NL2026569B1 - Target mediated endocytotic drug delivery - Google Patents

Target mediated endocytotic drug delivery Download PDF

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NL2026569B1
NL2026569B1 NL2026569A NL2026569A NL2026569B1 NL 2026569 B1 NL2026569 B1 NL 2026569B1 NL 2026569 A NL2026569 A NL 2026569A NL 2026569 A NL2026569 A NL 2026569A NL 2026569 B1 NL2026569 B1 NL 2026569B1
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cells
active compound
disease
complex
cell
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NL2026569A
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Hendrik Maring Markwin
Gerrit De Boer Albertus
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Crm Therapeutics B V
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Priority to NL2026569A priority Critical patent/NL2026569B1/en
Priority to PCT/EP2021/076545 priority patent/WO2022069419A1/en
Priority to EP21786122.8A priority patent/EP4221757A1/en
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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
    • A61K47/69—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
    • 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
    • A61K47/51—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Abstract

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.

Description

P100537NLOO 1 Target mediated endocytotic drug delivery
FIELD OF THE INVENTION 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 te “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.
BACKGROUND OF THE INVENTION 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.
It is noted that 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.
An issue with drug administration is that typically relatively large amounts of drugs are needed. One of the reasons is that drugs are administered at a location different from where they are supposed to be delivered; hence they typically need to travel through the human body. As a consequence administration is only partly effective and typically causes side-effects; any compound is, depending on the dosage, toxic to the human body.
Also the action of a drug is typically far from optimal. A reason thereto is that to be treated cells or organs are typically not in a state such that they are susceptible enough to the drug.
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 40 functionality and advantages.
P100537NLOO 2
SUMMARY OF THE INVENTION It is an object of the invention to overcome one or more limitations of the drug delivery of the prior art and to improve this. In a first aspect 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 a chain capable of forming a pore in an endosomal membrane under acidic conditions, in particular a T-chain (also referred to as T-part), preferably attached to the at least one targeting molecule, at least one second constituent comprising a liposome, the at least one liposome enclosing an amount of at least one active compound, which is also referred to as medicine, pharmaceutical drug, or simply drug, that is a drug used to diagnose, cure, or treat a disease, selected from lipophilic compounds, and from water-soluble compounds, in particular 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, daunorubicin, and doxorubicin (CAS 23214-92-8), vinblastin, docetaxel, paclitaxel (fig. 4, CAS No. 23214-92-8)), apoptin, apoptin-associated proteins (AAP), such as AAPl, AAP2, ARP3, AAP4, AAP5, and AAP6, HIV-tin, amytin, copaxotin, prednitin, lysotin, protectin, 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: 40 1) Reduction by a factor of 4-350 of the required amount of drug
P100537NLOO 3 by avoiding protein binding 2) Enhanced efficacy of disease treatment (increased % of the dose in target tissue), so a 20-30 times lower dose, 3) Reduction of drug distribution and less side effects 4) Partly circumventing drug resistance 5) Better control of disease treatment, modulated pharmacokinetics 6) Increased Quality of Life of patients. With respect to the compounds mentioned above the following is noted: A. DOXOTIN comprises doxorubicine-HCL B. APOPTIN comprises an expression plasmid encoding for Apoptin with the amino-acid sequence:
MNALQEDTPP GPSTVEFRPPT SSRPLETPHC REIRIGIAGI TITLSLCGCA NARAPTLRSA TADNSESTGE KNVPDLRTDQ PKPPSKKRSC DPSEYRVSEL KESLITTTPS RPRTAKRRIR
L E. HIV-TIN comprises 1) Kick and Kill with Apoptin and Doxotin 2) an expression plasmid translational providing transcriptional gene silencing (TGS) by e.g. 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. F. AMYTIN comprises an expression plasmid encoding for the protein neprilysin comprising the following amino acids: 1 MGKSESQMDI TDINTPKPKK KORWTPLEIS LSVLVLLLTI IAVTMIALYA
TYDDGICKSS 61 DCIKSAARLI QNMDATTEPC TDFFKYACGG WLKRNVIPET SSRYGNFDIL
RDELEVVLKD 121 VLOEPKTEDI VAVQKAKALY RSCINESAID SRGGEPLLKL LPDIYGWPVA
TENWEOKYGA 181 SWTAEKAIAQ LNSKYGKKVL INLFVGTDDK NSVNHVIHID QPRLGLPSRD
YYECTGIYKE 241 ACTAYVDFMI SVARLIRQEE RLPIDENQLA LEMNKVMELE KEIANATAKP
EDRNDPMLLY 301 NKMTLAQIQN NFSLEINGKP FSWLNFTNEI MSTVNISITN EEDVVVYAPE
YLTKLKPILT 361 KYSARDLQONL MSWREIMDLV SSLSRTYKES RNAFRKALYG TTSETATWRR
CANYVNGNME 421 NAVGRLYVEA AFAGESKHVV EDLIAQIREV FIQTLDDLTW MDAETKKRAE 40 EKALAIKERI
P100537NLOO 4 481 GYPDDIVSND NKLNNEYLEL NYKEDEYFEN IIQNLKEFSQS KQLKKLREKV
DKDEWISGAA 541 VVNAFYSSGR NQIVFPAGIL QPPFFSAQQS NSLNYGGIGM VIGHEITHGF
DDNGRNENKD 601 GDLVDWWTQQ SASNFKEQSQ CMVYQYGNFS WDLAGGQHLN GINTLGENIA DNGGLGQAYR661 AYQNYIKKNG EEKLLPGLDL NHKQLFFLNFE AQVWCGTYRP
EYAVNSIKTD VHSPGNEFRIT 721 GTLONSAEFS EAFHCRKNSY MNPEKKCRVW G. COPAXOTIN comprises a random polymer of L-ALANINE, L-GLUTAMIC ACID, L-LYSINE, and L-TYROSINE that structurally resembles MYELIN BASIC PROTEIN. 1} a typical example of glatiramer-acetate is:
EAYRAAEKAYAAKEAAKAKAEKKAAYAKAKAAKYEKKAKKAA 2) a typical example of an expression plasmid encoding glatiramer- acetate is: EAYKAAFEKAYAAKEAAKAKAEKKAAYAKAKAAKYEKKAKKAA H. PREDNITIN comprises methylprednisolone-hemi-acetate or dexamethasone-acetate (also referred to as Dexatin). I. LYSOTIN: comprises 1) For Fabry Disease: an expression plasmid encoding for alpha- galactosidase A 2) For Gaucher Disease: an expression plasmid encoding for glucocerebrosidase J. PROTECTIN comprises: tacrolimus and/or mycophenolate mofetil K. BACTERIOTIN comprises: doxycycline-HCl The present technology, also referred to as IQ-Targeted Mediated Drug Delivery (IQ-TMDD), is particularly applicable to the treatment of cancers, white blood-cell related diseases and diseases associated with inflammation The expression of the heparin-binding epidermal growth factor (HB-EGF) at cells makes drug delivery to these cells/organs very selective. It has been shown that 95% of the 20000 cancers express the HB-EGF cell receptor. Further, efficient delivery of drugs to specific cellular reservoirs is of particular importance for therapeutics that are not able to pass cellular barriers and that may have unwanted side effects in off-target tissues. Heparin- binding epidermal growth factor (HB-EGF) is expressed on leukocytes and may be targeted for specific drug delivery using a cross-reacting material such as CRM197, which is a non-toxic variant of diphtheria toxin and exogenous substrate for HB-EGF. 40 Inventors used fluorescently labelled cross-reacting material and
P100537NLOO 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. Cross-reacting 5 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.
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 first constituent, comprising the targeting molecule (square part), and the chain (triangular part). Optionally also a protein, 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.
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. Apoptin is considered a highly sophisticated gene therapy which triggers a mechanism which leads to programmed cell death (apoptosis) of e.g. only of cancer cells and of transformed cells, and not of healthy cells. Although the mechanism of Apoptin is proven and tested it turned out to be impossible to deliver the protein or its corresponding gene into cancer cells. By combining Apoptin with the present IQ-TMDD complex technology this issue is solved. In an example, a (solid) 40 cancer comprises mainly cancer cells, white blood cells of the
P100537NLOO 6 cancer micro-environment 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 micro-environment. 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.
In an alternative 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. In addition, it has been shown in rheumatoid arthritis (RA), that cellular stress like UV- and X-ray irradiation, caused a so-called SO0S-response, resembling a transient transforming state, that activates Apoptin which subsequently forces RA-fibroblast-like synoviocytes (FLS) to apoptosis. Here we propose a “Kick and Kill” treatment of AID’s, by firstly applying Doxotin as a stress stimulus subseguently followed by Apoptin treatment.
In a further alternative 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.
Further Precision Medicine for treatment of HIV is provided with the same technology. 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 Kill”- approach now provides a real cure. In all cases 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 40 (AB) protein which causes micro bleeds in the brain (CAA and
P100537NLOO 7 HCHWA-D). Neprilysin is a major Af-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 A8- areas in the brain, the present dual treatment approach is considered to breakdown Af 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 (AB) oligomers and monomers and rescue neuronal connections in the brain providing a rationale for the treatment of CAA, HCHWA-D and AD by Copaxone. Since, macrophages are tissue resident monocytes and both highly express the IQ-transport receptor, it is found that an 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 Af from the brain and brain vessels. This subsequently decreases disease progression at a lower dose and with less side effects.
Multiple Sclerosis (MS) is a chronic inflammatory neurodegenerative disease. Particularly, monocytes play an important role by remitting and relapsing MS. These 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 3 of the dose entering the target tissue increases by at least a factor 8 compared to the application of conventional non-targeted liposomes. This means that Prednitin is substantially more effective than the current treatment modalities of methylprednisolone. This results in an increased Quality of Life of MS-patients. Next to the treatment of MS, Prednitin can also be used in the treatment of inflammatory and auto-immune diseases.
Lysosomal storage disorders (LSD’s) are caused by the 40 deficiency of a single enzyme required for the breakdown of
P100537NLOO 8 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.
Treatment of transplant rejection. 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. The IQ-TMDD technology will substantially enhance this treatment, reduce side effect and control disease treatment resulting in an increased quality of life of these patient that have to take their treatment a lifetime long.
Treatment of intracellular bacteria. 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.
In a second aspect 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 40 dosages according to the invention, at least one first dosage
P100537NLOO
S 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. This is a major advantage compared to the application of drugs that are meant to treat only dividing cancer cells including healthy cells. Apoptin forces not only fast dividing cancer cells to apoptosis but also slowly dividing cancer cells, and even non-dividing cancer cells. In addition, it was found that Apoptin-induced apoptosis particularly occurs in cancer cells and not in non-transformed cells, the anti-apoptotic protein Bcl-2 accelerated Apoptin induced apoptosis in transformed mammalian cells, adenoviral vectors expressing Apoptin have been applied in mice reducing cancer growth when administered intra-cancer in human hepatoma or breast cancers, and non-viral Apoptin vectors have been successfully targeted to hepatocarcinoma cells via the asialoglycoprotein receptor, however this receptor is mainly present at liver (cancer) cells. This indicates that Apoptin treatment will substantially improve cancer treatment by decreasing side effects, increasing efficacy and survival, and subsequently improving the quality of life of patients. However, the Apoptin protein cannot naturally penetrate cancer cells and the same holds for genes and mRNA encoding Apoptin and therefore a delivery device is necessary.
In a third aspect the present invention relates to a method of applying a dosage according to the invention or a complex 40 according to the invention, further comprising applying localized
P100537NLOO 10 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. Advantages of the present description are detailed throughout the description.
DETAILED DESCRIPTION OF THE INVENTION In an exemplary embodiment of the present complex 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.
In an exemplary embodiment of the present complex the targeting molecule may comprise a non-toxic mutant of a diphtheria toxin.
In an exemplary embodiment 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.
In an exemplary embodiment of 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.
In an exemplary embodiment 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. Other autoinflammatory diseases that do not have clear genetic causes include adult-onset Still's disease, systemic-onset juvenile idiopathic arthritis, Schnitzler syndrome, and chronic recurrent multifocal osteomyelitis, Chronic prostatitis, Crohn's disease, Dermatitis, Diverticulitis, Encephalitis, Fibromyalgia, 40 Glomerulonephritis, Hepatitis, Hidradenitis suppurativa, HIV/AIDS,
P100537NLOO 11 Hypersensitivities, Inflammatory bowel diseases: Crohn’s Disease, Ulcerative Colitis, Interstitial cystitis, Lichen planus, Leukocyte defects, Mast Cell Activation Syndrome, Mastocytosis, Meningitis, Myopathies, Nephritis, Otitis, Parkinson's disease, Pelvic inflammatory disease, pancreatitis, Reperfusion injury, Rheumatic fever, Rheumatoid arthritis, Rhinitis, Sarcoidosis, Transplant rejection, Ulcerative colitis, Vasculitis, Cerebral Amyloid Angiopathy, HCHWA-D, Multiple Sclerosis, and COVID-19.
In an exemplary embodiment the present complex may be for use in a drug-delivery involving a passage of a cellular barrier.
In an exemplary embodiment of the present complex the targeting molecule may have dissociation constant Kd for the HB- EGF receptor of < 107% mole, preferably < 107? mole, and hence good association to said receptor. Therewith extremely good delivery is obtained.
In an exemplary embodiment of the present complex 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.
In an exemplary embodiment of the present complex the first constituent may be CRM197.
In an exemplary embodiment of the present complex 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.
In an exemplary embodiment of the present complex 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-0-Somes.
In an exemplary embodiment of the present complex the at least one water-soluble active compound may have a water-solubility of >0.1 mole/l, preferably >0.5mole/l1.
In an exemplary embodiment of the present complex the at least one lipophilic active compound may have a hexane-solubility of >0.1mole/1, preferably >0.5mole/l. A similar solubility is also obtained in octancl.
40 In an exemplary embodiment of the present complex the at least
P100537NLOO 12 one active compound may have a molecular weight of < 10 kDa, preferably < 5 kDa, such as < 2 kDa.
In an exemplary embodiment 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. 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 amino-group of arginine and forms citrulline thereby. Examples are Doxotin, and doxorubicin, vinblastine, docetaxel, paclitaxel, which therewith provide the KICK effect.
In an exemplary embodiment the present complex may comprise a second active compound, wherein the second active compound induces apoptosis of the transformed cell, such as apoptin (KILL).
In an exemplary embodiment of 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 (S0S), 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 incubating the extruded liposome to transfer the CRM197- PEG2000-DSPE to the SOS, and optionally purifying the incubated complex, such as on a Sepharose column, and optionally sterilizing the complex by extrusion, such as through a 0.22 pm filter. 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). These so-called SAINT-0-Somes have a diameter of 80- 40 100 nm, are as stable as conventionally formulated liposomes, and
P100537NLOO 13 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.
In an exemplary embodiment the present dosage may comprise comprising 0.01-1mg 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 .
In an exemplary embodiment 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.
In an exemplary embodiment 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.
In an exemplary embodiment of the at least two dosages 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 AAPl, AAP2, AAP3, AAP4, AAP5, and AAPS, and combinations thereof. Throughout the description and claims the term “apoptin is considered to relate to apoptin, apoptin- associated proteins (AAP), and similar compounds.
The invention will hereafter be further elucidated through the following examples which are exemplary and explanatory of nature and are not intended to be considered limiting of the invention. To the person skilled in the art it may be clear that many variants, being obvious or not, may be conceivable falling within the scope of protection, defined by the present claims.
SUMMARY OF THE FIGURES 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.
40 An example of the present complex is shown in fig. 2.
P100537NLOO 14 A considered mode of action of the present complex is shown in fig. 3.
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.
DETAILED DESCRIPTION OF FIGURES 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.
An example of the present complex is shown in fig. 2.
With respect to figure 3. The uptake of CRM197 by cells via the HB-EGF-like receptor (IQ-transporter) is shown. The B-chain of CRM197 binds to the IQ-transporter which transports CRM197 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).
CRM197 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. In addition, 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. CRM197 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 CRM197. Following internalization into the cell in a vehicle called an endosome, the T-chain of CRM197 40 changes its conformation due to the acidic environment and becomes
P100537NLOO 15 a pore in the endosomal membrane. This allows the endoscmal escape of particularly water-soluble drugs from the endosome into the cell. CRM197 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). CRM197 is a 60 kDa protein that has a high affinity for HB-EGF (Kd = 10% mol). CRM197 has already been successfully used widely. CRM197 is available in pre-clinical and GMP-grade.
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 CRM197 (4 and 40 ug). Cells were washed and intracellular fluorescence was determined.
EXAMPLES Many experiments have been carried out, showing positive effects of the present invention, as detailed below.
I9-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.
Experiments in mice with a human immune system, so-called HIS-mice, have shown that fluorescent CRM197 (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 40 and liver).
P100537NLOO 16 IQ-TMDD was applied to selectively deliver radicactive technetium to the cancer following injection into the belly of these hamsters. Localization of radicactivity was done by SPECT (single photon emission computed tomography) and CT (computed tomography) after 4 hrs. Apart from the presence of radicactivity 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 neck-cancers after sacrificing the animals.
Hamsters were injected intra-venously with control- or IQ- liposomes containing HRP (horse radish peroxidase, 40 kDa) and rhodamine-PE (red coloured dye). It is found that IQ-liposomes were extensively taken up by monocytes (which have much HB-EGF- like IQ-transporter) in-vitro and in-vivo. In contrast, following in-vitro incubation of control-liposomes with isolated monocytes only a slight red signal could be observed while in vivo there was no red signal at all. This can be explained by the a-specific binding of the liposomes to monocytes which can be seen in vitro but which is not strong enough to withstand the forces of shear stress (due to blood flow) in vivo.
Experiments in marmoset monkeys with multiple sclerosis (MS) show that following intravenous injection, CRM-FITC has been taken up by white blood cells in the blood compartment and transported to MS-lesions in marmoset monkey brain.
Human umbilical vein endothelial cells (HUVEC) were stimulated twice with LPS. The second stimulation was given 3h after the first challenge. Then quiescent respectively LPS activated HUVEC were incubated with anti-VCAM-1 (AbVCAM) and non- targeted dexamethasone containing SAINT-O-Somes (dex SOS). (A) Fluorescence microscopy images show the uptake of targeted SAINT- O-Somes by activated HUVEC after 4h incubation. The liposome membrane was labelled with DiI (red) and the nuclei of the cells were stained using Hoechst 33342 (blue). (B) Specificity of AbVCAM-1 SAINT-O-Some association to VCAM-1 was determined by co- incubation of cells with 50 times excess of anti-VCAM-1 monoclonal antibodies together with AbVCAM-1 SAINT-0-Somes. After 3h respectively 21h co-incubation, the association of SAINT-O-Somes 40 with activated HUVEC was quantified by flow cytometric analysis.
P100537NLOO 17 Data are presented as mean fluorescence intensity (MFI) values + SD of triplicate samples from one experiment. *, P < 0.05, AbVCAM- 1 SAINT-O-Scmes vs. non-targeted SAINT-O-Somes; #, P<0.05, association of AbVCAM-1 SAINT-0-Some with LPS stimulated HUVEC vs. unstimulated HUVEC; &, P<0.05, significant difference between with or without excess anti-VCAM-1 antibodies.
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-0-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.
Since 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.
In addition, the occurrence of drug resistance due to drug efflux-transporters is largely circumvented due to the receptor-mediated uptake into the cells.
Similarly, side effects are reduced due to the targeted delivery, the incorporation of doxorubicin into liposomes and the substantially smaller dose of the drug.
It is found that treatment of AML cells with a solution of doxorubicin is much more efficient than treatment with non- targeted liposomes.
This is caused by the fact that these liposomes are not taken up by these cells and that doxorubicin is slowly released from these liposomes and subsequently slowly entering AML cells.
Such a delivery is suboptimal in cancer treatment and may also result in a fast development of drug resistance.
Materials and methods General procedures Technical procedures for the preparation of IQ-medicines 40 Preparation IQ-Saint-O-Somes-liposomes (SOS)
P100537NLOO 18 IQ-S0S-liposomes is prepared in a three-step procedure. , 1) Introduction of SH-groups at CRM197 SH-groups is introduced at CRM197 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. 2) Preparation of SOS liposomes 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 filters having a pore size of 80 nm, using a high pressure extruder. 3) Transfer of CRM197 micelles to SOS Micelles 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 incorporation of therapeutics genes into expression plasmids 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 S0S-liposomes.
Subsequently CRM197 is attached to the S0S-liposomes as indicated above.
Cell viability assay by Fan et al., 2018 An 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 ul dimethyl sulfoxide is used to dilute the formazan crystals.
The optical density value of each sample is measured at 490 nm using a 40 plate reader.
All determinations is carried out in sextuplicate.
P100537NLOO 19 Cell apoptosis assay by Fan et al., 2018 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 ul 1X binding buffer containing 5 ul Annexin V and 5 nl propidium iodide (PI) for 30 min at room temperature in the dark.
Subsequently, 400 ul Annexin V binding buffer is added and the cells is analysed using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA}, according to the manufacturer's protocol; 230,000-gated events is acquired from each sample.
Early stage apoptotic cells is stained with fluorescein isothiocyanate (FITC) Annexin V, but not PI; whereas late stage apoptotic cells and necrotic cells is stained positively for FITC Annexin V and PI.
In vitro X-ray treatment of cells 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. 1) The development of the Apoptin-genes 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.
In addition, preliminary work has already shown that glioblastoma cells undergo Apoptin-induced apoptosis and that the CRM197 targeting technology has demonstrated to be effective in the delivery of a Green Fluorescent Protein (GFP) plasmid and the Apoptin plasmid into glioblastoma cells. 2) Preparation of IQ-SOS containing the Apoptin gene The plasmids encoding Apoptin or control GFP-gene is incorporated into PEGylated Saint-O-Somes. 3) In-vitro experiments 40 The efficacy of cancer cell killing by plasmid delivery of
P100537NLOO 20 Apoptin and the control GFP-gene by IQ-S0S, is studied in vitro in cultured selected AML cells.
Cells from two AML cell lines and a hamster DDT-MF2 cancer cell line are incubated for 74 h with the Apoptin-gene/mRNA- or control-GFP SOS to investigate the Apoptin- induced apoptosis. 4) Cell viability assay by Fan et al., 2018 An 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 KGaR, Darmstadt, Germany). MTT solution is then added to each well and incubated for 3 h at 37°C; 100 ul 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. 5) Cell apoptosis assay 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 ul 1X binding buffer containing 5 pl Annexin V and 5 ul propidium iodide (PI) for 30 min at room temperature in the dark.
Subsequently, 400 ul Annexin V binding buffer is added and the cells are analysed using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol; 230,000-gated events are acquired from each sample.
Early stage apoptotic cells are stained with fluorescein isothiocyanate (FITC) Annexin V, but not PI; whereas late stage apoptotic cells and necrotic cells are stained positively for FITC Annexin V and PI. “Kick and Kill” precision treatment of Cancers Treatment of cancers Cancer treatment is in many cases limited due to poor treatment efficacy, many side-effects and the occurrence of drug resistance.
In addition, 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, 40 circulation, micro-metastasis formation and metastatic
P100537NLOO 21 colonization. Many investigations have shown that 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.
Here an innovative and unique approach for a total treatment of cancers is given that provides the chance at a cure and an increased Quality of Life of patients. It comprises the application of the “Kick” by Doxotin and the “Kill” by Doxotin and Apoptin. Since 95% of the 20000 cancer investigated express the IQ-transport receptor (see Diseases with expression of the IQ- transport receptor), many cancers can be treated by applying this “Kick and Kill” approach.
Cancer-micro-environment Almost all (white blood) cells in the CME express the IQ- transport receptor, therefore the CME is treated by applying the “Kick and Kill” approach.
Transformed cells Cancer cells but also cells that have a changed behaviour due to the interference by viruses, gene-mutations 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.
Treatment of Cancer blood vessels Cancer blood vessels suffer from 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.
40 Total cancer treatment
P100537NLOO 22 Since the IQ-transport receptor is expressed at cancer cells including metastatic cells, white blood cells of the cancer micro- environment (CME) and the inflammatory endothelial cells of the cancer blood vessels, 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.
In addition, 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. “Kick and Kill” by Doxotin and Apoptin The unique combination of Apoptin and Doxotin will provide a precision treatment of cancers with superior efficacy because: 1) Apoptin forces cancer and transformed cells to apoptosis bypassing the p53 pathway.
In addition, it has been shown in cancer prone cells, that cellular stress like UV- and X- ray irradiation, cause a so-called SOS-response resembling a transient transforming state, that activates Apoptin, forcing these cells to apoptosis. 2) Doxorubicin is a DNA damaging drug that kills cells and is therefore a severe stress factor to cells.
In addition, it has been shown that doxorubicin is a strong SOS signal to cells that forces cells into a temporary “transformed” state.
A similar approach is applied to auto-immune diseases, with similar results.
Citrullination of proteins is caused by the increased activity of the enzyme peptidyl arginine deaminase, particularly type IV (PADI4). This enzyme converts the amino acid arginine into citrulline.
In addition, it has been shown that PADI4 rescues auto-immune cells, like fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA), from apoptosis, due to inhibition of the cellular apoptotic (programmed cell death) machinery via p2l and p53. This means that these cells cannot go into apoptosis anymore and will continuously produce citrullinated proteins causing symptoms associated with RA and other AID’s. “Kick and Kill” Treatments 40 1) Treatment with growth stimulation and Apoptin
P100537NLOO 23 In vitro experiments have shown that stimulation of fibroblast-like synoviocytes (FLS) with 40% human serum followed by Apoptin treatment also forced these cells to apoptosis. This treatment is not directly feasible in humans but maybe this can be replaced by treatment of these cells with an HB-EGF Precision Medicine, that stimulates growth by activation of the epidermal growth factor receptor (EGFR) with HB-EGF.
2) Treatment with X-ray and Apoptin It has been shown that X-ray treatment of “transformed” fibroblasts subsequently followed by Apoptin treatment forced these cells to apoptosis while healthy fibroblast survived. This opens the way to treat fibroblast FLS in RA in this way. This is an attractive treatment since X-ray can be applied locally with an adjusted dose followed by administration of the Apoptin Precision Medicine.
3) Treatment with HIF-10-mRNA or -plasmid and Apoptin Fan et al. have shown that treating FLS cells by inhibiting PADI4 with siRNA will enhance apoptosis of these cells. It is well known that PADI4 blocks the p53 route leading to apoptosis. Thus restauration of the p53 route enhances the apoptosis of cells. Since Apoptin bypasses this route, we do not need to block the PADI4 route. Therefore we can treat FLS cells with a stressor like the heat-shock-inducible factor-alpha (HIF-a) followed by the Apoptin Precision Medicine that forces these cells to apoptosis.
4) Treatment with Doxotin and Apoptin It has been shown that RA-FLS are transformed cells. This means that applying an extra stress stimulus (SOS-signal) followed by Apoptin will force these cells to apoptosis. 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.
Methods Cell viability assay An MTT assay is performed to analyse cell viability. Cells, obtained from RA-animals (preferentially hamsters), is plated in 96-well plates at 5xz103 cells/100 ml medium overnight prior to the 40 experiment. The cell viability in each well is examined using MTT
P100537NLOO 24 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 assay 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 ul 1X binding buffer containing 5 ul Annexin V and 5 ul propidium iodide (PI) for 30 min at room temperature in the dark.
Subsequently, 400 ul Annexin V binding buffer is added and the cells is analysed using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol; 230,000-gated events is acquired from each sample.
Early stage apoptotic cells is stained with fluorescein isothiocyanate (FITC) Annexin V, but not PI; whereas late stage apoptotic cells and necrotic cells is stained positively for FITC Annexin V and PI.
In vitro X-ray treatment of cells 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.
A. “Kick and Kill” precision treatment of Viral Diseases Viral Diseases Many viral diseases are difficult to treat since these viruses are hiding in cells that preserve as a viral reservoir.
The most important cells in this respect are monocytes and macreophages.
Monocytes and macrophages as viral targets and reservoirs To serve as a viral reservoir cells must meet the following characteristics: . have a sufficient lifespan 40 . be able to avoid apoptosis
P100537NLOO 25 . be able to avoid the immune response . have sufficient interactions with other cell populations 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.
As soon as the virus has entered monocytes, its RNA or DNA will induce the production of: . new virus particles 15 . proteins that activate the apoptotic route for the dissemination of new virus particles . proteins that enhance the inflammatory activity of the monocytes attracting new monocytes . proteins that reduce the inflammatory activity of monocytes to avoid the immune response . proteins that downregulate the apoptotic route for protecting the virus in viral reservoirs like macrophages 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, alveclar 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. “Kick and Kill” approach This innovative and unique approach provides the eradication of cells expression the IQ-transport receptor.
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 40 “Kicking” these with Doxotin (containing doxorubicin) and the
P100537NLOO 26 “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-tin - Precision medicine to treat HIV/AIDS 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: Non-nucleoside reverse transcriptase inhibitors (NNRTIs) Nucleoside reverse transcriptase inhibitors (NRTIs) Protease inhibitors (PIs) Fusion inhibitors CCR5 antagonists Integrase strand transfer inhibitors (INSTIs) Post-attachment inhibitors 3) “Kick and Kill” approach 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.
Presently, the potential applications, the Doxitin/Apoptin “Kick and Kill’ treatment and the combined Transcriptional Gene Silencing (TGS) and C46 treatment are preferred. Starting from this point of view the following experiment is done: 1) TGS/C46 treatment a) 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 40 c) Testing of an IQ-TMDD-plasmid for TGS and C46 treatment
P100537NLOO 27 in an animal model with HIV (no rats or mice, preferably macaques) 2) Doxotin/Apoptin “Kick and Kill” treatment a) Investigations on Doxotin and Apoptin in the eradication of HIV-infected cells in vitro.
b) Investigations on Doxotin and Apoptin in the eradication of HIV in animals (macaques).
CD4+-SupTl cells were treated with CRM197-Alexa488 (squares) or BSA-Alexa488 (triangles). The exterior fluorescent signal was quenched using Trypan blue allowing distinction between binding and internalization (black symbols) or internalization alone (white symbols). From 2 to 6 hrs of incubation with CRM197- Alexa488, the total (binding + internalization) signal was significantly higher than the internalization signal alone (*p<0.001). The data shown are based on n=3 per condition and are representative of 3 independent experiments with similar outcomes.
G. Amytin —- precision medicine for treatment of CAA, HCHWA-D and AD 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 Dual Disease Treatment Modality 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. In addition, 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: 1) direct treatment by targeting the IQ-S0S-NEP1-gene precision medicine to the inflamed brain-vascular endothelial cells. There these systems is internalized into the endothelial cells via the IQ-transport receptor (HB-EGF). Subsequently neprilysin is expressed at the surface of these cells which will lead to breakdown of AB monomers and oligomers there.
2) indirect treatment by targeting monocytes. Monocytes, but also other white blood cells like neutrophils, have already a very high expression of the IQ-transport receptor. Following intravenous administration of the IQ-SOS-NEPl-gene precision 40 medicine, monocytes and other white blood cells is loaded with the
P100537NLOO 28 NEPl-gene and will express the NEP1 enzyme at the surface of the cell. When these cells migrate into the inflamed disease areas in the brain of AD-, CAA- or HCHWA-D-patients, they can break down the AB monomers and oligomers and delay or stop the disease progression.
In a series of recent experiments on several cell types (including porcine LLC-PK1, primate COS1 and human glioblastoma cells), we have demonstrated the receptor-mediated uptake of Green Fluorescent Protein genes (plasmids) by our IQ-targeting technology. For this purpose 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 IO-PEI system. Incubation with various cells (e.g. primate COS-1 cells, porcine LLC-PK1 cells and human glioblastoma cells) showed uptake as demonstrated by intracellular fluorescence while replacement of the IQ-transport molecule by a control protein (horse radish peroxidase) and addition of excess the free IQ-molecule did not result in any intracellular fluorescence. Since we will also target NEPl-gene to inflammatory brain endothelial cells we want to apply a special type liposomes called Saint-0-Somes that are able to incorporate high mRNA and gene loadings. The figure below shows schematically the various steps resulting ultimately in the intracellular delivery of the NEP1- gene.
1) NEP1-plasmid PIRESneo-NEP (AA-sequence available at NCBI) encoding human Neprilysin protein is constructed by subcloning the NotI/EcoRI firefly cDNA fragment from pBKS vector into the polylinker of pIRESneo-vector. Gene DNA is amplified in DH 5 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.
1) IQ-NEP1-S0S-liposomes These is prepared in a two-step procedure (see Preparation IQ9-S0S-liposomes}.
Experiments 1) Ex-vivo experiments Investigating the presence of HB-EGF at small and middle 40 sized blood vessels in the post-mortem brain of CAA patients:
P100537NLOO 29 Coupes is made of post-mortem brains of CAA and HCHWA-D patients, washed 3 times with PBS and fixed in ice-cold 4% paraformaldehyde. Subsequently coupes is washed with T-PBS (PBS and 0.1% Tween 20, Sigma-Aldrich), blocked with 10% rabbit normal serum (Vector Laboratories, Burlingame, CA, USA) and an Avidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA, USA) following the manufacturer's protocol. 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.
2) In-vitro experiments a) In-vitro investigation on the targeted uptake of IQ- GFP-plasmid-liposomes by endothelial cells and monocytes: following preparation of IQ-GFP-liposomes, an in vitro culture of human brain endothelial cells is used to study the uptake of IQ- GFP-plasmid-liposomes following an inflammatory stimulus by investigating the degree of fluorescence produce by the expressed GFP-protein.
b) In-vitro investigations on the endothelial targeting of the NEP1-plasmid by IQ-liposomes: Following preparation of the IQ- NEP-liposomes, an in vitro culture of human brain endothelial cells is used to study the uptake of the IQ-NEPl-liposomes following an inflammatory stimulus. Expression of the NEPl-protein is estimated by measurement of the NEPl-enzymatic activity.
c) Measurement of Neprilysin-1 enzymatic activity: Neprilysin activity is measured in whole cell or tissue homogenates using glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide (GAAPN) as a substrate. Protein content is quantified.
Precision medicine for MS, AD, CAA and HCHWA-D Applications of Glatiramer acetate 1) Glatiramer acetate (GA) in MS GA (also known as Copolymer 1, Cop-1, or Copaxone) is an immunomodulator currently used to treat multiple sclerosis. GA is 40 approved in the United States to reduce the frequency of relapses,
P100537NLOO 30 but not for reducing the progression of disability. However, observational studies, but not randomized controlled trials, suggest that it may reduce progression of disability. While a conclusive diagnosis of multiple sclerosis requires a history of two or more episodes of symptoms and signs, glatiramer acetate is approved to treat a first episode anticipating a diagnosis. It is also used to treat relapsing-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 is the antigen in the myelin sheaths of the neurons that stimulates an autoimmune reaction in people with MS, so the peptide may work as a decoy for the attacking immune cells.
The molecular weight of these co-polymerised amino acids varies from 5.5 to 9 kD. GA seems to prevent T-cell activation by competitively binding to the major histocompatibility (MHC) class IT molecules thereby preventing the presentation of other antigens and hinder T-cell activation. Its efficacy in multiple sclerosis is related to the shift in T-cell response from the pro- inflammatory to the anti-inflammatory pathway. In addition, it provides also neuroprotection due to the GA-activated secretion of brain-derived neurotrophic factor in the brain. Further, GA seems to be also active in immune-mediated diseases like graft-versus- host diseases and inflammatory bowel disease (colitis).
2) Glatiramer acetate in CAA, HCHWA-D and Alzheimer’s Disease (AD) CAA comprises cerebral amyloid angiopathy and HCHWA-D is similar but advances much faster. Both diseases are characterized by accumulation of amyloid-beta (A8) in the middle and smaller blood vessels in the brain.
Recently it has been shown that co-culturing neurones with bone-marrow derived macrophages protected synapses against Ap fibrils, moreover, immune activation of macrophages with GA conferred further protection against oligomers. Fibrils were cleared by intracellular endosomal proteolysis and by extracellular MMP-9 enzymatic degradation. In vivo studies with GA-stimulated macrophages or monocytes confirmed the in-vitro 40 data. This indicates that activated macrophages can clear Ap
P100537NLOO 31 oligomers and fibrils, rescue synapses which provides therefore a rationale for the treatment of CAA, HCHWA-D and AD. Materials and Methods 1) Preparation of Copaxotin See Preparation IQ-SOS-TMDD.
2) Testing efficacy of Copaxotin The efficacy in clearing AB 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/macrophages.
3) Dosing of Copaxotin It is found that the necessary dose of 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 1x/day or 40 mg subcutaneously 3x/week with intervals of 48 h.
b) Children from 12 year: 20 mg subcutaneously 1x/day.
H. Precision medicine for treatment of multiple scleroses (MS) Multiple sclerosis (MS) 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. Next to microglia, white blood cells, like monocytes, macrophages and dendrocytes, play a key role in the disease course of 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 CRM197 in the blood compartment. In addition, Meena and Cools (2019) have recently shown that dendrocytes are also able to migrate into the brain following inflammation in the brain. This occurs via the Blood- Brain Barrier (located in the smallest blood vessels in the 40 brain), the Choroid-Plexus (located in the ventricles in the
P100537NLOO 32 brain) and via the meningeal blood vessels (located at the outside of the brain). Under non-inflammatory conditions this migration is very less but under inflammatory conditions like in MS, strongly enhanced.
MS-Treatment modality 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.
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.
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.
In contrary, when applied as targeted IQ-liposomes (IQ-MPRED-30S) in blood and in target tissue following i.v.-administration.
No protein binding, all drug is available to reach the target site.
The target concentration is 600 ng/ml.
As a consequence, 10 to 100 times as high at the target.
Materials and Methods Preparation of IQ-S0S containing methylprednisolone (Prednitin) Methylprednisolone-hemi-acetate is incorporated into PEGylated Saint-O0-Somes.
See Preparation IQ-S0S-liposomes. 4) Other applications of Prednitin a) Inflammatory diseases like (rheumatoid) arthritis, multiple sclerosis, bacterial inflammation, viral inflammation, multiple sclerosis, meningitis, encephalitis, ulcerative colitis, 40 Crohn’ s Disease, etc.
P100537NLOO 33 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.
J. LYSOTIN - Precision Medicine for Treatment of Lysosomal Storage Diseases Lysosomal Storage Diseases (LSD’s) 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.
Cells and organs are treated with 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 40 limited to the space outside the cells because the enzyme as such
P100537NLOO 34 cannot pass cellular membranes and therefore cannot reach its optimal intracellular environment for activity. So inventors have developed an IQ-precision medicine comprising the selectivity of the IQ-targeting technology and the application of an LSD-enzyme, an LSD-mRNA or an LSD-plasmid formulated into a novel SOS drug delivery system. Firstly we will focus on the development of an IQ-TMDD-precision medicine (Lysotin) that is able to target the gene encoding glucocerebrosidase (Gaucher Disease) or alpha- galactosidase A (Fabry Disease) to LSD affected cells.
Since T-cells, macrophages, dendritic cells and Langerhans cells (skin dendritic cell) have at average a lifetime of at least two weeks, a required dosing regimen could be typically once per two weeks.
Transplant rejection.
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. Protectin may contain immunosuppressive drugs like tacrolimus, mycophenolate mofetil (= mephenolic acid) and prednisolone or combinations thereof.
Experiments The following experiments are to investigate the effectivity of Protectin in downregulating the activity of white blood cells particularly, T-cells, B-cells, monocytes and macrophages: 1) In-vitro investigation on the downregulation of T-cells, B- cells, monocytes and macrophages by Protectin following an inflammatory stimulation 2) In-vivo investigation on the downregulation of T-cells, B- cells, monocytes and macrophages by Protectin in hamsters following an inflammatory stimulus.
40 Treatment of intracellular bacteria by Bacteriotin.
P100537NLOO 35 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-EGF cell receptor is expressed by the infected cells and therefore antibiotics manufactured inte IQ-TMDD medicines can be brought into the infected cells to treat these bacteria effectively. Moreover, since bacteria hide in white blood cells including monocytes and macrophages cells for long time survival, and these cell highly express the HB-EGF cell receptor, they can be substantially more effective treated with Bacteriotin containing antibiotics like doxycycline, isoniasid, rifampicin, etc., by applying our IQ-TMDD technology.
Experiments The following experiment is performed: 1) In-vitro investigations on the killing of bacteria infected monocytes and macrophages by treating these cells with Bacteriotin 2) In- vitro investigations on the killing of bacteria infected endothelial cells by treating these cells with Bacteriotin 3) In-vivo investigations on the killing of bacteria in bacteria infected animals (hamsters) by treating these animals with Bacteriotin.
For the purpose of searching the following section is added, which may be considered embodiments of the present invention, and of which the subsequent section represents a translation into Dutch.
1. Complex for target mediated endocytotic drug delivery comprising as constituents at least one first constituent comprising a targeting molecule capable of interacting with a heparin-binding epidermal growth factor (HB-EGF) cell receptor, the HB-EGF cell receptor capable of forming an endosome in a cell, and a chain capable of forming a pore in an endosomal membrane under acidic conditions, in particular a T-chain, preferably attached to the at least one targeting molecule, at least one second constituent comprising a liposome, the at least one liposome enclosing an amount of at least one active compound selected from 40 lipophilic compounds, and from water-soluble compounds, in
P100537NLOO 36 particular from compounds that cause DNA strand breaks, or interact with DNA by intercalation and inhibition of macromolecular biosynthesis, such as cytostatic drugs and cytelytic drugs, such as anthracyclines, such as doxotin, Daunorubicin, and doxorubicin, vinblastin, docetaxel, paclitaxel, apoptin, HIV-tin, amytin, copaxotin, prednitin, lysotin, protectin, and bacteriotin, from genes or sequences thereof, from proteins, and from RNA-sequences, preferably a sequence with <30 nucleotides.
2. Complex according to embodiment 1, wherein the first constituent comprises 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.
3. Complex according to any of embodiments 1-2, wherein the targeting molecule comprises a non-toxic mutant of a diphtheria toxin.
4. Complex according to any of embodiments 1-3, comprising 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.
5. Complex according to any of embodiments 1-4, comprising 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.
6. Complex according to any of embodiments 1-5, for use in the treatment of cancers, such as solid Cancers, White Blood Cell 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, other autoinflammatory diseases that do not have clear genetic causes including adult-onset Still's 40 disease, systemic-onset juvenile idiopathic arthritis,
P100537NLOO 37 Schnitzler syndrome, and chronic recurrent multifocal osteomyelitis, Chronic prostatitis, Crohn's disease, Dermatitis, Diverticulitis, Encephalitis, Fibromyalgia, Glomerulonephritis, Hepatitis, Hidradenitis suppurativa, HIV/ AIDS, Hypersensitivities, Inflammatory bowel diseases, Crohn’s Disease, Ulcerative Colitis, Interstitial cystitis, Lichen planus, Leukocyte defects, Mast Cell Activation Syndrome, Mastocytosis, Meningitis, Myopathies, Nephritis, Otitis, Parkinson's disease, Pelvic inflammatory disease, pancreatitis, Reperfusion injury, Rheumatic fever, Rheumatoid arthritis, Rhinitis, Sarcoidosis, Transplant rejection, Ulcerative colitis, Vasculitis, Cerebral Amyloid Angiopathy, HCHWA-D, Multiple Sclerosis, and COVID-19.
7. Complex according to any of embodiments 1-6, for use in a drug- delivery involving a passage of a cellular barrier.
8. Complex according to any of embodiments 1-7, wherein the targeting molecule has dissociation constant Kd for the HB-EGF receptor of < 105 mole, preferably < 107% mole.
9. Complex according to any of embodiments 1-8, wherein the at least one targeting molecule and or at least one liposome do not interact with endogencus ligands.
10. Complex according to any of embodiments 1-9, wherein the first constituent is CRM197.
11. Complex according to any of embodiments 1-10, wherein the at least one liposome comprises 5-65 wt.% active compound, preferably 10-60 wt.% active compound, more preferably 20-50 wt.% active compound, even more preferably 30-50 wt.3 active compound, such as 40-45 wt.% active compound, wherein percentages are based on a total weight of the liposome and active compound.
12. Complex according to any of embodiments 1-11, wherein the at least one liposome is selected from SAINT molecules, such as SAINT 18, as well as molecules comprising SAINT-molecules, such as saint-0-Somes.
13. Complex according to any of embodiments 1-12, wherein the at least one water-soluble active compound has a water-solubility of >0.1mole/1, preferably >0.5mole/l, or wherein the at least one lipophilic active compound has a hexane-solubility of >0.1lmole/1, preferably >0.5mole/l.
40 14. Complex according to any of embodiments 1-13, wherein the at
P100537NLOO 38 least one active compound has a molecular weight of < 10 kDa, preferably < 5 kDa, such as < 2 kDa.
15. Complex according to any of embodiments 1-14, comprising a first active compound, wherein the first active compound brings a cell in a transformed state, such as doxotin, doxorubicin, vinblastin, docetaxel, paclitaxel (KICK), and/or comprising a second active compound, wherein the second active compound induces apoptosis of the transformed cell, such as apoptin (KILL).
16. Complex according to any of embodiments 1-15, wherein 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 mRNA, therewith incorporating the active compound in the liposome, such as in a Saint-0O-some (80S), -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 -incubating the extruded liposome to transfer the CRM197- PEG2000-DSPE to the S0S, and optionally -purifying the incubated complex, such as on a Sepharose column, and optionally sterilizing the complex by extrusion, such as through a 0.22 pm filter.
17. At least one dosage comprising a complex according to any of embodiments 1-16.
18. At least one dosage according to embodiment 17 comprising
0.01-1mg complex according to any of embodiments 1-15 per ml cancer, preferably 0.02-0.1 mg/ml, such as 0.04-0.07 mg/ml.
19. At least one dosage according to any of embodiments 17-18 for treatment of a tumour, of a white blood cell disease, of a virus infection, such as a HIV-infection, and of white blood 40 cells, in particular macrophages and monocytes.
P100537NLOO 39
20. At least two dosages according to any of embodiments 17-19, 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.
21. At least two dosages according to embodiment 20, wherein the first active compound is selected from compounds that cause DNA strand breaks, or interact with DNA by intercalation and inhibition of macromolecular biosynthesis, as cytostatic drugs and cytolytic drugs, such as anthracyclines, such as doxotin, and doxorubicin, vinblastin, docetaxel, paclitaxel, and combinations thereof, and/or wherein the second active compound is selected from apoptin, AAP1, AAP2, AAP3, AAP4, AAPS, AAPS, and combinations thereof.
22. Method of applying a dosage according to any of embodiments 17-21 or a complex according to any of embodiments 1-16, further comprising applying localized UV-light, localized X-ray radiation, localized heat-shock, or a combination thereof.
100537 sequence listing v2
SEQUENCE LISTING <110> CRM Therapeutics B.V. <120> Target mediated endocytotic drug delivery <130> 100537NL <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 750 <212> DNA <213> plasmid <400> 1 MetGlyLysSerGluSerGlnMetAspIle ThrAspIleAsnThrProLysProLysLys LysGlnArgTrpThrProLeuGluIleSer LeuSerValLeuValLeuLeuLeuThrIle IleAlaValThrMetIleAlaLeuTyrAla ThrTyrAspAspGlyIleCysLysSerSer 60 AspCysIleLysSerAlaAlaArgLeuIle GlnAsnMetAspAlaThrThrGluProCys ThrAspPhePheLysTyrAlaCysGlyGly TrpLeuLysArgAsnValIleProGluThr SerSerArgTyrGlyAsnPheAspIleLeu ArgAspGluLeuGluValValLeuLysAsp 120 ValLeuGlnGluProLysThrGluAspIle ValAlaValGlnLysAlaLysAlaLeuTyr ArgSerCysIleAsnGluSerAlaIleAsp SerArgGlyGlyGluProLeuLeuLysLeu LeuProAspIleTyrGlyTrpProValAla ThrGluAsnTrpGluGlnLysTyrGlyAla 189 SerTrpThrAlaGluLysAlaIleAlaGln LeuAsnSerLysTyrGlyLysLysValLeu IleAsnLeuPheValGlyThrAspAspLys AsnSerValAsnHisValIleHisIleAsp GlnProArgLeuGlyLeuProSerArgAsp TyrTyrGluCysThrGlyIleTyrLysGlu 240 AlaCysThrAlaTyrValAspPheMetIle SerValAlaArgLeuIleArgGlnGluGlu ArgLeuProIleAspGluAsnGlnLeuAla LeuGluMetAsnLysValMetGluLeuGlu LysGluIleAlaAsnAlaThrAlaLysPro GluAspArgAsnAspProMetLeuLeuTyr 300 AsnLysMetThrLeuAlaGlnIleGlnAsn AsnPheSerLeuGluIleAsnGlyLysPro PheSerTrpLeuAsnPheThrAsnGluIle MetSerThrValAsnIleSerIleThrAsn GluGluAspValValValTyrAlaProGlu TyrLeuThrLysLeuLysProIleLeuThr 360 LysTyrSerAlaArgAspLeuGlnAsnLeu MetSerTrpArgPheIleMetAspLeuVal SerSerLeuSerArgThrTyrLysGluSer ArgAsnAlaPheArgLysAlaLeuTyrGly ThrThrSerGluThrAlaThrTrpArgArg CysAlaAsnTyrValAsnGlyAsnMetGlu 420 AsnAlaValGlyArgLeuTyrValGluAla AlaPheAlaGlyGluSerLysHisValVal GluAspLeuIleAlaGlnIleArgGluVal PheIleGlnThrLeuAspAspLeuThrTrp MetAspAlaGluThrLysLysArgAlaGlu GluLysAlaLeuAlaIleLysGluArgIle 480 GlyTyrProAspAspIleValSerAsnAsp AsnLysLeuAsnAsnGluTyrLeuGluLeu AsnTyrLysGluAspGluTyrPheGluAsn IleIleGlnAsnLeuLysPheSerGlnser LysGlnLeuLysLysLeuArgGluLysVal AspLysAspGluTrpIleSerGlyAlaAla 549 ValValAsnAlaPheTyrSerSerGlyArg AsnGlnIleValPheProAlaGlyIleLeu GlnProProPhePheSerAlaGlnGlnSer AsnSerLeuAsnTyrGlyGlyIleGlyMet ValIleGlyHisGluIleThrHisGlyPhe AspAspAsnGlyArgAsnPheAsnLysAsp 600 GlyAspLeuValAspTrpTrpThrGlnGln SerAlaSerAsnPheLysGluGlnSerGln CysMetValTyrGlnTyrGlyAsnPheSer TrpAspLeuAlaGlyGlyGlnHisLeuAsn GlyIleAsnThrLeuGlyGluAsnIleAla AspAsnGlyGlyLeuGlyGlnAlaTyrArg 660 AlaTyrGlnAsnTyrIleLysLysAsnGly GluGluLysLeuLeuProGlyLeuAspLeu Pagina 1
100537 sequence listing v2 AsnHisLysGlnLeuPhePheLeuAsnPhe AlaGlnValTrpCysGlyThrTyrArgPro GluTyrAlaValAsnSerIleLysThrAsp ValHisSerProGlyAsnPheArgIleIle 720 GlyThrLeuGlnAsnSerAlaGluPheSer GluAlaPheHisCysArgLysAsnSerTyr MetAsnProGluLysLysCysArgValTrp
750
<2105 2 <211> 121 <212> DNA <213> plasmid <400> 2 MetAsnAlaLeuGlnGluAspThrProPro GlyProSerThrValPheArgProProThr SerSerArgProLeuGluThrProHisCys ArgGluIleArgIleGlyIleAlaGlyIle ThrIleThrLeuSerLeuCysGlyCysAla AsnAlaArgAlaProThrLeuArgSerAla 60 ThrAlaAspAsnSerGluSerThrGlyPhe LysAsnValProAspLeuArgThrAspGln ProLysProProSerLysLysArgSerCys AspProSerGluTyrArgValSerGluLeu LysGluSerLeuIleThrThrThrProSer ArgProArgThrAlaLysArgArgIleArg 120 Leu
121 <2105 3 <211> 42 <212> DNA <213> plasmid <400> 3 GluAlaTyrLysAlaAlaGluLysAlaTyr AlaAlalysGluAlaAlalysAlalysAla GluLysLysAlaAlaTyrAlaLysAlaLys AlaAlaLysTyrGluLysLysAlaLysLys AlaAla
42 Pagina 2

Claims (22)

P100537NLOO 40 ConclusiesP100537NLOO 40 Conclusions 1. Complex voor doel-bemiddeld endocytotisch medicijnafgifte omvattend als bestanddelen ten minste één eerste bestanddeel omvattend een doelmolecuul dat in staat is tot interactie met een heparinebindende epidermale groeifactor (HB-EGF), waarbij de HB-EGF-cel receptor in staat is een endosoom in een cel te vormen, en een keten die onder zure omstandigheden een porie kan vormen in een endosomaal membraan, met name een T-keten, bij voorkeur vastgehecht aan het ten minste één doelmolecuul, ten minste een tweede bestanddeel dat een liposoom omvat, waarbij ten minste een liposoom omsluit een hoeveelheid van ten minste één actieve verbinding gekozen uit lipofiele verbindingen, en uit in water oplosbare verbindingen, in het bijzonder uit verbindingen die DNA-bundelbreuken veroorzaken, of die met DNA intercaleren door intercalatie en remming van macromoleculaire biosynthese, zoals cytostatica en cytolytische geneesmiddelen, zoals anthracyclines, zoals doxotine, daunorubicine, en doxorubicine, vinblastine, docetaxel, paclitaxel, apoptine, HIV-tin, amytine, copaxotine, prednitine, lysotine, protectine, en bacteriotine, uit genen of sequenties daarvan, uit eiwitten, en uit RNA-sequenties, bij voorkeur een sequentie met <30 nucleotiden.A complex for target-mediated endocytic drug delivery comprising as components at least one first component comprising a target molecule capable of interacting with a heparin binding epidermal growth factor (HB-EGF), wherein the HB-EGF cell receptor is capable of forming an endosome in a cell, and a chain capable of forming a pore in an endosomal membrane under acidic conditions, in particular a T chain, preferably attached to the at least one target molecule, at least a second moiety comprising a liposome, wherein at least one liposome encompasses an amount of at least one active compound selected from lipophilic compounds, and from water-soluble compounds, in particular from compounds which cause DNA bundle breaks, or which intercalate with DNA by intercalation and inhibition of macromolecular biosynthesis, such as cytostatics and cytolytic drugs, such as anthracyclines, such as doxotine, daunorubicin, and doxorubicin, vinblastine, docetaxel, paclitaxel, apoptin, HIV tin, amytin, copaxotin, prednitine, lysotine, protectin, and bacteriotin, from genes or sequences thereof, from proteins, and from RNA sequences, preferably a sequence with <30 nucleotides. 2. Complex volgens conclusie 1, waarbij het eerste bestanddeel een eiwit (A-deel) omvat met een molecuulgewicht van > 40 kDa, bij voorkeur > 50 kDa, zoals >55 kDa, en bij voorkeur met een molecuulgewicht van < 100 kDa, bij voorkeur < 70 kDa, zoals <65 kDa, en bij voorkeur vastgehecht aan de keten.The complex of claim 1, wherein the first component comprises 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, in preferably <70 kDa, such as <65 kDa, and preferably chain attached. 3. Complex volgens een van de conclusies 1-2, waarbij het doelmolecuul een niet-giftige mutant van een difterietoxine omvat.The complex of any one of claims 1-2, wherein the target molecule comprises a non-toxic mutant of a diphtheria toxin. 4. Complex volgens een van de conclusies 1-3, omvattend als derde bestanddeel een spacer die aan het liposoom en aan het eerste bestanddeel is bevestigd, zoals aan de keten of aan het eiwit, bij voorkeur een spacer met een molecuulgewicht van >400 Da, zoals > 1 kDa, bij voorkeur een PEG-spacer, zoals PEG 2000.Complex according to any one of claims 1 to 3, comprising as a third component a spacer attached to the liposome and to the first component, 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. 5. Complex volgens een van de conclusies 1-4, omvattend 2-100 eerste bestanddelen per tweede bestanddeel, bij voorkeur 10-90 eerste bestanddelen per tweede bestanddeel, bij voorkeur 20-80, zoals 40-70 eerste bestanddelen per tweede bestanddeel.Complex according to any one of claims 1-4, comprising 2-100 first components per second component, preferably 10-90 first components per second component, preferably 20-80, such as 40-70 first components per second component. 40 40 6. Complex volgens een van de conclusies 1-5, voor gebruik bijComplex according to any one of claims 1-5, for use in P100537NLOO 41 de behandeling van kanker, zoals vaste kankersoorten, witte bloedcelziekten, lysosomale stapelingsziekten, virusziekten, transplantatie en ziekten die verband houden met ontstekingen, in het bijzonder Acne vulgaris, allergieën, de ziekte van Alzheimer, Ankylose spondylitis, Astma, Atherosclerose, auto-immuunziekten, zoals coeliakie, diabetes mellitus type 1, Graves! ziekte, inflammatoire darmziekte, multiple sclerose, psoriasis, (reumatoïde) artritis, en systemische lupus erythematosus, auto inflammatoire aandoeningen, zoals familiaire mediterrane, aphtheuze stomatitis, faryngitis en cervicale adenitis, andere auto inflammatoire aandoeningen die geen duidelijke genetische oorzaken hebben zoals onder andere de ziekte van de volwassen- ontsteker, systemische-ontsteking van de jeugd, het Schnitzler- syndroom, en chronisch terugkerende multifocale osteomyelitis, Chronische prostatitis, ziekte van Crohn, Dermatitis, Diverticulitis, Encefalitis, Fibromyalgie, Glomerulonephritis, Hepatitis, Hydradenitis suppurativa, HIV/AIDS, Overgevoeligheden, Ontstekingsverschijnselen, Ziekte van Crohn, Ulceratieve Colitis, Interstitiële cystitis, Lichen planus, Leukocyt defecten, Mastcel Activatie Syndroom, Mastocytose, Meningitis, Myopathie, Nefritis, Otitis, Ziekte van Parkinson, Bekkenontsteking, pancreatitis, Reperfusie-letsel, reumatische koorts, reumatoïde artritis, rinitis, Sarcoïdose, afstoting van transplantaties, colitis ulcerosa, vasculitis, cerebrale amyloïde angiopathie, HCHWA-D, multiple sclerose en COVID-19.P100537NLOO 41 the treatment of cancers such as solid cancers, white blood cell diseases, lysosomal storage diseases, viral diseases, transplantation and diseases related to inflammation, especially Acne vulgaris, allergies, Alzheimer's disease, Ankylosis spondylitis, Asthma, Atherosclerosis, autoimmune disease immune diseases, such as celiac disease, diabetes mellitus type 1, Graves! disease, inflammatory bowel disease, multiple sclerosis, psoriasis, (rheumatoid) arthritis, and systemic lupus erythematosus, autoinflammatory conditions such as familial Mediterranean, aphthous stomatitis, pharyngitis, and cervical adenitis, other autoinflammatory conditions that have no apparent genetic causes such as the adult inflammatory disease, childhood systemic inflammation, Schnitzler syndrome, and chronic recurrent multifocal osteomyelitis, Chronic prostatitis, Crohn's disease, Dermatitis, Diverticulitis, Encephalitis, Fibromyalgia, Glomerulonephritis, Hepatitis, Hydradenitis suppurativa, HIV/AIDS Hypersensitivities, Inflammatory Symptoms, Crohn's Disease, Ulcerative Colitis, Interstitial Cystitis, Lichen planus, Leukocyte Defects, Mast Cell Activation Syndrome, Mastocytosis, Meningitis, Myopathy, Nephritis, Otitis, Parkinson's Disease, Pelvic Inflammation, Pancreatitis, Reperfusion Injury, Rheumatic Fever, rheumatoid arthritis tis, rhinitis, sarcoidosis, transplant rejection, ulcerative colitis, vasculitis, cerebral amyloid angiopathy, HCHWA-D, multiple sclerosis, and COVID-19. 7. Complex volgens een van de conclusies 1-6, voor gebruik in een geneesmiddel-afgifte omvattend een passage van een cellulaire barrière.A complex according to any one of claims 1-6 for use in a drug delivery comprising a passage of a cellular barrier. 8. Complex volgens een van de conclusies 1-7, waarin het doelmolecuul een dissociatieconstante Kd heeft voor de HB-EGF- receptor van < 10% mol, bij voorkeur < 10% mol.Complex according to any one of claims 1-7, wherein the target molecule has a dissociation constant Kd for the HB-EGF receptor of <10% mol, preferably <10% mol. 9. Complex volgens een van de conclusies 1-8, waarin ten minste één doelwitmolecuul en ten minste één liposoom geen interactie vertonen met endogene liganden.The complex of any one of claims 1-8, wherein at least one target molecule and at least one liposome do not interact with endogenous ligands. 10. Complex volgens een van de conclusies 1-9, waarin het eerste bestanddeel CRM197 is.A complex according to any one of claims 1-9, wherein the first component is CRM197. 11. Complex volgens een van de conclusies 1-10, waarin het ten minste één liposoom 5-65 gew.% actieve verbinding omvat, bij voorkeur 10-60 gew.% actieve verbinding, meer bij voorkeur 20-50 40 gew.% actieve verbinding, nog meer bij voorkeur 30-50 gew.%A complex according to any one of claims 1-10, wherein the at least one liposome comprises 5-65 wt% active compound, preferably 10-60 wt% active compound, more preferably 20-50 wt% active compound compound, even more preferably 30-50 wt% P100537NLOO 42 actieve verbinding, zoals 40-45 gew.% actieve verbinding, waarin de percentages betrokken zijn op een totaal gewicht van het liposoom en de actieve verbinding.P100537NL00 42 active compound, such as 40-45% by weight active compound, wherein the percentages are based on a total weight of the liposome and the active compound. 12. Complex volgens een van de conclusies 1-11, waarin het ten minste één liposoom is gekozen uit SAINT-moleculen, zoals SAINT 18, en uit moleculen die SAINT-moleculen omvatten, zoals saint-0- somes.A complex according to any one of claims 1-11, wherein the at least one liposome is selected from SAINT molecules, such as SAINT 18, and from molecules comprising SAINT molecules, such as saint-0-somes. 13. Complex volgens een van de conclusies 1-12, waarin de ten minste één in wateroplosbare werkzame stof een wateroplosbaarheid heeft van >0,1 mol/l, bij voorkeur >0,5mol1/1, of waarin ten minste één lipofiele werkzame stof een hexaanoplosbaarheid heeft van >0,1mol/1, bij voorkeur >0,5mol/1.A complex according to any one of claims 1-12, wherein the at least one water-soluble active ingredient has a water solubility of >0.1 mol/l, preferably >0.5 mol/l, or wherein at least one lipophilic active ingredient has a hexane solubility of >0.1mol/l, preferably >0.5mol/l. 14. Complex volgens een van de conclusies 1-13, waarin de ten minste een actieve verbinding een molecuulgewicht van < 10 kDa heeft, bij voorkeur < 5 kDa, zoals < 2 kDa.A complex according to any one of claims 1-13, wherein the at least one active compound has a molecular weight of < 10 kDa, preferably < 5 kDa, such as < 2 kDa. 15. Complex volgens een van de conclusies 1-14, omvattend een eerste actieve verbinding, waarin de eerste actieve verbinding een cel in een getransformeerde toestand brengt, zoals doxotine, doxorubicine, vinblastine, docetaxel, paclitaxel (KICK), en/of omvattend een tweede actieve verbinding, waarin de tweede actieve verbinding apoptose van de getransformeerde cel induceert, zoals apoptine (KILL).A complex according to any one of claims 1-14, comprising a first active compound, wherein the first active compound brings a cell into a transformed state, such as doxotin, doxorubicin, vinblastine, docetaxel, paclitaxel (KICK), and/or comprising a second active compound, wherein the second active compound induces apoptosis of the transformed cell, such as apoptin (KILL). 16. Complex volgens een van de conclusies 1-15, waarin het ten minste één liposoom is verkregen door het reageren met een spacer in een aprotische oplossing, zoals van Mal-PEG2000-DSPE, DSPE-PEG2000, SAINT-C-18, POPC en cholesterol in een molaire verhouding van 1:4:18:37:40 in chloroform:methanol (9:1, v/v) daarmee een lipidenmengsel verschaffend, het drogen van het lipidenmengsel, zoals onder gereduceerde stikstof druk, hydratatie van het gedroogde lipidenmengsel, zoals in buffer, zoals bij een pH van 6-9, in het bijzonder pH 6,7, waarbij de buffer een actieve verbinding omvat, zoals Apoptine, een gen of mRNA, waarbij de actieve verbinding in het liposoom wordt opgenomen, zoals in een Saint-O-some (SOS), het op grootte brengen van het verkregen liposoom, bijvoorbeeld door extrusie door polycarbonaatfilters, bijvoorbeeld met een poriegrootte van 60-100 nm, zoals 80 nm, bij voorkeur met 40 behulp van een hogedruk extrudeerder, enA complex according to any one of claims 1-15, wherein 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) thereby 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 at pH 6-9, especially pH 6.7, wherein the buffer comprises an active compound, such as Apoptin, a gene, or mRNA, wherein the active compound is incorporated into the liposome, such as in a Saint-O-some (SOS), sizing the resulting liposome, e.g. by extrusion through polycarbonate filters, e.g. with a pore size of 60-100 nm, such as 80 nm, preferably using a high pressure extruder, and P100537NLOO 43 het incuberen van het geëxtrudeerde liposoom om de CRM197- PEG2000-DSPE over te brengen naar de S05, en eventueel het zuiveren van het geïncubeerde complex, zoals op een Sefarose-kolom, en optioneel steriliseren van het complex door middel van extrusie, zoals door een 0,22 um filter.P100537NL00 43 incubating the extruded liposome to transfer the CRM197-PEG2000-DSPE to the SO5, optionally purifying the incubated complex, such as on a Sepharose column, and optionally sterilizing the complex by extrusion, such as through a 0.22 µm filter. 17. Ten minste één dosering die een complex omvat volgens een van de conclusies 1-16.At least one dosage comprising a complex according to any one of claims 1-16. 18. Ten minste één dosering volgens conclusie 17 omvattend uit een complex van 0,01-1mg volgens een van de conclusies 1-15 per ml kanker, bij voorkeur 0,02-0,1 mg/ml, zoals 0,04-0,07 mg/ml.At least one dosage according to claim 17 comprising a complex of 0.01-1mg according to any one of claims 1-15 per ml cancer, preferably 0.02-0.1 mg/ml, such as 0.04-0 .07 mg/ml. 19. Ten minste één dosering volgens een van de conclusies 17-18 voor de behandeling van een tumor, van een witte bloedcelziekte, van een virusinfectie, zoals een Hiv-infectie, en van witte bloedcellen, in het bijzonder macrofagen en monocyten.At least one dosage according to any one of claims 17-18 for the treatment of a tumor, of a white blood cell disease, of a viral infection, such as an HIV infection, and of white blood cells, in particular macrophages and monocytes. 20. Ten minste twee doseringen volgens een van de conclusies 17- 19, waarbij ten minste een eerste dosering een eerste actieve verbinding omvat die een cel in een getransformeerde toestand kan brengen, en ten minste een tweede dosering die een tweede actieve verbinding omvat voor het induceren van apoptose in de getransformeerde cel.At least two dosages according to any one of claims 17-19, wherein at least a first dosage comprises a first active compound capable of bringing a cell into a transformed state, and at least a second dosage comprising a second active compound for induce apoptosis in the transformed cell. 21. Ten minste twee doseringen volgens de conclusie 20, waarin de eerste actieve verbinding is gekozen uit verbindingen die DNA- bundelbreuken veroorzaken, of met DNA intercaleren door intercalatie en reming van macromoleculaire biosynthese, zoals cytostatica en cytolytische geneesmiddelen, zoals anthracyclines, zoals doxotine, en doxorubicine, vinblastine, docetaxel, paclitaxel en combinaties daarvan, en/of waarbij de tweede werkzame stof is gekozen uit apop-tin, AAPl, AAP?, AAP3, AAP4, AAP5, AAPS, en combinaties daarvan.At least two dosages according to claim 20, wherein the first active compound is selected from compounds that cause DNA bundle breaks, or intercalate with DNA by intercalating and inhibiting macromolecular biosynthesis, such as cytostatics and cytolytic drugs, such as anthracyclines, such as doxotine, and doxorubicin, vinblastine, docetaxel, paclitaxel and combinations thereof, and/or wherein the second active agent is selected from apoptin, AAP1, AAP™, AAP3, AAP4, AAP5, AAPS, and combinations thereof. 22. Werkwijze voor het toepassen van een dosering volgens een van de conclusies 17-21 of een complex volgens een van de conclusies 1-16, verder omvattend uit het toepassen van gelokaliseerd Uv- licht, gelokaliseerde röntgenstraling, gelokaliseerde hitteschok, of een combinatie daarvan.A method of applying a dosage according to any one of claims 17-21 or a complex according to any one of claims 1-16, further comprising applying localized UV light, localized X-rays, localized heat shock, or a combination thereof. .
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