EP4221757A1 - Zielvermittelte endozytotische wirkstofffreisetzung - Google Patents

Zielvermittelte endozytotische wirkstofffreisetzung

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Publication number
EP4221757A1
EP4221757A1 EP21786122.8A EP21786122A EP4221757A1 EP 4221757 A1 EP4221757 A1 EP 4221757A1 EP 21786122 A EP21786122 A EP 21786122A EP 4221757 A1 EP4221757 A1 EP 4221757A1
Authority
EP
European Patent Office
Prior art keywords
cells
active compound
complex according
disease
compounds
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21786122.8A
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English (en)
French (fr)
Inventor
DE Albertus Gerrit BOER
Markwin Hendrik MARING
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Crm Therapeutics BV
Original Assignee
Crm Therapeutics BV
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Filing date
Publication date
Priority claimed from NL2026569A external-priority patent/NL2026569B1/en
Priority claimed from NL2027601A external-priority patent/NL2027601B1/en
Application filed by Crm Therapeutics BV filed Critical Crm Therapeutics BV
Publication of EP4221757A1 publication Critical patent/EP4221757A1/de
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/62—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 non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—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
    • 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
    • A61K47/6905—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 the form being a colloid or an emulsion
    • A61K47/6911—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 the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/006—Biological staining of tissues in vivo, e.g. methylene blue or toluidine blue O administered in the buccal area to detect epithelial cancer cells, dyes used for delineating tissues during surgery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
    • A61K49/0084—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion liposome, i.e. bilayered vesicular structure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1806—Suspensions, emulsions, colloids, dispersions
    • A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1217—Dispersions, suspensions, colloids, emulsions, e.g. perfluorinated emulsion, sols
    • A61K51/1234—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is in the field of Intelligent Drug Delivery. Targeted medicines have been developed thereto, using a Targeted Mediated Drug Delivery-technology. This technology leads drugs selectively and specifically into diseased cells/organs, in contrast to “dumping” drugs into the body. This technology is found to substantially increase the efficacy of disease treatment and reduce side-effects while much smaller amounts of drug(s) are required. In addition, a novel innovative approach is used that can be applied in the treatment of e.g. cancers, auto-immune diseases, viral diseases, transplantation, and diseases associated with inflammation.
  • the present invention is in the field of Intelligent Drug Delivery.
  • the drug delivery relates to a medication, which is also referred to as a medicine, a pharmaceutical drug, or simply drug. In general it may be used to diagnose, cure, treat, or prevent disease.
  • the present invention in particular relates to drug delivery and drug therapy.
  • drugs can be classified in various ways. In view of the present invention an important distinction is between small-molecule drugs, such as obtained by chemical synthesis, biopharmaceuticals, such as recombinant proteins, vaccines, RNA, and gene therapy, and more complex molecules and the like. Also a mode of action, and route of administration, may be different.
  • HB-EGF Heparin-binding epidermal growth factor
  • CCM crossreacting material
  • CRM197 used fluorescently labelled CRM197 and CRM197-coated liposomes to investigate their potential use for drug delivery to leukocytes. They demonstrate that CRM197-guided systems are efficiently taken up by human leukocytes in vitro. CRM 197 was also found to specifically target leukocytes in vivo in mice with components of the human immune system (HIS mice) and hamsters. Monocytes represent the most prominent subset of leukocytes that showed highly specific CRM197- mediated uptake. They therefore propose the application of CRM 197 as a novel targeting approach in diseases that require the selective treatment of monocytes.
  • US 2010/129437 Al recites methods of targeted drug delivery of antiviral compounds, including, chemical agents (like nucleoside analogues or protease inhibitors) and nucleic acid based drugs (like DNA vaccines, antisense oligonucleotides, ribozymes, catalytic DNA (DNAzymes) or RNA molecules, siRNAs or plasmids encoding thereof).
  • the invention relates to targeted drug delivery of antiviral compounds to intracellular target sites within cells, tissues and organs, in particular to target sites within the central nervous system (CNS), into and across the blood-brain barrier, by targeting to internalizing uptake receptors present on these cells, tissues and organs.
  • CNS central nervous system
  • the antiviral compounds are conjugated to ligands that facilitate the specific binding to and internalization by these receptors.
  • Liposomes enclosing antiviral compounds are conjugated to CRM 197 through a PEG spacer.
  • US 2010/209494 Al recites molecular medicine and pharmacology. More specifically, it relates to liposomes and their use as delivery vehicle for therapeutic compounds.
  • a liposome comprising at least one lipid bilayer enclosing an interior compartment, wherein said lipid bilayer comprises at least one synthetic pyridinium -derived amphiphile, for instance a Saint-molecule.
  • the present invention therefore relates to a drug delivery, which solves one or more of the above problems and drawbacks of the prior art, providing reliable results, without jeopardizing functionality and advantages.
  • the present invention relates to a complex, i.e. a chemical compound in which the constituents are more intimately associated with one and another than in a simple mixture, for target mediated endocytotic drug delivery comprising as constituents at least one first constituent comprising a targeting molecule, in the description and figures also referred to as B-part, capable of interacting with a heparin-binding epidermal growth factor (HB-EGF) cell receptor, including similar growth factors, so HB-EGF like growth factors, the HB-EGF cell receptor capable of forming an endosome in a cell, and directly or indirectly attached to the targeting molecule a chain capable of forming a pore in an endosomal membrane under acidic conditions, such as at a pH of ⁇ 6, in particular a pH ⁇ 5.6, more in particular a pH ⁇ 5.0, in particular a T-chain (also referred to as T-part),
  • HB-EGF heparin-binding epidermal growth factor
  • Apoptin apoptin-associated proteins (AAP), such as AAP1, AAP2, AAP3, AAP4, AAP5, and AAP6, HIV-tin, amytin, copaxotin, prednitin, lysotin, protectin, vaditin, gadotin, filgotin, and bacteriotin, from genes or sequences thereof, from proteins, and from RNA-sequences, such as mRNA, siRNA and shRNA, and genes encoding mRNA, siRNA and shRNA, preferably a sequence with ⁇ 30 nucleotides, such as 20-27 nucleotides.
  • the present complex substantially enhances the prior art treatment of diseases by equipping conventional drugs and “personalized medicines” with a homing device that brings drugs into diseased cells.
  • the present complex and dosage provide the following improvements of disease treatment:
  • DOXOTIN comprises doxorubicin-HCL
  • APOPTIN comprises an expression plasmid encoding for Apoptin with the amino-acid sequence: MNALQEDTPP GPSTVFRPPT SSRPLETPHC REIRIGIAGI TITLSLCGCA NARAPTLRSA TADNSESTGF KNVPDLRTDQ PKPPSKKRSC DPSEYRVSEL KESLITTTPS RPRTAKRRIR L
  • E. HIV-TIN comprises
  • TGS transcriptional gene silencing
  • Prom-A directed at promotor-region of the virus and viral entry inhibition by delivery of shRNA to express C46 at the membrane of white blood cells.
  • shRNA may be applied.
  • AMYTIN comprises an expression plasmid encoding for the protein neprilysin comprising the following amino acids:
  • COPAXOTIN comprises a random polymer of L-ALANINE, L-GLUTAMIC ACID, L-LYSINE, and L-TYROSINE that structurally resembles MYELIN BASIC PROTEIN.
  • mRNA may be applied.
  • PREDNITIN comprises methylprednisolone-hemi-acetate or dexamethasone-acetate (also referred to as Dexatin).
  • LYSOTIN comprises
  • J. PROTECTIN comprises: tacrolimus and/or my cophenolate mofetil
  • BACTERIOTIN comprises: doxycycline -HC1
  • Vaditin® comprises: 2-amido benzimidazole (diABZI-2 ; CAS Number 934-32-7)
  • Gadotin comprising Gd, capable of neutron capture, and used as enhancer for e.g. CT, SPECT, MRI, in scans and diagnosis, and for therapy of tumours.
  • P-Glucocerebrosidase also called acid P-glucosidase, D-glucosyl-N-acyl sphingosine glucohydrolase, or GCase
  • GAA Homo sapiens glucosyl ceramidase beta
  • transcript variant 1 mRNA sequence
  • Filgotin comprises Filgotinib, used for treatment of rheumatoid arthritis.
  • IQ-TMDD IQ-Targeted Mediated Drug Delivery
  • HB-EGF heparin-binding epidermal growth factor
  • Inventors used fluorescently labelled cross-reacting material and cross-reacting material -coated liposomes to investigate their potential use for drug delivery to leukocytes.
  • Inventors demonstrated that cross-reacting material -guided systems are efficiently taken up by human leukocytes in vitro.
  • Crossreacting material was also found to specifically target leukocytes in vivo in mice with components of the human immune system (HIS mice) and hamsters.
  • Monocytes represent the most prominent subset of leukocytes that showed highly specific cross-reacting material-mediated uptake (see e.g. fig. 1).
  • the application of cross-reacting material as a novel targeting approach in diseases that require the selective treatment of monocytes is therefore established.
  • FIG. 2 An example of the present complex is shown in fig. 2. Therein a circular liposome is shown, enclosing an active compound, identified as drug. Attached to the liposome is the present at least one first constituent, comprising the targeting molecule (square part), and the chain (triangular part). Optionally also a protein, attached to the present chain at one end and to the liposome (at least one second constituent), typically a non-toxic mutant of a diphtheria toxin is shown (circular part). The first constituent is attached to the second constituent as an example by a polyethylene glycol (PEG) molecule or a polyethylene-maleimide-DSPE. A considered mode of action of the present complex is shown in fig. 3.
  • PEG polyethylene glycol
  • DPE polyethylene-maleimide-DSPE
  • Doxotin is found to be an effective and cost-efficient targeted medicine containing doxorubicin, vinblastin, docetaxel, paclitaxel, which is selectively delivered directly into e.g. cancer cells and white blood cells of the cancer micro-environment by the applied IQ-TMDD technology.
  • Doxotin combines a 25 times lower dose of doxorubicin, vinblastin, docetaxel, paclitaxel in combination with targeted delivery resulting in at least a 7 times enhanced delivery of doxorubicin, vinblastin, docetaxel, paclitaxel with less side effects compared to conventional non-targeted liposomes.
  • a (solid) cancer comprises mainly cancer cells, white blood cells of the cancer microenvironment and the endothelial cells of the cancer blood vessels.
  • the present invention is in particular suited for treatment of an outer surface of these (solid) cancers as well as of the cancer microenvironment. All these cells express the HB-EFG-transport receptor. Since Doxotin can force these cells into a transformed state which selectively activates Apoptin forcing these cells to apoptosis, the combination of Doxotin and Apoptin is found to treat the whole cancer including metastatic cells that may be released during treatment. This “Kick and Kill” approach will therefore substantially enhance the Quality of Life of cancer patients.
  • Apoptin provides a unique and specific treatment of inflammatory diseases like auto-immune diseases (AID’s). Apoptin can force transformed cells following a stress stimulus to apoptosis, bypassing the p53 apoptosis pathway.
  • AID auto-immune diseases
  • RA rheumatoid arthritis
  • SOS-response a transient transforming state
  • Apoptin RA-fibroblast-like synoviocytes
  • a precision Treatment of Viral Diseases is provided.
  • Many viral diseases are found difficult to treat since the responsible viruses are hiding in cells that preserve as a viral reservoir.
  • the most important cells in this respect are monocytes and macrophages. Since monocytes and macrophages highly express the IQ-transport receptor, it is possible to eradicate these viruses by treating these cells with the “Kick and Kill” approach.
  • HIV-1/2 enters CD4+-T-cells, dendritic cells and macrophages via their CXCR4 and CCR5 co-receptors.
  • the eradication of HIV- cells by a Doxotin/Apoptin -“Kick and KilF’-approach now provides a real cure.
  • the present non-viral and precision treatment of HIV is more effective and selective than the prior art HIV treatments.
  • Cerebral Amyloid Angiopathy (CAA), and “Katwijk Disease” (HCHWA-D) are characterized by accumulation of the amyloid-beta (Ap) protein which causes micro bleeds in the brain (CAA and HCHWA-D).
  • Neprilysin is a major A -monomer and -oligomer degrading enzyme.
  • Amytin is a precision medicine for the targeted delivery of the neprilysin gene to brain blood vessels and to monocytes in the blood compartment. Since monocytes move to inflammatory Ap-areas in the brain, the present dual treatment approach is considered to breakdown Ap and decrease or stop the further development of CAA, HCHWA-D and possibly also AD, in particular when using Amytin.
  • Glatiramer acetate (sold as Copaxone) is an immunomodulator medication currently used to treat multiple sclerosis. Recently it has been shown that macrophages activated by Glatiramer acetate, are able to remove amyloid-beta (AP) oligomers and monomers and rescue neuronal connections in the brain providing a rationale for the treatment of CAA, HCHWA-D and AD by Copaxone.
  • AP amyloid-beta
  • IQ- TMDD containing Glatiramer acetate is able to deliver Glatiramer acetate selectively and more efficiently to macrophages and therefore reduces side-effects and enhances the removal of Ap from the brain and brain vessels. This subsequently decreases disease progression at a lower dose and with less side effects.
  • MS Multiple Sclerosis
  • monocytes play an important role by remitting and relapsing MS.
  • white blood cells take care of the immune surveillance of the brain and move to inflammatory sites.
  • Prednitin provides the targeted delivery of methylprednisolone to monocytes in the blood compartment, to reduce or stop their inflammatory activities in the brain. By doing so Prednitin enhances disease treatment, at a lower dose with less side effects.
  • the dose of methylprednisolone needed is reduced at least by a factor of 30 while the % of the dose entering the target tissue increases by at least a factor 8 compared to the application of conventional non-targeted liposomes.
  • Prednitin is substantially more effective than the current treatment modalities of methylprednisolone. This results in an increased Quality of Life of MS-patients.
  • Prednitin can also be used in the treatment of inflammatory and autoimmune diseases.
  • Lysosomal storage disorders are caused by the deficiency of a single enzyme required for the breakdown of compounds in cellular organelles called lysosomes. When these enzymes are defective or function poorly, compounds will accumulate in these cells and tissues/organs. LSDs affect mostly children and they often die at a young and unpredictable age, many within a few months or years of birth. Many other children die of these diseases following years of suffering from various symptoms of their particular disorder. Because LSD’s are also associated with inflammation, the cells and organs that are affected by a certain LSD, express the IQ-receptor (HB-EGF). This means that these cells and organs can be treated by application of IQ-targeted gene therapy (Lysotin). Prior art treatment modalities have serious problems in delivering therapeutics into the diseased cells and organs. Lysotin fulfils these needs by the selective delivery of genes for the treatment of Gaucher Disease and Fabry Disease. This considerably enhances the Quality of Life of these patients.
  • Protectin is a precision medicine for the selective delivery of immunosuppressive drugs like tacrolimus, mycophenolate mofetil (prodrug of mephenolic acid) and prednisolone to white blood cells like monocytes, macrophages, T-cells, etc., that are involved in transplant rejection.
  • immunosuppressive drugs like tacrolimus, mycophenolate mofetil (prodrug of mephenolic acid) and prednisolone to white blood cells like monocytes, macrophages, T-cells, etc.
  • Bacteria that live intracellularly are difficult to treat since many antibiotics poorly pass cellular membranes. Moreover, bacteria may hide for a long time in white blood cells including monocytes, macrophages and T-cells, and may therefore infect the body again during periods of immune suppression. Since inflammatory cells and white blood cells express the IQ-transport receptor it is possible to treat these bacteria very efficiently by applying Bacteriotin containing the antibiotic doxycycline.
  • the present invention relates to at least one dosage comprising a complex according to the invention.
  • the dosage is typically applied intravenously.
  • the dosage and complex are in particular intended for a two-step approach, referred to as “kick” and “kill”.
  • An example thereof relates to a least two dosages according to the invention, at least one first dosage comprising a first active compound capable of bringing a cell in a transformed state, an at least one second dosage comprising a second active compound for inducing apoptosis to the transformed cell. It has been found particularly difficult, if not impossible, to deliver apoptin to a cell with prior art techniques. Promising techniques have been available, but none of these to the knowledge of the inventors was successful in terms of delivery of apoptin.
  • Apoptin provides a unique and specific treatment of e.g. cancers. It was found that Apoptin, such as a from chicken anaemia virus (CAV)-derived 13.6 kDa protein, induced “programmed cell death” (apoptosis) in human transformed (cancer) cells independent of the tumour suppressor protein p53. Initially, it was thought that the development of cancers was caused mainly by enhanced cell proliferation. However, it was shown that a decreased level of apoptosis also contributes to cancer formation. Therefore, apoptosis can be exploited for the treatment of cancers. Apoptin forces cancer cells and transformed cells but not healthy cells to apoptosis.
  • CAV chicken anaemia virus
  • the present invention relates to a method of applying a dosage according to the invention or a complex according to the invention, further comprising applying localized UV-light, localized X-ray radiation, localized heat-shock, or a combination thereof.
  • the application of localized radiation is considered to be an alternative to the kick-step.
  • the first constituent may comprise a protein (A-part) with a molecular weight of > 40 kDa, preferably > 50 kDa, such as >55 kDa, and preferably with a molecular weight of ⁇ 100 kDa, preferably ⁇ 70 kDa, such as ⁇ 65 kDa, and preferably attached to the chain.
  • the targeting molecule may comprise a nontoxic mutant of a diphtheria toxin.
  • the present complex may comprise as third constituent a spacer attached to the liposome and to the first constituent, such as to the chain or to the protein, preferably a spacer with a molecular weight of >400 Da, such as > 1 kDa, preferably a PEG-spacer, such as PEG 2000.
  • the present complex may comprise 2-100 first constituents per second constituent, preferably 10-90 first constituents per second constituent, more preferably 20-80, such as 40-70 first constituents per second constituent.
  • the present complex may be for use in the treatment of cancers, such as solid Cancers, White Blood Cell Diseases, auto-immune diseases, Lysosomal Storage Diseases, viral diseases, transplantation, and diseases associated with inflammation, in particular Acne vulgaris, Allergy, Alzheimer’s Disease, Ankylosing spondylitis, Asthma, Atherosclerosis, Autoimmune diseases, such as celiac disease, diabetes mellitus type 1, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, (rheumatoid) arthritis, and systemic lupus erythematosus, Autoinflammatory diseases, such as familial Mediterranean, aphthous stomatitis, pharyngitis, and cervical adenitis.
  • cancers such as solid Cancers, White Blood Cell Diseases, auto-immune diseases, Lysosomal Storage Diseases, viral diseases, transplantation, and diseases associated with inflammation, in particular Acne vulgaris,
  • the present complex may be for use in a drug-delivery involving a passage of a cellular barrier.
  • the targeting molecule may have dissociation constant Kd for the HB-EGF receptor of ⁇ 10’ 6 mole, preferably ⁇ 10’ 8 mole, and hence good association to said receptor. Therewith extremely good delivery is obtained.
  • the at least one targeting molecule and or at least one liposome do not interact with endogenous ligands, i.e. ligands present in e.g. the blood stream, and therefore arrive at the intended location in the body.
  • the at least one active compound may be coupled to the at least one first constituent, to the present targeting molecule, to the present chain, to the at least one second constituent comprising a liposome, or a combination thereof.
  • the at least one active compound is selected from fluorescent compounds, such as cyanines.
  • the at least one active compound is selected from isotopes, such as radionuclides selected from a group consisting of 64 Cu, 67 Cu, 67 Ga, 68 Ga, 70 Ga, 72 Ga, 89 Zr, 90 Y, 95 Zr, " m Tc, in In, 114 In, 123 I, 124 I, 153 Gd, 159 Gd, and 177 Lu, wherein the radionuclide is optionally present as a cation, such as with a valence of 0, 1, 2, 3, 4, 6, or 7, such as Cu + , Cu 2+ , Cu 3+ , Cu 4+ , Ga + , Ga 2+ , Ga 3+ , Gd + , Gd 2+ , Gd 3+ , I + , I 3+ , In + , In 2+ , In 3+ , Lu 3+ , Tc 4+ , Tc 6+ , Tc 7+ , Zr + , Zr 2 ⁇ Zr
  • the at least one active compound is selected from ferromagnetic compounds, such as Fe, Co, Ni, Gd, and combinations thereof.
  • the at least one active compound is selected from a ferromagnetic or ferrimagnetic alloy comprising magnetic components (A,B), wherein component A and/or component B comprise(s) at least one magnetic material selected from Group 3-12, Period 4-6 elements, such as Fe, Co, Ni, Gd, and combinations thereof, such as FePd, FeCo and FePt.
  • the at least one active compound component A and/or component B comprise(s) a material selected from lanthanoids, scandium, yttrium, and combinations thereof, such as from Sc, Y, Sm, Gd, Dy, Ho, Er, Yb, Tb, such as Tb, and combinations thereof.
  • the present complex is for use in imaging, such as in magnetic resonance imaging (MRI), computer tomography (CT), Single-photon emission computed tomography (SPECT), and fluorescence.
  • MRI magnetic resonance imaging
  • CT computer tomography
  • SPECT Single-photon emission computed tomography
  • the present complex is for use in surgery or treatment, such as in removal of tissue, such as removal of cancerous tissue.
  • the first constituent may be CRM 197.
  • the at least one liposome may comprise 5-65 wt.% active compound, preferably 10-60 wt.% active compound, more preferably 20-50 wt.% active compound, even more preferably 30-50 wt.% active compound, such as 40-45 wt.% active compound, wherein per-centages are based on a total weight of the liposome and active compound.
  • the at least one liposome may be selected from SAINT molecules, such as shown in EP-0755924, such as SAINT 18, as well as molecules comprising SAINT-molecules, such as saint-O-Somes.
  • the at least one water-soluble active compound may have a water-solubility of >0.1 mole/1, preferably >0.5mole/l.
  • the at least one lipophilic active compound may have a hexane-solubility of >0.1mole/l, preferably >0.5mole/l. A similar solubility is also obtained in octanol.
  • the at least one active compound may have a molecular weight of ⁇ 10 kDa, preferably ⁇ 5 kDa, such as ⁇ 2 kDa.
  • the present complex may comprise a first active compound, wherein the first active compound brings a cell in a transformed state, for instance in a state wherein transformed cells are not able anymore to go into apoptosis due to an inadequate p53 intracellular cascade, which can be caused by (tumour-suppressor) gene-mutations, and viral and bacterial infections.
  • the first active compound brings a cell in a transformed state, for instance in a state wherein transformed cells are not able anymore to go into apoptosis due to an inadequate p53 intracellular cascade, which can be caused by (tumour-suppressor) gene-mutations, and viral and bacterial infections.
  • Examples are cancers, Epstein-Barr Virus mediated auto-immune disease (e.g. MS) that inhibits p53 via Peptidyl- arginine-deiminase-IV (PADI4), and immortalized cells.
  • PADI4 is an enzyme that removes an aminogroup of arginine
  • the present complex may comprise a second active compound, wherein the second active compound induces apoptosis of the transformed cell, such as apoptin (KILL).
  • KILL apoptin
  • the present complex may comprise the at least one liposome is obtained by reacting with a spacer in an aprotic solution, such as from Mal-PEG2000-DSPE, DSPE- PEG2000, SAINT-C-18, POPC and cholesterol in a molar ratio of 1:4: 18:37:40 in chloroform methanol (9: 1, v/v) therewith providing a lipid mixture, drying the lipid mixture, such as under reduced nitrogen pressure, hydration of the dried lipid mixture, such as in buffer, such as with a pH of 6-9, in particular pH 6.7, the buffer comprising an active compound, such as Apoptin, a gene or RNA, therewith incorporating the active compound in the liposome, such as in a Saint-O-some (SOS), sizing the obtained liposome, such as through extrusion through polycarbonate filters, such as having a pore size of 60-100 nm, such as 80 nm, preferably using a high pressure extruder, and
  • SOS comprise a new class of lipid based drug delivery devices that are formulated with a cationic amphiphile, l-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride (SAINT-C18).
  • SAINT-O-Somes have a diameter of 80-100 nm, are as stable as conventionally formulated liposomes, and have a superior release of their content at pH conditions that liposomes encounter when they are endocytosed by cells. They are particularly suited for the efficient delivery of hydrophobic as well as more lipophilic drugs.
  • the present dosage may comprise comprising 0.01-lmg complex according to any of claims 1-15 per ml cancer, preferably 0.02-0.1 mg/ml, such as 0.04-0.07 mg/ml .
  • the present dosage may be for treatment of a tumour, of a white blood cell disease, of a virus infection, such as a HIV-infection, and of white blood cells, in particular macrophages and monocytes.
  • the present dosage may comprise at least one first dosage comprising a first active compound capable of bringing a cell in a transformed state, and at least one second dosage comprising a second active compound for inducing apoptosis to the transformed cell.
  • the first active compound may be selected from compounds that cause DNA strand breaks, or interact with DNA by intercalation and inhibition of macromolecular biosynthesis, such as cytostatic drugs and cytolytic drugs, such as anthracyclines, such as Doxotin, and doxorubicin, vinblastine, docetaxel, paclitaxel, and combinations thereof, and/or wherein the second active compound may be selected from apoptin, and apoptin-associated proteins (AAP), such as AAP1, AAP2, AAP3, AAP4, AAP5, and AAP6, and combinations thereof.
  • AAP apoptin-associated proteins
  • Fig. 1 shows pre-Clinical research executed by Blood-Brain Barrier (BBB) Research Group of the Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University on animal models provided proof of concept of the IQ-TMDD.
  • BBB Blood-Brain Barrier
  • LACDR Leiden/Amsterdam Center for Drug Research
  • Fig. 4 shows a chemical formula of doxorubicin.
  • Fig. 5 shows expression of affinity towards HB-EGF at cancers.
  • Fig. 6 shows white blood cells involved in schematics of to be treated diseases with inflammation.
  • Fig. 7 shows experimental results.
  • Fig. 8 shows (left) : nuclei of cells in MS-lesions blue coloured with Hoechst. Cells with larger and smaller nuclei can be observed; Right figure: same as left figure but under fluorescent light. Cells with fluorescent CRM197 are associated with cells having larger nuclei, most probably monocytes.
  • Fig. 9 shows Cross, frontal and length section of SPECT, CT, and merged pictures of " m Tc- CRM197 distribution in hamsters following administration in the belly.
  • Fig. 1 shows pre-Clinical research executed by Blood-Brain Barrier (BBB) Research Group of the Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University on animal models provided proof of concept of the IQ-TMDD.
  • BBB Blood-Brain Barrier
  • LACDR Leiden/Amsterdam Center for Drug Research
  • FIG. 3 The uptake of CRM 197 by cells via the HB-EGF-like receptor (IQ- transporter) is shown.
  • the B-chain of CRM 197 binds to the IQ-transporter which transports CRM 197 into the cell forming a vesicle (endosome).
  • the vesicle becomes acidic (H+) and the T-chain of CRM197 forms a pore in the vesicle wall which allows the A-chain to escape from the vesicle (endosomal escape) into the cell interior (cytoplasm).
  • CRM 197 is a non-toxic mutant of the diphtheria toxin that binds to the heparin-binding-epidermal growth factor like growth factor (HB-EGF).
  • HB-EGF is an early responsive gene that is highly activated in cancers and in cells under inflammatory disease conditions. This disease-induced presence of HB-EGF is a major advantage since it provides a very selective delivery of medicines to diseased cells by the present IQ-TMDD technology that subsequently results in an enhanced disease treatment.
  • HB-EGF is also present at white blood cells (WBC) which opens the possibility to treat WBC-related diseases also.
  • WBC white blood cells
  • these cells can be used as a local drug delivery platform for the treatment of diseases associated with inflammation since WBC migrate to inflammatory sites in the body.
  • CRM 197 binds selectively to HB-EGF, and no other compounds otherwise being present bind to this receptor. This implies that there is no competition by other compounds for this receptor that may hinder the cellular uptake of CRM 197.
  • the T-chain of CRM197 changes its conformation due to the acidic environment and becomes a pore in the endosomal membrane. This allows the endosomal escape of particularly water-soluble drugs from the endosome into the cell.
  • CRM 197 uses an effective and safe, non-toxic endogenous transport mechanism called receptor- mediated endocytosis (IQ-transport receptor) with proven cargo-carrying properties, including an intrinsic endosomal escape mechanism.
  • the IQ-transport receptor has no endogenous ligands and thus neither competition from endogenous ligands, nor blockade of transport of essential nutrients to cells is to be expected. With respect to safety of CRM197, no in vitro or in vivo toxic effects were observed.
  • CRM197 is well characterized (i.e., known receptor binding domain, conjugation sites, manufacturing process).
  • Fig. 4 shows a chemical structure of doxorubicin.
  • Fig. 5 shows HB-EGF expression of various cancers (The Cancer Genome Atlas).
  • Fig. 6 shows two “families” of white blood cells that have been differentiated from a common precursor cell, the multipotential hematopoietic stem cell and that are involved with diseases associated with inflammation.
  • Fig. 7 shows CRM197-FITC and FITC uptake by U87MG (human glioblastoma cells): unconjugated FITC was not internalized by the cells. Cells were incubated for 0, 2, 4, 8 and 24 h with FITC or with FITC-labelled CRM 197 (4 and 40 ug). Cells were washed and intracellular fluorescence was determined.
  • IQ-transport receptor-mediated gene delivery (Green Fluorescent Protein plasmid (pGFP) was successfully applied in COS (monkey kidney-fibroblast)-cells, LLC-PK1 (porcine kidney-epithelial)-cells and human glioblastoma (brain cancer)-cells.
  • HIS-mice Experiments in mice with a human immune system, so-called HIS-mice, have shown that fluorescent CRM 197 (CRM-FITC) and not Bovine-Serum -Albumin (BSA-FITC) is taken up by monocytes and dendrocytes in blood and in various tissues (bone marrow, spleen and liver).
  • CCM-FITC fluorescent CRM 197
  • BSA-FITC Bovine-Serum -Albumin
  • IQ-TMDD was applied to selectively deliver radioactive technetium to the cancer following injection into the belly of these hamsters. Localization of radioactivity was done by SPECT (single photon emission computed tomography) and CT (computed tomography) after 4 hrs. Apart from the presence of radioactivity in the kidneys and bladder (due to elimination from the blood by the kidneys), it can be seen that radioactivity has been accumulated in the neck of these hamsters. This indicates that Technetium has been selectively taken up by cancer cells. This was confirmed by analysing the neckcancers after sacrificing the animals.
  • HUVEC Human umbilical vein endothelial cells
  • AbVCAM anti-VCAM-1
  • dex SOS non-targeted dexamethasone containing SAINT-O- Somes
  • E-selectin targeted SOS containing p65 -siRNA inhibited NF-kB activity and reduce inflammation in vascular glomerular nephritis.
  • Doxotin is a IQ-Precision Medicine (IQ-TMDD) containing doxorubicin manufactured into an Saint-O-Some.
  • This Precision Medicine is targeted to cancer cells by the IQ-transport molecule and internalized by cancer cells via the IQ-transport receptor and provides a selective and substantially more effective treatment of cancers. Simulations have shown that the required dose will decrease by a factor of 25, while the amount of drug taken up by cancer cells will increase from 1.6 to 11.1 % of the administered dose.
  • Doxotin allows a three-fold treatment of a cancer, including the cancer cells, the white blood cells in the cancer micro-environment and the endothelial cells of the cancer blood vessels (see Treatment of Solid Cancers), Doxotin treatment of a cancer is substantially more efficacious than conventional treatment modalities.
  • the occurrence of drug resistance due to drug effluxtransporters is largely circumvented due to the receptor-mediated uptake into the cells.
  • side effects are reduced due to the targeted delivery, the incorporation of doxorubicin into liposomes and the substantially smaller dose of the drug.
  • IQ-SOS-liposomes is prepared in a three -step procedure.
  • SH-groups is introduced at CRM 197 by the SATA-reaction (N-succinimidyl S-acetyl thioacetate).
  • SATA-modified-CRM197 is allowed to react with micelles containing PEG2000-maleimide- DSPE and PEG2000-DSPE.
  • SOS is prepared from Mal-PEG2000-DSPE, DSPE-PEG2000, SAINT-C-18, POPC and cholesterol in a molar ratio of 1:4: 18:37:40 in chloroform methanol (9: 1, v/v).
  • the lipid mixture is dried under reduced nitrogen pressure followed by hydration in buffer, pH 6.7, containing the Apoptin-gene or -mRNA.
  • the so-formed SOS is sized through extrusion through polycarbonate fdters having a pore size of 80 nm, using a high pressure extruder.
  • mice and SOS are incubated to transfer the CRM197- PEG2000-DSPE to the SOS.
  • SOS is purified on a Sepharose column and sterilized by extrusion through a 0.22 pm filter. Characterization is performed with respect to size, lipid content, protein content, gene content and stability according to established procedures (Kowalski et al., 2015).
  • the DNA plasmid encoding to the various proteins and RNA’s, as indicated in Active compounds in the described precision medicines, is bought from BaseClear (Leiden, The Netherlands) according to the required sequences and cloned into the mammalian expression vector pcDNA3.1(+) (Invitrogen).
  • the plasmids is incorporated into SOS-liposomes.
  • CRM197 is attached to the SOS-liposomes as indicated above.
  • MTT assay is performed to analyse cell viability.
  • Cells is plated in 96-well plates at 5x103 cells/100 ml medium overnight prior to the experiment.
  • the cell viability in each well is examined using MTT colorimetric assay (5 mg/ml; cat. no. M2003; Sigma- Aldrich; Merck KGaA, Darmstadt, Germany).
  • MTT solution is then added to each well and incubated for 3 h at 37°C; 100 pl dimethyl sulfoxide is used to dilute the formazan crystals.
  • the optical density value of each sample is measured at 490 nm using a plate reader. All determinations is carried out in sextuplicate.
  • Cell apoptosis is performed by using Annexin V Apoptosis Detection kit APC (eBioscience; Thermo Fisher Scientific, Inc.). Cells (2x105 cells/well) is cultured in 6-well plates until they reached 70-80% confluence, after which the cells is collected by trypsinization, washed twice with ice-cold Annexin V binding buffer, and stained with 300 pl IX binding buffer containing 5 pl Annexin V and 5 pl propidium iodide (PI) for 30 min at room temperature in the dark.
  • Annexin V Apoptosis Detection kit APC eBioscience; Thermo Fisher Scientific, Inc.
  • the X-ray source is an Andrex 225 SMART apparatus (Andrex St, Copenhagen, Denmark), which is used at 200 kV and 4 mA with a 1-mm Al fdter.
  • the applied dose is 5 Gy.
  • Dose and dose rate is monitored with a PTW dosimeter. After irradiation, the stored medium is added, and the irradiated cultures is incubated for DNA transfection.
  • DNA plasmids The DNA plasmid encoding apoptin are bought from BaseClear (Leiden, The Netherlands) according to the apoptin sequence used by Danen-Van Oorschot and cloned into the mammalian expression vector pcDNA3. 1(+) (Invitrogen). The Apoptin plasmid is tested in human cancer cells following transfection with Lipofectin to study cell viability and Apoptin protein expression.
  • glioblastoma cells undergo Apoptin-induced apoptosis and that the CRM 197 targeting technology has demonstrated to be effective in the delivery of a Green Fluorescent Protein (GFP) plasmid and the Apoptin plasmid into glioblastoma cells.
  • GFP Green Fluorescent Protein
  • the plasmids encoding Apoptin or control GFP-gene is incorporated into PEGylated Saint-O- Somes.
  • MTT assay is performed to analyse cell viability.
  • Cells are plated in 96-well plates at 5x103 cells/100 ml medium overnight prior to the experiment.
  • the cell viability in each well is examined using MTT colorimetric assay (5 mg/ml; cat. no. M2003; Sigma- Aldrich; Merck KGaA, Darmstadt, Germany).
  • MTT solution is then added to each well and incubated for 3 h at 37°C; 100 pl dimethyl sulfoxide is used to dilute the formazan crystals.
  • the optical density value of each sample is measured at 490 nm using a plate reader. All determinations are carried out in sextuplicate.
  • Cell apoptosis are performed by using Annexin V Apoptosis Detection kit APC (eBioscience; Thermo Fisher Scientific, Inc.). Cells (2x105 cells/well) are cultured in 6-well plates until they reached 70-80% confluence, after which the cells are collected by trypsinization, washed twice with ice-cold Annexin V binding buffer, and stained with 300 pl IX binding buffer containing 5 pl Annexin V and 5 pl propidium iodide (PI) for 30 min at room temperature in the dark.
  • Annexin V Apoptosis Detection kit APC eBioscience; Thermo Fisher Scientific, Inc.
  • Cancer treatment is in many cases limited due to poor treatment efficacy, many side-effects and the occurrence of drug resistance.
  • cancer mortality is for more than 90% due to metastasis. This is mainly due to cancer cell dissemination via blood vessels which plays a vital role in the metastasis cascade and predominantly involves local invasion, intravasation, circulation, micro-metastasis formation and metastatic colonization.
  • vessel targeting to kill cancers leads to tumour metastasis since these treatments are mostly focused at one target, e.g. blood vessels, leaving open the escape of metastasis into the body.
  • Cancer cells but also cells that have a changed behaviour due to the interference by viruses, genemutations or other modifications are called “transformed” cells. These cells are not able to change their aberrant cellular status to normal. When these modifications result in blocking of the ultimate route to escape from this situation, e.g. the p53 mediated apoptotic route, this will cause chronic diseases like cancers, and auto-immune diseases (see also “Kick and Kill” precision treatment of Auto-Immune Diseases). The “Kick and Kill” approach is able to force also “ transformed cell” to apoptosis.
  • HB-EGF hypoxia which is a strong inflammatory stimulus and induces the expression of HB-EGF. It has been shown that in vitro, HB-EGF is involved in cancer blood vessel formation and in vivo present at cancer blood vessel endothelium of adeno cystic carcinoma. These cells are treated with the “Kick and Kill” approach since Doxotin will kill and/or bring the endothelial cells into a temporary transformed state that activates Apoptin forcing these cells to apoptosis. This all will result in a collapse of the cancer blood vessels and a cut-off from the nutrient supply of the cancer and subsequently in killing of the whole cancer.
  • the “Kick and Kill” approach is very beneficial to cancer treatment.
  • the combinatorial treatment of cancers by Doxotin followed by Apoptin will kill and/or bring all these cells into a transformed state, that will activate Apoptin which subsequently will force only these cells to apoptosis including metastatic cells.
  • repeated administration of the non-toxic Apoptin will take care of the anti -cancer surveillance of the blood compartment since Apoptin will force these metastatic cancer cells to apoptosis and healthy cells not. Therefore, the “Kick and Kill” approach provides a treatment of the whole cancer and therefore a substantial chance at a cure.
  • Doxorubicin is a DNA damaging drug that kills cells and is therefore a severe stress factor to cells.
  • doxorubicin is a strong SOS signal to cells that forces cells into a temporary “transformed” state.
  • RA-FLS are transformed cells. This means that applying an extra stress stimulus (SOS-signal) followed by Apoptin will force these cells to apoptosis.
  • SOS-signal extra stress stimulus
  • Doxorubicin is a DNA damaging drug and presents a severe stress factor to cells. This opens the way to force FLS to apoptosis by firstly “kicking” these cells with Doxotin (a doxorubicin containing Precision Medicine) followed by “killing” these cells by Apoptin. This “Kick and Kill” approach will effectively eradicate AID affected cells and reduce or may even stop disease progression.
  • MTT assay is performed to analyse cell viability.
  • Cells obtained from RA-animals (preferentially hamsters), is plated in 96-well plates at 5x103 cells/100 ml medium overnight prior to the experiment.
  • the cell viability in each well is examined using MTT colorimetric assay (5 mg/ml; cat. no. M2003; Sigma- Aldrich; Merck KGaA, Darmstadt, Germany).
  • MTT solution is then added to each well and incubated for 3 h at 37°C; 100 pl dimethyl sulfoxide is used to dilute the formazan crystals.
  • the optical density value of each sample is measured at 490 nm using a plate reader. All determinations is carried out in sextuplicate.
  • Cell apoptosis is performed by using Annexin V Apoptosis Detection kit APC (eBioscience; Thermo Fisher Scientific, Inc.).
  • FLS (2x105 cells/well from RA-rats) is cultured in 6-well plates until they reached 70-80% confluence, after which the cells is collected by trypsinization, they is washed twice with ice-cold Annexin V binding buffer, and stained with 300 pl IX binding buffer containing 5 pl Annexin V and 5 pl propidium iodide (PI) for 30 min at room temperature in the dark.
  • the X-ray source is an Andrex 225 SMART apparatus (Andrex St, Copenhagen, Denmark), which is used at 200 kV and 4 mA with a 1-mm Al filter.
  • the applied dose is 5 Gy.
  • Dose and dose rate is monitored with a PTW dosimeter. After irradiation, the stored medium is added, and the irradiated cultures is incubated for DNA transfection.
  • Monocytes are white blood cells that are by definition non-dividing cells with a short half-life (hours) that limits viral replication making it almost impossible. Monocytes are continuously produced and broadly present in the blood stream where they can be exposed to viruses. In addition, monocytes- macrophages have several appealing characteristics as a target for viral infection, thus viruses have found ways to avoid the limitations and adapt these cells for their replication.
  • RNA or DNA will induce the production of:
  • monocytes At the end of their lifetime monocytes will die following apoptosis or differentiate into macrophages. The latter may become resident in various tissues and are then called histiocytes, Kupffer cells, Hofbauer cells, alveolar macrophages and microglia, etc.
  • the virus may infect both monocytes and macrophages. Therefore, these cells may function as a viral factory but also cause spread of the virus over the whole body since monocytes and macrophages are able to penetrate tissue barriers. Moreover, macrophages have a long lifetime and function therefore as the main viral reservoirs from which the virus may be reactivated when the immune system is depressed.
  • Doxorubicin is a DNA damaging drug and presents a severe stress factor to cells. This opens the way to force virus infected cells to apoptosis by “Kicking” these with Doxotin (containing doxorubicin) and the “Killing” them by Apoptin. This will eradicate the virus infected monocytes and macrophages including the virus. In addition, the “Kick and Kill” approach is not sensible for the escape of the virus due to mutations which happens with many viruses.
  • HIV/AIDS Human Immunodeficiency Virus (HIV-1/2) is currently well-managed and achieves plasma viral suppression with existing combination antiretroviral therapy (cART). However, next to life-long treatment including side effects, high demands are put to patient compliance.
  • the present treatment of HIV involves the administration of various drugs:
  • NRTIs Non-nucleoside reverse transcriptase inhibitors
  • NRTIs Nucleoside reverse transcriptase inhibitors
  • Integrase strand transfer inhibitors INSTIs
  • HIV-DNA Since the HIV-DNA is integrated into the host DNA, once integrated, the proviral DNA is replicated along with cellular DNA during cycles of cell division, as with any cellular gene. Particularly, resident macrophages function as viral reservoirs (see Infected cells and HIV-reservoirs).
  • One approach for achieving HIV cure has been termed ‘Kick and Kill’ (or ‘Shock and Kill’). Here, the reservoir is stimulated to reverse latency, and the now targetable cells are killed. In theory such “kick and kill” approach can eradicate HIV from the body.
  • TGS/C46 treatment 1) Construction of a IQ-TMDD-plasmid for TGS and C46 treatment b) Testing of an IQ-TMDD-plasmid plasmid that provides TGS and C46 expression in human CD4+-T-cells following lipofectin transfection c) Testing of an IQ-TMDD-plasmid for TGS and C46 treatment in an animal model with HIV (no rats or mice, preferably macaques)
  • Neprilysinl-gene encoding the neprilysin protein and manufactured in an IQ-TMDD precision medicine, is delivered into the inflamed brain endothelial cells and monocytes to decrease or stop disease progression of CAA or HDHWA-D and maybe AD patients
  • AD Since AD, CAA and HCHWA-D are associated with inflammation this will cause upregulation of HB-EGF at inflammatory cells including the endothelium of the blood vessels in the brain.
  • monocytes that migrate to inflammatory sites in the brain also express under normal conditions already largely the IQ-transport receptor. This opens the possibility for a Dual Treatment Modality:
  • monocytes but also other white blood cells like neutrophils, have already a very high expression of the IQ-transport receptor.
  • monocytes and other white blood cells is loaded with the NEPl-gene and will express the NEP1 enzyme at the surface of the cell.
  • these cells migrate into the inflamed disease areas in the brain of AD-, CAA- or HCHWA-D-patients, they can break down the A monomers and oligomers and delay or stop the disease progression.
  • IQ-transport molecule was coupled to a polymer (polyethylene imide (PEI)) which has a positive charge. Since plasmids are negatively charged they is electrostatically attached to the IQ-PEI system. Incubation with various cells (e.g.
  • NEP 1 -plasmid pIRESneo-NEP (AA-sequence available at NCBI) encoding human Neprilysin protein is constructed by subcloning the Notl/EcoRI firefly cDNA fragment from pBKS vector into the polylinker of pIRESneo-vector.
  • Gene DNA is amplified in DHD5 and isolated and purified by using QIAGEN Gene Mega Kits. Purity is confirmed by gel electrophoresis followed by ethidium bromide staining and the DNA concentration is measured in terms of the UV absorption at 260 nm.
  • Coupes is incubated overnight at 4 °C with polyclonal goat anti HB-EGF (1: 100, Santa Cruz Biotechnology, USA) in T-PBS. Coupes is washed 3 times with T-PBS for 5 min and incubated at room temperature with biotinylated rabbit anti-goat polyclonal antibody (1:500, Jackson, USA) for 55min. Next, slides is incubated with Streptavidin-Texas Red Rhodamine (1:2000, Vector Laboratories, Burlingame, CA, USA) for 20min. Cells is rinsed with deionized water and embedded in mounting medium for fluorescence with DAPI (Vectashield, Vector Technologies, Burlingame, CA). HB-EGF expression will then be assessed using fluorescent microscopy.
  • DAPI Vectashield, Vector Technologies, Burlingame, CA
  • GA also known as Copolymer 1, Cop-1, or Copaxone
  • Copolymer 1, Cop-1, or Copaxone is an immunomodulator currently used to treat multiple sclerosis.
  • GA is approved in the United States to reduce the frequency of relapses, but not for reducing the progression of disability.
  • glatiramer acetate is approved to treat a first episode anticipating a diagnosis. It is also used to treat re lapsing -remitting multiple sclerosis. It is administered by subcutaneous injection.
  • GA is a mixture of random-sized peptides that are composed of the four amino acids found in myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) in particular, glutamic acid, lysine, alanine, and tyrosine.
  • MBP myelin basic protein
  • PBP proteolipid protein
  • MOG myelin oligodendrocyte glycoprotein
  • G myelin oligodendrocyte glycoprotein
  • GA seems to prevent T-cell activation by competitively binding to the major histocompatibility (MHC) class II molecules thereby preventing the presentation of other antigens and hinder T-cell activation.
  • MHC major histocompatibility
  • Its efficacy in multiple sclerosis is related to the shift in T-cell response from the pro-inflammatory to the antiinflammatory pathway.
  • it provides also neuroprotection due to the GA-activated secretion of brain-derived neurotrophic factor in the brain.
  • GA seems to be also active in immune -mediated diseases like graft-versus-host diseases and inflammatory bowel disease (colitis).
  • CAA comprises cerebral amyloid angiopathy and HCHWA-D is similar but advances much faster. Both diseases are characterized by accumulation of amyloid-beta (Ap) in the middle and smaller blood vessels in the brain.
  • the efficacy in clearing Ap monomers and oligomers is performed according to Li et al., 2020, following incubation of Ap monomers and oligomers with Copaxotin activated and non-activated human monocytes/macrophage s .
  • Copaxotin can be reduced considerably since only white blood cells, mainly monocytes and macrophages, is treated.
  • the present dosing is: a) Adults: 20 mg subcutaneously 1 x/day or 40 mg subcutaneously 3 x/week with intervals of 48 h. b) Children from 12 year: 20 mg subcutaneously 1 x/day.
  • MS Multiple sclerosis
  • MS is a chronic inflammatory neurodegenerative disease of the central nervous system.
  • the early stage is characterized by relapses and the later stage, by progressive disability.
  • white blood cells like monocytes, macrophages and dendrocytes, play a key role in the disease course of MS.
  • monocytes, macrophages and dendrocytes play a key role in the disease course of MS.
  • MS There are nearly 1 million people in the United States with MS and about 2.5 million people globally .
  • a fundamental problem in the treatment of MS is the lacking ability to selectively deliver therapeutically active drugs to MS-areas in the brain with high efficiency.
  • We have shown (see Brain penetration of monocytes and dendrocytes in MS-marmoset monkey) that white blood cells (most probably monocytes) are able to penetrate MS-lesions in marmoset brain (Callithrix jacchus with experimental autoimmune encephalomyelitis, an animal model for MS) after being loaded with fluorescent CRM 197 in the blood compartment.
  • Meena and Cools 2019 have recently shown that dendrocytes are also able to migrate into the brain following inflammation in the brain.
  • Monocytes, macrophages and dendrocytes express the IQ-transport receptor (HB-EGF), therefore, these cells can be treated with an IQ-targeted mediated delivery of methylprednisolone (Prednitin). Because mainly these cells contribute to the inflammatory processes in the brain, their pro- inflammatory activity can be inhibited by decreasing the release of pro-inflammatory compounds from these cells in the brain.
  • Prednitin methylprednisolone
  • Methylprednisolone is widely used in the treatment of MS and has already shown to be effective. It binds to a receptor inside cells in contrast to most other anti-MS-drugs like interferon-beta that binds to an extra-cellular target. The drug is off-patent.
  • Prednitin By applying Prednitin, we expect to increase the efficacy of MS-treatment, to reduce side-effects and to increase the quality of life of these patients. Moreover, Prednitin can also be applied in the treatment of other diseases associated with inflammation such as (rheumatoid) arthritis, meningitis, encephalitis, ulcerative colitis, Crohn’s Disease, etc.
  • Methylprednisolone in blood and in target tissue with protein binding in blood (78%) and free drug (22%) is available to reach the target.
  • the target concentration is 600 ng/ml.
  • IQ-MPRED-SOS targeted IQ-liposomes
  • target concentrations of the present active compounds are in the order of 100-1000 ng/ml, such as 200-500 ng/ml.
  • Prednitin a) Inflammatory diseases like (rheumatoid) arthritis, multiple sclerosis, bacterial inflammation, viral inflammation, multiple sclerosis, meningitis, encephalitis, ulcerative colitis, Crohn’s Disease, etc. b) Since dexamethasone is more potent in activation of the glucocorticoid receptor it should be investigated if this drug is more applicable than methylprednisolone. c) Methylprednisolone has shown to be effective in the treatment of Covid- 19 , also dexamethasone.
  • LSD LSD’s are a group of about 50 rare inherited metabolic disorders that result from defects in lysosomal function. Lysosomes are cellular vesicles with enzymes that digest large molecules and pass the fragments on to other parts of the cell for recycling. This process requires several critical enzymes. If one of these enzymes is defective, because of a mutation, the large molecules accumulate within the cell, eventually killing it. LSD’s usually occur as a consequence of deficiency of a single enzyme required for the metabolism of lipids, glycoproteins (sugar-containing proteins), or so-called mucopolysaccharides. Individually, LSDs occur with incidences of less than 1: 100,000; however, as a group, the incidence is about 1:5,000 - 1: 10,000. Most of these disorders are autosomal recessively inherited such as Niemann- Pick disease, type C, but a few are X-linked recessively inherited, such as Fabry disease and Hunter syndrome (MPS II).
  • Symptoms may include developmental delay, movement disorders, seizures, dementia, deafness and/or blindness. In other diseases, hepatosplenomegaly, pancytopenia, pulmonary and cardiac problems, and bone manifestation are prominent symptoms and signs.
  • IQ-precision medicines containing the functional enzyme, or mRNA or a plasmid (encoding for the functional enzyme) that is able to produce the functional enzyme.
  • An interesting feature of IQ-targeted medicines is that following internalization by the affected cells into endosomes these subsequently fuses with the lysosomes. This means that the functional enzyme is transported by IQ-precision medicines to the right part of the cell (see fig. below). This is a much more advanced and effective delivery of the functional enzyme compared to the present delivery of the enzyme following intravenous infusion. In the latter case the activity of this enzyme is limited to the space outside the cells because the enzyme as such cannot pass cellular membranes and therefore cannot reach its optimal intracellular environment for activity.
  • T-cells, macrophages, dendritic cells and Langerhans cells have at average a lifetime of at least two weeks, a required dosing regimen could be typically once per two weeks.
  • Transplant Rejection is an adaptive immune response via cellular immunity, inducing apoptosis of target cells, as well as humoral immunity (mediated by activated B cells secreting antibody molecules), though the action is joined by components of innate immune response (phagocytes and soluble immune proteins).
  • White blood cells particularly T-cells and B-cells including monocytes and macrophages, have been recognized also as important cells in transplant rejection.
  • Applying the IQ-TMDD technology it is possible to treat white blood cells, including T-cells, B-cells, monocytes and macrophages, selectively and very efficiently with immunosuppressive drugs, at a lower dose and with less side-effect while optimizing treatment also due to modulated pharmacokinetics of the drug delivery system.
  • Bacteria that live intracellularly are difficult to treat since many antibiotics poorly pass cellular membranes. However, because these diseases (like tuberculosis, Lyme Disease, sexually transmitted diseases, Rocky Mountain Spotted fever, Q-fever, Legionellosis) are associated with inflammation, the HB-EGL cell receptor is expressed by the infected cells and therefore antibiotics manufactured into IQ- TMDD medicines can be brought into the infected cells to treat these bacteria effectively.
  • these diseases like tuberculosis, Lyme Disease, sexually transmitted diseases, Rocky Mountain Spotted fever, Q-fever, Legionellosis
  • bacteria hide in white blood cells including monocytes and macrophages cells for long time survival, and these cell highly express the HB-EGL cell receptor, they can be substantially more effective treated with Bacteriotin containing antibiotics like doxycycline, isoniasid, rifampicin, etc., by applying our IQ-TMDD technology.
  • IQ-targeted radio-labels also enhances the diagnostic as well as the therapeutic efficacy of these compounds in cancer patients.
  • Gadolinium is paramagnetic at room temperature. Paramagnetic ions enhance nuclear relaxation rates, which makes gadolinium useful for magnetic resonance imaging (MRI). Solutions of organic gadolinium complexes are used as intravenous MRI contrast agent to enhance images in medical magnetic resonance imaging and magnetic resonance angiography (MRA) procedures.
  • MRA magnetic resonance angiography
  • Gd is complexed with DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10- tetraacetic acid) or a comparable complexing compound and injected intravenously. In such a situation the Gd compounds stays outside cells.
  • DOTA l,4,7,10-tetraazacyclododecane-l,4,7,10- tetraacetic acid
  • the present IQ-targeted approach offers the possibility that Gd is internalized by the target cells (cancer cells but also inflammatory cells).
  • Gd in a such a targeted imaging agent
  • complexation of Gd3+ with preferably DOTA (dodecane-tetra acetic acid) and subsequent directly coupling to CRM 197 (via SATA reagent into an thioester) will provide an MRI imaging agent (see fig. 2b wherein the dot-parts represent GD).
  • CRM 197 will be coated with DOTA-Gd which will decrease the affinity of the HB-EGF receptor.
  • incorporation of Gd-complex preferably macrocyclic DOTA
  • Avoiding the binding of many molecules to CRM197 will prevent that the binding affinity for HB-EGF will decrease.
  • imaging can be considerably enhanced since more Gd can be delivered via an IQ-Gd-SOS liposome at the target site. This is beneficial since MRI has a contrast sensitivity of about 10-6 mol.
  • the IQ-Gd- SOS will be taken up by the target tissue and since Gd-complex is hydrophilic, this will stay for a longer time in this tissue thereby enhancing the difference between the target signal and that of the environment.
  • IQ-Gd-SOS An additional advantage of the IQ-Gd-SOS is that Gd is incorporated into an SOS which will reduce the systemic toxicity of Gd.
  • Fig. 2c shows IQ (CRM197)-targeted-SOS liposome containing gadolinium (Gd) for MRI imaging.
  • Dual labelled agents have the advantage that these can be applied for diagnostic total body imaging together with regional (fluorescent) image guided surgery.
  • a nonradioactive agent that can be used for whole and regional body imaging may be advantageous.
  • such an agent contains an MRI detectable compound (e.g. Gd) and a fluorescent dye like CF790, or Alexa Fluor 790, or a NIRF Dye).
  • the fluorescent groups preferably have their emission wavelength in the near infrared (750-900 nm) since these agents have the greatest potential for clinical translation, offer the lowest background due to the lack of autofluorescence at excitation wavelength > 750 nm and consequently possess the greatest sensitivity.
  • the IQ-targeted-SOS-imaging agent preferably comprises the following parts: a) an SOS liposome containing Gd-complexed with DOTA b) an SOS liposome with an incorporated fluorescent dye like CF790 or Alexa Fluor 790 c) an SOS liposome labelled at the outside with the CRM197 as the targeting agent.
  • ⁇ Wm Tc-CRM 197 was administered into the belly of these hamsters. It can be seen that after 16 h, next to the accumulation in the cancer, the radioactivity is present in the belly, but also in the kidneys and bladder of these animals. This means that 99mTc-CRM197 is also relatively fast excreted by the kidneys, which is important with respect to the exposition to radiation. Since the cells in the CME are mainly white blood cells and all express in varying degrees HB-EGF, this means that the cancers cells as well as the CME will be imaged by CRM197-targeted-imaging.
  • the IQ-technology can also be used for the targeted delivery of diagnostic as well as therapeutic radiolabels including ⁇ -, - and > -emitting agents. These could be applied next to chemo- and immunotherapies .
  • Filgotinib has a plasma half-life of 7 h. It has an IC50 of 629 nM or 297 ng/ml by inhibiting JAK1 and a 30-fold selectivity for JAK1 over JAK2 -dependent signalling in human whole blood.
  • Filgotinib is fast converted by esterases into its active metabolite GS-829845 and both compounds can enter the target cells.
  • Filgotinib delivers filgotinib into the target cells avoiding metabolization in the blood compartment. Further, the cellular uptake is not any more dependent upon lipophilicity (and protein binding), but on the rate of receptor mediated uptake. Subsequently, the intracellular availability of filgotinib is dependent on its endosomal/lysosomal degradation and the intra-cellular esterases, which also determines the intracellular availability of filgotinib and its metabolite GS-829845. Filgotinib is also effective in the treatment of colitis ulcerosa. It provides an increased efficacy of treatment of e.g. rheumatoid arthritis, at a lower dose, with less side effects.
  • Total RNA is extracted with Trizol (Invitrogen). 200ng of total RNA from each sample is used for Illumina mRNA profiling. For real-time qPCR analysis, total RNA is reverse-transcribed into cDNA using oligo dT and Superscriptase III (Life Technologies) and real-time qPCR was performed in triplicate using SYBR green PCR on a 7900HT real-time PCR system (Applied Biosystems, CA).
  • IL- la is constitutively present as a bioactive precursor inside a wide range of cells. It is present, for example, in epithelial cells of the lungs, keratinocytes of the skin, and vascular endothelial cells. During necrosis resulting in cell death, the bioactive IL- la precursor is released, furthermore, IL-la is also present on the surface of monocytes and B lymphocytes.
  • IL- 1 P is produced by more specific subsets of cells; it is a product of monocytes, tissue macrophages, and dendritic cells.
  • STAT1 phosphorylation is induced by IFN-a and IFN-y.
  • STAT1 U2OS cells (Invitrogen, catalogue no. K1469) were preincubated with compound at 37°C for 1 h, treated with 30,000 U/ml IFN-aB2 (PBL IFN source, catalogue no. I l l 15-1) or 20 ng/ml IFN-y (PeproTech, catalogue no. 300-02, lot 010827) at 37°C for 1 h, lysed (lysis buffer containing 2 nM Tb-Ab; Invitrogen) according to manufacturer’s protocol, and incubated at RT for 60 min.
  • pSTATl was detected by time-resolved fluorescence resonance energy transfer (PerkinElmer).
  • siRNA small interfering RNA

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