to proteolysis.
• the invention concerns a composition, which comprises nanoparticles and use thereof for the encapsulation of therapeutically active substances inside nanoparticles having a specific coat.
• the particles are chemically formed so as to prevent high intracellular absorption. Encapsulation requires a direct bond between the nanoparticle and the therapeutically active substance.
• the pharmaceutical composition comprises nanoparticles with high affinity towards cancer cells and contains at least one therapeutic substance selected from a group comprising e.g. bleomycin.
• the objective of this invention is development of conditions which, which would enable the use of a dodecahedric virus-like particle vector for therapeutic purposes and also preparation and characterisation of a pharmaceutical composition, which contains a vector carrying a low molecular weight therapeutic substance to be used in human therapy.
• the dodecahedron is a potentially polyvalent vector specific for cancer cells.
• the vector has adenoviral endosomolytic activity and, therefore, it penetrates into cellular cytosol easily.
• Dd has high affinity to ⁇ v integrins.
• the integrins recognise the RGD motif (arginine-glycine-aspartic acid).
• the adenoviral dodecahedron with 60 RGD motifs is probably the most specific ligand for ⁇ v integrins.
• the Applicant assumed that the Dd can selectively supply therapeutic agents inside endothelial cells from, which newly grown tumour blood vessels are formed. Therefore, the Applicant decided to use such virus-like particles for the transfer of low-molecular weight therapeutic agents, expecting that the use of a targeted therapeutic Dd conjugate would lead to increased bioavailability and limited adverse effects of low-molecular weight drugs, in particular anti-proliferation factors, in particular glycopeptides, including anti-cancer antibiotics, such as bleomycin.
• low-molecular weight drugs in particular anti-proliferation factors, in particular glycopeptides, including anti-cancer antibiotics, such as bleomycin.
• rDd dodecahedra
• Ad3 adenovirus serotype 3
• rDds transduce 100% cells in cell cultures, and they have ability to transduce cells non-permissive for Ad3 as well.
• HS heparin sulphate
• HS interacts with positively charged protein fragments and it seems that such fragments are form in the Dd due to the proximity of penton base proteins in the VLP. Therefore, dodecahedron penetration occurs not only via viral receptors, but also through omnipresent heparin sulphates.
• the recombinant dodecahedric particle (rDd) is obtained with high yield in insect cells in the baculovirus system. The yield is comparable to that described for the most efficient bacterial protein expression systems, being 10 mg rDd per 100 mL of cell culture. rDds have been heretofore purified by sucrose gradient ultracentrifugation. This enables elimination of cellular proteins, but fails to do so with nucleic acids, most likely attached to the VLP surface.
• the present invention relates to a polyvalent virus-like particle vector, characterised in that it constitutes a recombinant adenoviral dodecahedron particle comprising adenoviral pentons or adenoviral penton base proteins, with an encapsulated or covalently linked low- molecular weight therapeutic substance in at least two copies, wherein the therapeutic substance is an anti-proliferative agent, preferably an anti-cancer agent, wherein the adenoviral dodecahedron originates from a mammalian, especially human, virus.
• the delivered low-molecular weight therapeutic substance is an antiproliferative agent, preferably a glycopeptide, in particular an anti-cancer agent, preferably belonging to the bleomycin family, according to Formula I,
• bleomycin A5 preferably bleomycin A5, according to Formula II.
• the low-molecular weight therapeutic substance is preferably encapsulated in or linked to the polyvalent recombinant adenoviral dodecahedron particle by cross-linking with a homo- or heterobifunctional chemical compound, preferably using carbodiimide (EDC), to amine groups or cysteine residues of the dodecahedron or else at the N-terminus or C-terminus of the penton base protein in the dodecahedron.
• EDC carbodiimide
• the penton base protein monomer carries between O and 2 BLM particles, with significant majority of monomers containing one BLM molecule, preferably one Dd molecule containing 60 base protein monomers carries at least 30 BLM residues.
• the linked or encapsulated low-molecular therapeutic substance is an unstable drug, such as anti-cancer drugs (medicine is art of healing -Wikipedia), preferably bleomycin, drug against neurodegenerative diseases, preferably 3,4-dihydroxyphenyl-l- alanine (L-DOPA), drug against tuberculosis and intercellular parasites, preferably isoniazid, anti-asthmatic agents, preferably salbutamol, intravenous anaesthetic agents, preferably thiopental, drugs against pathogenic organisms, preferably drugs against toxoplasmosis, leishmaniasis, trypanosomiasis and rickettsiosis.
• anti-cancer drugs medicine is art of healing -Wikipedia
• bleomycin drug against neurodegenerative diseases, preferably 3,4-dihydroxyphenyl-l- alanine (L-DOPA), drug against tuberculosis and intercellular parasites
• anti-asthmatic agents preferably salbuta
• the adenoviral dodecahedron carries the low-molecular weight therapeutic substance that is unstable at storage or in mammalian serum or else in the presence of intracellular eukaryotic enzymes.
• the linkage of the low-molecular weight therapeutic substance with the adenoviral dodecahedron ensures increased bioavailability of the therapeutic substances, in particular drugs against pathogenic organisms.
• the linkage of the low-molecular weight therapeutic substance with the adenoviral dodecahedron ensures increased bioavailability of the therapeutic substances, in particular therapeutic substances responsible for serious adverse effects.
• the cytotoxically effective BLM concentration delivered with the Dd is at least
• Another subject embodiment of the invention is a process for the manufacture of a virus-like particle vector, characterised in that the recombinant adenoviral dodecahedron particle originates from a mammalian, especially human, virus, and that it is produced in insect cells and, subsequently, it is purified using ultracentrifugation in sucrose concentration gradient and, subsequently, on an ion-exchange column, thus obtaining a fraction of pure rDds and, subsequently, to the resulting recombinant adenoviral dodecahedron comprising pentons or penton base proteins, at least two copies of the low- molecular weight therapeutic substance are encapsulated or attached covalently by chemical cross-linking, wherein the therapeutic substance is an anti-proliferation agent, preferably an anti-cancer drug.
• the low-molecular weight therapeutic substance is an anti-proliferation agent, preferably a glycopeptide, in particular an anti-cancer drug, preferably belonging to the bleomycin family according to Formula I, preferably bleomycin A5 according to Formula II.
• an anti-proliferation agent preferably a glycopeptide, in particular an anti-cancer drug, preferably belonging to the bleomycin family according to Formula I, preferably bleomycin A5 according to Formula II.
• the attached low-molecular weight therapeutic substance is placed through encapsulation inside the vector or attached by chemical cross-linking on the vector surface with a homo- or heterobifunctional chemical compound, preferably using carbodiimide (EDC).
• EDC carbodiimide
• the low-molecular weight therapeutic substance is attached to amine groups or cysteine residues of the dodecahedron or else at the N-terminus or C-terminus of the penton base protein in the dodecahedron.
• virus-like particle vector which is a recombinant adenoviral dodecahedron, constituting a conjugate of the recombinant adenoviral dodecahedron particle formed from pentons or penton base proteins, with at least two copies of an encapsulated or covalently linked low-molecular weight therapeutic substance, wherein the therapeutic substance is an antiproliferative agent, preferably an anti-cancer drug, wherein the adenoviral dodecahedron originates from a mammalian, especially human, virus for the delivery of therapeutic agents into tissues, preferably for the delivery of low-molecular weight therapeutic substances, preferably anti-cancer agents, into mammalian cancer tissues.
• the attached low-molecular weight therapeutic substance is an anti-proliferation agent, preferably a glycopeptide, in particular an anti-cancer drug, preferably belonging to the bleomycin family according to Formula I,
• At least two copies of the low-molecular weight therapeutic agent are encapsulated or linked to the recombinant adenoviral dodecahedron particle by cross- linking with a homo- or heterobifunctional chemical compound, preferably using carbodiimide (EDC), attached to amine groups or cysteine residues of the dodecahedron or else at the N-terminus or C-terminus of the penton base protein in the dodecahedron.
• EDC carbodiimide
• the penton base protein monomer carries between O and 2 BLM particles, with significant majority of monomers carrying one BLM molecule, preferably one Dd molecule in the conjugate containing 60 base protein monomers carries at least 30 BLM residues.
• the transferred low-molecular therapeutic substance is an unstable drug, such as anti-cancer agents, preferably bleomycins, drugs against neurodegenerative diseases, preferably 3,4-dihydroxyphenyl-l-alanine (L-DOPA), drugs against tuberculosis and intercellular parasites, preferably isoniazid, anti-asthmatic agents, preferably salbutamol, intravenous anaesthetic agents, preferably thiopental, drugs against pathogenic organisms, preferably drugs against toxoplasmosis, amoebiasis, leishmaniasis, trypanosomiasis and rickettsiosis.
• anti-cancer agents preferably bleomycins
• drugs against neurodegenerative diseases preferably 3,4-dihydroxyphenyl-l-alanine (L-DOPA)
• L-DOPA 3,4-dihydroxyphenyl-l-alanine
• drugs against tuberculosis and intercellular parasites preferably isoniazid
• the transferred therapeutic substance is unstable at storage, in mammalian serum or else in the presence of intracellular eukaryotic enzymes.
• the linkage of the low-molecular weight therapeutic substance with the adenoviral dodecahedron ensures increased bioavailability of the therapeutic agents, in particular therapeutics against pathogenic organisms.
• the linkage of the low-molecular weight therapeutic substance with the adenoviral dodecahedron ensures increased bioavailability of the therapeutic substances, in particular therapeutic substances responsible for serious adverse effects.
• the effective cytotoxic BLM concentration delivered with the Dd is at least 50 times as low as in the case of free bleomycin.
• Another embodiment of the present invention is a pharmaceutical composition, characterised in that it contains a recombinant polyvalent adenoviral dodecahedron particle formed from pentons or penton base proteins , which carries at least two copies of the low- molecular weight therapeutic substance, wherein the therapeutic substance is an anti- proliferation agent, preferably an anti-cancer agent, wherein the adenoviral dodecahedron originates from a mammalian, especially human, virus.
• the transferred low-molecular weight therapeutic substance is an anti- proliferation agent, preferably a glycopeptide, in particular an anti-cancer drug, preferably belonging to the bleomycin family, according to Formula I,
• At least two copies of the low-molecular weight therapeutic substance are encapsulated or linked to the recombinant adenoviral dodecahedron by cross-linking with a homo- or heterobifunctional chemical compound, preferably using carbodiimide (EDC), to amine groups or cysteine residues of the dodecahedron or else at the N-terminus or C- terminus of the penton base protein in the dodecahedron.
• EDC carbodiimide
• the penton base protein monomer carries between 0 and 2 BLM particles, with significant majority of monomers containing one BLM molecule, preferably one Dd molecule containing 60 base protein monomers carries at least 30 BLM residues.
• the attached low-molecular therapeutic substance is an unstable molecule, such as anti-cancer drugs, preferably bleomycins, agents against neurodegenerative diseases, preferably 3,4-dihydroxyphenyl-l-alanine (L-DOPA), drugs against tuberculosis and intercellular parasites, preferably isoniazid, anti-asthmatic agents, preferably salbutamol, intravenous anaesthetic drugs, preferably thiopental, drugs against pathogenic organisms, preferably drugs against toxoplasmosis, amoebiasis, leishmaniasis, trypanosomiasis and rickettsiosis.
• anti-cancer drugs preferably bleomycins, agents against neurodegenerative diseases, preferably 3,4-dihydroxyphenyl-l-alanine (L-DOPA), drugs against tuberculosis and intercellular parasites, preferably isoniazid, anti-asthmatic agents, preferably salbutamol, intravenous anaesthetic
• the adenoviral dodecahedron delivers the low-molecular weight therapeutic substance, unstable in the free form at storage or in mammalian serum or else in the presence of intracellular eukaryotic enzymes.
• the attachment of the low-molecular weight therapeutic substance to the adenoviral dodecahedron ensures increased bioavailability of the therapeutic substances, in particular drugs against pathogenic organisms.
• the attachment of the low-molecular weight therapeutic substance to the adenoviral dodecahedron ensures increased bioavailability of the therapeutic substances, in particular therapeutic substances responsible for serious adverse effects.
• the Dd-BLM conjugate inhibits the proliferation of cancer cells.
• the effective cytotoxic BLM concentration delivered with the Dd is at least 50 times as low as that of free bleomycin.
• Figure 1 shows a scheme of the adenovirus, penton and two dodecahedra (Dd).
• Figure 2 shows Dd stability analysed using dynamic light scattering (DLS) technique. Dd thermal stability depending on pH and ionic strength. Dd in 150 mM NaCl was tested using DLS technique at various pH values at temperature increments of 2°C every 2 min between 12 and 65°C.
• B Electrophoresis analysis of Dd and pentameric bases (Pb) in CAPS buffer (pH 9) and in carbonate buffer (pH 10). Some samples were subjected to temperature changes simulating DLS conditions (marked with DLS).
• C DLS analysis carried out using Dd samples in PBS in various ionic strength conditions. Average values from 3 apparatus readings are shown.
• Figure 3 shows Dd stability during lyophilisation, inside HeLa cells and in human serum, as well as Dd reconstitution from free penton base (Pb) proteins.
• Purified Dds were dialysed overnight at 4°C against water or 150 mM aqueous ammonium sulphate. Mannitol (0.4%) and sucrose (0.4%) were added to the samples marked "cryoprotectant +". Dd samples were frozen at -8O 0 C or dried with speed-vac or freeze-dried. The dried samples were reconstituted in the initial water volume. The samples were centrifuged for 30 min at 13000 rpm, and the condition of proteins in the supernatant was analysed using agarose gel electrophoresis.
• Lanes 2 and 3 correspond to human serum incubated for 2 hours at 4 or 37°C, respectively.
• D Purified Pbs were dialysed either at 4°C against 50 mM phosphate buffer, pH 6.6, containing 750 mM ammonium sulphate (left-hand panel), or at a temperature of 37°C against 100 mM phosphate buffer, pH 7.5 (right-hand panel). The samples were centrifuged for 30 min at 13000 rpm, and the proteins in the supernatant were analysed using agarose gel electrophoresis under non-denaturing conditions.
• Lane 1 contains the starting Pb preparation used for Dd reconstitution; lane 2 corresponds to free Pbs with 750 mM ammonium sulphate added before reconstruction; lane 3 contains free pentameric bases after 2-hour dialysis (two dialysis buffer changes). Lanes 4 and 6 correspond to Dd reconstructed during 4-day dialysis at 4 and 37°C, respectively. Lanes 7 and 8 correspond to Dd, and sample 8 contains 750 mM ammonium sulphate added before reconstruction. Figure 4 shows the cytotoxicity of bleomycin delivered by the Dd. Bleomycin has been chemically attached to the Dd (as discussed in Example FV). (A) Analysis of the Dd-BLM conjugate using mass spectrometry.
• Figure 5 shows the effect of Dd-BLM activity on HeLa cells.
• A Cells treated with Dd or Dd-BLM (1 ⁇ g sample) were analysed under a confocal microscope using a Dd-recognising antibody (red signal, white/grey on black and white photographs). Cell nuclei were stained with DAPI solution. The lowermost row shows cells after 50-hour treatment, without nuclear staining. The scale bar corresponds to 20 ⁇ m.
• B shows the effect of Dd-BLM activity on HeLa cells.
• the virus-like particle vector developed according to the invention made it possible to achieve better penetration of hydrophilic anti-proliferation therapeutic agents, especially glycopeptides, such as anti-cancer antibiotics, in particular such as bleomycin, through cell membranes.
• hydrophilic anti-proliferation therapeutic agents especially glycopeptides, such as anti-cancer antibiotics, in particular such as bleomycin
• the use of the Dd for the delivery of therapeutic agents most likely means at the same time specific targeting of such agents to newly grown blood vessels, which supply nutrients to neoplastic tumours.
• the RGD motif interacts with ⁇ v integrins whose levels are elevated only in the endothelial cells, which constitute newly grown vessels, which supply blood to the cancer tissue (Chen, 2006).
• the motif is located in the penton base protein of, which the Dd is composed; therefore, the Dd, which contains 60 RGD motifs is a highly specific ligand for ⁇ v integrins and, simultaneously, it has strong ability to penetrate cells owing to its endoosmolytic activity and affinity to heparin sulphates.
• Dd bleomycin (BLM) antibiotic, a low-molecular weight therapeutic agent
• Dd-BLM The biological (cytotoxic) effect of a Dd-BLM preparation, which carried numerous antibiotic copies was tested on human cancer cells in in vitro cultures. It appeared that the chemical cross-linking reaction between the vector and BLM did not reduce its cell penetration ability. Furthermore, the antibiotic's cytotoxic activity was retained. Namely, Dd-BLM, when penetrating into human HeLa cells in in vitro cultures, degrades nuclear DNA, similarly to free bleomycin. It was proved that the cytotoxically effective concentration of the antibiotic delivered with the Dd was approx. 100-fold lower than that used with free BLM. More than 60% human cancer cells (HeLa) in in vitro cultures were destroyed after the administration of the Dd-BLM conjugate, ,which was proved using the MTT cytotoxicity test (Fig. 4C). The cytotoxic effects were not observed either in the case of dodecahedron or free bleomycin administration in doses equivalent to those carried by the Dd-BLM conjugate.
• Dd-BLM efficiently penetrates through cell membranes using receptors recognised by either Dd or BLM. Most likely, the vector undergoes gradual proteolysis in the cytoplasm of human HeLa cells, as a result of, which peptides are released with attached bleomycin, wherein the BLM-peptides penetrate into the nucleus in, which the antibiotic, bleomycin in this case, is active.
• the cytotoxic BLM activity is known to result from DNA damage.
• Phosphorylation of the C-terminal region of the H2AX histone in higher eukaryotic cells is one of chromatin modifications in response to double-strand DNA breaks.
• a specific antibody, which recognises the phosphorylated H2AX histone form was used as the probe for detecting DNA damage.
• Dd-BLM when penetrating into human cancer cells in in vitro cultures, degrades nuclear DNA, similarly to free bleomycin.
• Dd being a recombinant protein (rDd)
• rDd recombinant protein
• the overexpression is 10 mg of rDd per 100 mL of cell suspension. This overexpression yield is comparable to that achieved in the most efficient bacterial systems (Song et al., 2008).
• rDds have been heretofore purified by saccharose gradient ultracentrifugation. The stage made it possible to eliminate low- and medium-molecular weight cell proteins, but failed to do so with nucleic acids, most likely attached to the rDd surface.
• the vector particle retains integrity during dialysis, after freezing and thawing, in speed-vac drying and during freeze-drying in the presence of a cryoprotectant (Fig. 3A).
• the high vector stability makes rDd handling and storage easier.
• the rDd was found to retain integrity in conditions, which simulate its in vivo use; namely, it was stable in human serum at a temperature of 37°C for at least 2 hours (Fig. 3B). The results make it possible to use the rDd as a vector for various applications and in various environmental conditions.
• the properties of the Dd discussed above imply the potential of the nanoparticle to be used as a vector for the delivery of therapeutic agents to human tissues.
• the first example concerns bleomycin, an anti-cancer antibiotic.
• the Applicant found that bioavailability of the antibiotic increased owing to the use of the Dd as the vector; this should enable the use of reduced doses and, in consequence, reduce adverse effects of its activity.
• studies in the mouse cancer model will be conducted. If the Dd-BLM preparation used in the model system, such as mice with implanted human brain tumour, proves at least as efficacious as BLM delivery by electrochemotherapy used previously, this will make it possible to suggest using the Dd-BLM conjugate in human anti-cancer treatment. Therefore, the use of bleomycin in anti-cancer treatment could be limited to the administration of a Dd-BLM preparation without any need to use electric shock, which frequently requires complete anaesthesia.
• a recombinant baculovirus which comprised the penton base protein gene of the human serotype 3 adenovirus (Ad3) was used (Fender et al., 1997).
• the amplification of the recombinant baculovirus carrying the base protein gene was carried out in a monolayer cell culture of Spodoptera frugiperda (Sf21). The cells were cultured in TClOO medium containing 5% foetal bovine serum (Invitrogen).
• Trichoplusia ni cells also known as High Five, HF
• Trichoplusia ni cells also known as High Five, HF
• gentamycin 50 mg/L
• amphotericin B 0.25 mg/L
• Trichoplusia ni cells were infected with the recombinant baculovirus at the MOI (multiplicity of infection) of 4 infectious units per one cell. 48 hours after the infection, the cells were harvested and lysed by freezing and thawing three times. The supernatant obtained after lysate clarification was centrifuged in 15 - 40% sucrose gradient (Fender at al., 1997).
• VLP product recovered in 30-40%sucrose, was contaminated with cellular proteins and nucleic acids. Final Dd purification was achieved by chromatography on an ion-exchange column as a result of, which dodecahedra were prepared as a homogeneous fraction. The oligomeric status of the particles and purity level of the resulting product were analysed in native agarose gels, using electron microscopy and in denaturing polyacrylamide gels.
• the stability and solubility of purified Dd particles was tested.
• the purified rDds were dialysed against various buffers (with 3 changes of each) and, subsequently, incubated at 30 or 37°C. After incubation, the samples were centrifuged and proteins in the supernatant were analysed using agarose gel electrophoresis.
• the Dd remains dissolved at 4°C and pH of 4.0 to 10.9, in the presence of 150 mM NaCl. Without NaCl, the Dd does not remain in solution and it disappears from the supernatant during centrifugation. Therefore, NaCl in physiological concentration protects Dds against denaturation.
• DLS dynamic light scattering
• CAPS is an organic buffer, which may cause aggregation by interacting with surface hydrophobic fragments.
• T m Dd melting temperature
• Fig. 2C structural stabilisation
• Dd samples (5 ⁇ g aliquots ) concentrated by ultrafiltration in Microcon (Millipore), were incubated in human serum (SL) at a temperature of 4°C for 2 hours and at 37°C for 15 min or 2 hours.
• the Dd retains integrity in conditions, which simulate its potential in vivo use; namely, it is stable in freshly prepared human serum at a temperature of 37°C for at least two hours (Fig. 3C).
• a homogeneous fraction of free pentameric bases (Pb) was obtained during purification on an ion-exchange column.
• the purified rPbs were dialysed against 50 mM pH 6.6 or pH 7.5 phosphate buffers containing 750 mM ammonium sulphate with several buffer changes.
• the samples were centrifuged and the oligomeric status of proteins in the supernatant was analysed using agarose gel electrophoresis.
• association of dodecahedra from pentameric bases occurs. Owing to dodecahedron in vitro reconstruction from their constituent parts in the presence of low-molecular weight compounds, it is possible to obtain a vector, which contains a therapeutic substance encapsulated in the virus-like particle.
• Bleomycin A 5 hydrochloride (Hangzhou Xiangyuan Co., Ltd., China) was chemically attached to previously purified rDd particles during a two- stage conjugation procedure using carbodiimide (EDC) and succinic acid ester (s-NHS) (Pierce, Rockford IL, USA).
• EDC carbodiimide
• s-NHS succinic acid ester
• Dodecahedra at a concentration of 27 nM were activated in the 0.1 M pH 6.0 MES buffer containing 0.5 M NaCl, in the presence of 0.31 mM EDC and 5 mM s-NHS. Conjugation with bleomycin (23 mM) was carried out for two hours at room temperature upon gentle stirring.
• the reaction was terminated by adding hydroxylamine to a final concentration of 10 mM.
• the reagents used and unbound bleomycin were eliminated during 24-hour dialysis with four changes of 20 mM pH 7.5 Tris buffer containing 150 mM NaCl and 5% glycerol.
• Bleomycin quantity attached to the Dd was determined using mass spectrometry technique. The analysis was carried out using a Perseptive Biosystems mass spectrometer (Framingham, MA), by way of a pulse nitrogen laser at a wavelength of 337 nm. The samples were concentrated in ZipTipC4 (Millipore) and extracted with saturated sinapinic acid solution prepared in 80% mixture of aqueous acetonitrile (vol./vol.) comprising 0.3% trifluoroacetic acid according to the manufacturer's instructions. The eluent mixture was transferred onto a steel plate and dried on air. The apparatus was calibrated using bovine albumin (Biosystems) with a molecular weight of 66431 Da.
• the penton base protein monomer (of, which Dd comprises) carries between 0 and two BLM particles (the BLM molecular weight is 1400) with significant majority of monomers containing one BLM molecule (Fig. 4A).
• the data indicate that one Dd molecule, which contains 60 base protein monomers carries 60 BLM residues on average.
• Tests using dynamic light scattering technique (DLS) proved that the melting temperature of the Dd-BLM conjugate is very similar to that of the initial dodecahedron, which indicates that the cross-linking reaction does not change the biophysical properties of the vector.
• the results of the studies underscore vector polyvalency, wherein one Dd particle is able to provide multiple copies of the therapeutic substance.
• HeLa human cancer cells were treated with the Dd-BLM conjugate prepared according to the invention. Similarly to free bleomycin, the Dd-BLM conjugate led to the inhibition of cancer cell proliferation. What was the most important, the cytotoxically effective BLM concentration delivered with the Dd was 100 times as low as in the case of free bleomycin.
• the cytotoxic Dd-BLM activity was quantitatively evaluated in vitro using the MTT test (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).
• MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
• MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
• the subsequent stage included microscopic examination of human cancer cells subjected to Dd-BLM treatment.
• HeLa cells (5 x 10 4 ) were plated onto special coverslips.
• various amounts of pure Dd, Dd-BLM conjugate or free bleomycin were applied onto the cells; all samples were suspended in the serum-free EMEM medium.
• foetal bovine serum was added to a final concentration of 10%.
• the cells were washed with cold PBS and subsequently fixed and permeabilised for 10 min in 100% cold methyl alcohol.
• the Dd-BLM conjugate induces the occurrence of enlarged cells, which is visible 30 hours after conjugate application, being even more pronounced at a later time. Fifty hours after Dd-BLM application, the Dd signal is present throughout the cell, which indicates that nuclear membrane integrity has been destroyed (one of cell death symptoms).
• cytotoxic, BLM activity is known to result from DNA damage (Mir et al., 1996).
• Phosphorylation of the C-terminal region of the H2AX histone in higher eukaryotic cells is one of chromatin modifications in response to double-strand DNA breaks (Kinner et al., 2008).
• a specific antibody, which recognises the phosphorylated histone form (anti- ⁇ - H2AX; Calbiochem, Darmstadt, Germany) was used as the probe for detecting DNA damage.
• anti- ⁇ - H2AX Calbiochem, Darmstadt, Germany
• the Dd-BLM conjugate when penetrating into cells, degrades nuclear DNA, which is indicated by the presence of the signal from the specific antibody.
• Application of free bleomycin has a similar effect (Fig. 5B, row: BLM).
• the effect of the action of 0.08 ⁇ M BLM-containing conjugate is stronger than damage induced by free BLM at a concentration of 8 ⁇ M and, therefore 100 times as high (Fig. 5B, rows: Dd-BLM and BLM).
• Electrochemotherapy results of cancer treatment using enhanced delivery of bleomycin by electroporation.

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