PKC INHIBITORS FOR THE TREATMENT OF SEPTIC CHOLESTASIS WITH CTM TARGETING

Abstract

The invention relates to inhibitors of the PKC signaling pathway for use in the treatment of septic cholestasis, wherein the inhibitors are targeted into the liver by a selective nanostructured delivery system, wherein the selective nanostructured delivery system comprises at least one carbohydrate targeting moiety and at least one polymer and/or at least one lipid and/or at least one virus-like particle.

Claims

1.-13. (canceled)

14. A nanostructured delivery system, comprising: an inhibitor of a protein kinase C (PKC) signaling pathway; at least one carbohydrate targeting moiety; and a carrier, wherein the nanostructured delivery system is adapted for treating septic cholestasis by lowering or inhibiting PKC in liver cells.

15. The nanostructured delivery system of claim 14, wherein the carbohydrate targeting moiety is covalently attached to the carrier.

16. The nanostructured delivery system of claim 15, wherein the inhibitor of the PKC signaling pathway is encapsulated by the carrier.

17. The nanostructured delivery system of claim 14, wherein the inhibitor of the PKC signaling pathway is covalently attached to the carbohydrate targeting moiety or to the carrier.

18. The nanostructured delivery system of claim 14, wherein the at least one carbohydrate targeting moiety is selected from the group of N-acetyl-galactosamine (GalNAc), galactose, lactose, mannose, glucosamine, asialofetuin, pullulan, arabinogalactan, glycyrrhizin, glycyrrhetinic acid, or any derivative thereof.

19. The nanostructured delivery system of claim 14, wherein the at least one carbohydrate targeting moiety is adapted to bind to a recognizing unit located on the liver.

20. The nanostructured delivery system of claim 19, wherein the recognizing unit is a receptor.

21. The nanostructured delivery system of claim 20, wherein the receptor is a lectin, an asialoglycoprotein receptor (ASGPR) or an aka Ashwell-Morell receptor.

22. The nanostructured delivery system of claim 14, wherein the inhibitor of the PKC signaling pathway is selected from the group of a PKC inhibitor, a PI3 kinase inhibitor, a MAPK inhibitor, a PLC inhibitor, a DAG level reducing agent, siRNA, shRNA, miRNA, a modified oligo analogue, an antisense construct, or RNAse H.

23. The nanostructured delivery system of claim 22, wherein the inhibitor of the PKC signaling pathway is selected from the group of a bisindolylmaleimide, a staurosporine, a midostaurin, UCN-01, sotrastaurin, enzastaurin, ruboxistaurine, tivantinib, enzastaurin, Go 6983, K252a, ANA-12, lestaurtinib, stauprimide, CEP-701, Arcyriaflavin a, or Bisindolylmaleimids 1-XII aka BIM 1-XII.

24. The nanostructured delivery system of claim 22, wherein the inhibitor of the PKC signaling pathway is a PI3 kinase inhibitor, the PI3 kinase inhibitor being selected from the group of copanlisib, idelalisib, wortmannin derivatives, bryostain derivatives, taselisib, omipalisib, AS605240, GSK1059615, buparlisib, alpelisib, pictilisib, serabilisib, dactolisib, dihydrosphingosine, calphostin C, or melittin.

25. The nanostructured delivery system of claim 14, wherein the inhibitor of the PKC signaling pathway directly or indirectly inhibits or reduces an activity of PKC or PKC subtypes.

26. The nanostructured delivery system of claim 14, wherein the carrier includes at least one polymer, or at least one lipid, or at least one virus-like particle, or any combination thereof.

27. The nanostructured delivery system of claim 26, wherein the carrier includes at least one polymer, the at least one polymer being an organic polymer, an inorganic polymer, a hydrophobic polymer, a hydrophilic polymer, an amphiphilic polymer, an anionic polymer, a cationic polymer, or any combination thereof.

28. The nanostructured delivery system of claim 27, wherein the at least one polymer is selected from the group of a polyester, a polyacrylate, a polystyrene, a polyamide, a polyurethane, a polyacrylonitrile, a polytetrafluoroethylene, a silicone, a polyethylene glycol, a polyethylene oxide, a polyoxazoline, a polysaccharide, or any copolymer thereof, or any combination thereof.

29. The nanostructured delivery system of claim 27, wherein the at least one polymer is selected from the group of PLGA, PLA, PCL, PGA, PDMAEMA, PMMA, PMAA, PEI, PEtOx, PEG, HPMA, APMA, PVP, hydrolyzed PVP, or any combination thereof.

30. The nanostructured delivery system of claim 26, wherein the carrier includes at least one lipid, the at least one lipid being selected from the group of a saturated fatty acid, an unsaturated fatty acid, a cholesterol derivative, a phospholipid, a sphingolipid, a lipoprotein, a glycolipid, or any combination thereof.

31. The nanostructured delivery system of claim 26, wherein the carrier includes at least one virus-like particle, the at least one virus-like particle being derived from a virus selected from the group of Bacteriophage MS2, Bacteriophage Ob, Enterobacteria phage P22, Cowpea mosaic virus (CPMV) Cowpea Chlorotic Mottle Virus (CCMV), hepatitis B virus carries (HBVc), or Adeno associated virus (AAV).

32. The nanostrucured delivery system of claim 14, wherein the carbohydrate targeting moiety is a multivalent construct.

33. The nanostructured delivery system of claim 14, wherein the inhibitor of the PKC signaling pathway includes a PI3-kinase inhibitor, the at least one carbohydrate targeting moiety is a trivalent construct, and the carrier is a nanostructured polymer.

Description

[0076] The invention will be illustrated in more detail with reference to the Figures, which not have to be understood to limit the scope of the invention.

[0077] FIG. 1 shows a schematic representation of different inventive constructs; monovalent and multivalent (at present trivalent) CTM on ligand or ligand construct (also nanostructured carrier). Attachment is achieved via a spacer or a linker moiety.

[0078] FIG. 2 shows examples for lectine-binding moieties useful for hepatocyte targeting. “R” represents a possible connection point for the delivery system (polymer, virus-like particle, lipid or a genetic construct).

[0079] FIG. 3 demonstrates a general synthetic approach for the synthesis/attachment of lectin-binding carbohydrate moieties to agents, agent constructs, carrier polymers, virus-like particles or linkers.

[0080] FIG. 4 demonstrates exemplified methods to introduce and/or change functional groups for the attachment of an agent/agent construct, polymers and targeting moieties.

[0081] FIG. 5 shows strategies for the direct coupling of nucleic acid material to a CTM. FIG. 5A: 3′EndLabeling strategy mainly for DNA-like constructs; FIG. 5B: 5′EndLabeling strategy for DNA, RNA or modified nucleotides.

[0082] FIG. 6 demonstrates examples for the connection strategies between agent, or agent construct with polymer and/or targeting moiety and/or linker.

[0083] FIG. 7 shows an exemplary building block to generate/prepare various different nanostructured delivery systems, useful for the treatment of septic cholestasis. FIG. 7a shows a variety of potential compounds, which reduce PKC activity (A); FIG. 7b shows some carbohydrate targeting moieties (CTMs) (B); FIG. 7c shows examples for targeted nanostructured delivery systems (C).

[0084] FIG. 8 shows the synthetic route to carboxy- and amine-functionalized GalNAc (CTM1) derivatives. For the design of monomeric carbohydrate-based targeting units, the synthetic route to GalNAc derivatives is shown; basically this scheme can be adopted to other carbohydrate derivatives. The carboxy-terminated CTM can be coupled to any amine-terminated carrier, polymer, lipid, protein or drug, whereas the amine-terminated CTM (CTM1) can be coupled to any carrier, polymer, lipid, protein or drug via regular peptide coupling, known for a skilled person (e.g. EDC/NHS); exemplified in FIG. 11.

[0085] FIG. 9 shows a scheme for the synthesis of a trivalent Gal-NAc construct with maleimide linker to couple the construct to a thiol group as shown in FIG. 5.

[0086] FIG. 10 shows a scheme for the synthesis of an amino-terminated trivalent GalNAc construct.

[0087] FIG. 11 shows the coupling of amino-terminated GalNAc (CTM1) to the terminal carboxylic acids of PLGA.

[0088] FIG. 12 demonstrates methods for the preparation of nanoparticles by emulsion, double emulsion and nanoprecipitation. FIG. 12A: Emulsion and double emulsion; FIG. 12B: Nanoprecipitation.

[0089] FIG. 13 shows toxicity of targeted nanoparticles vs free drugs (BIM-1, midostaurin and AS605240) in L929 mouse fibroblasts.

[0090] FIG. 14 shows Kaplan-Meier-Schatzer plots indicating the survival of mice in a peritoneal contamination and infection (PCI) model using two different batches of stool.

[0091] FIGS. 15, 16 and 17 show the survival rates of mice treated with PKC activity lowering compounds in Kaplan-Meier-Schatzer plots. The figures show the effects of the drug and the targeted nanostructured particles on healthy animals (sham) and on animals with PCI.

[0092] The carbohydrate moiety-triggered endocytosis according to the invention can be adopted for the tissue specific transport of the agent. To this end, the agent of interest is coupled with a linker/spacer, which contains the ASGPR-specific recognition ligand or ligand construct. According to the invention, the inhibitors of the PKC signaling pathway are coupled either directly or with a spacer comprising the ASGRP-specific recognition ligand as shown in FIG. 1 or ligand construct to the polymer or a nanostructured delivery system (i.e. polymer particle).

[0093] The ASPGR-specific recognition ligand might be GalNAc or another liver-specific lectin recognition ligand as shown in FIG. 2. These recognition ligands are responsible for the targeted delivery and cell/tissue/organ specificity of the agent, agent construct or carrier. As outlined in FIG. 2, such recognition ligands preferably are derivatives of carbohydrates. They are useful for hepatocyte targeting. The shown molecules are preferred for the construction of a CTM, but also larger molecules like pullulan- or arabinogalactan derivatives (as shown in FIG. 7b) could be used.

[0094] To couple the ASPGR-specific carbohydrate based recognition moiety to a drug (agent), drug construct (agent construct) or carrier, different approaches can be applied. Depending on the functional groups present in the respective drug, drug construct or inventive nanostructured delivery system and the respective recognition ligand, the most suitable method has to be evaluated. In case of carbohydrate derivatives, suitable leaving groups (e.g. acetates) are preferably further activated by TMS-OTf or HBr and subsequently substituted by various nucleophiles, like alcohols, amines, thiols or C-nucleophiles as demonstrated in FIG. 3.

[0095] In case, the direct coupling is difficult due to a lack of suitable connection points, suitable functional groups are preferably introduced according to generally known functional group interconversion methods as shown in FIG. 4 to link the agent/agent construct, or nanostructured delivery system to the carbohydrate targeting moiety. FIG. 4 shows the interconversion of carboxylic acid to amine, alcohol to carboxylic acid and alcohol to maleimide. The carboxylic acids are suitable for coupling with amines and vice versa, whereas maleimides can be coupled to thiols.

[0096] The carbohydrate targeting moiety (e.g., ASPGR-recognition moiety) can either be attached directly, or via additional spacers to increase the distance between targeting moiety and the agent/agent construct, polymer and/or delivery system. For the preparation of Gal-NAc PLGA (CTM1-PLGA), useful as nanocarrier, an exemplified synthesis is shown in FIG. 11. Further disclosure is given in Example 3. GalNAc labelled PLGA (FIG. 11 and Example 3) is useful for the encapsulation of the PKC-inhibiting agent preferably by nanoprecipitation as, for example, described in Example 4, emulsion or double emulsion.

[0097] Alternatively, the carbohydrate targeting moiety CTM (here GalNAc) can be coupled to the final particle (nanostructured delivery system) after encapsulation of the inhibitor of the invention. In this case, the inhibitor of the PKC signaling pathway is encapsulated accordingly into a non-targeted carrier system. After the preparation of the nanoparticle, the functional groups in the polymer are activated and coupled to the CTM analogously to the coupling as shown in FIG. 11. Depending on the functional groups on the polymer and the drug used, different coupling strategies can be applied. Such coupling strategies are well-known in the art; preferred coupling strategies usable according to the invention are shown in FIG. 6.

[0098] This approach can be adopted to small molecules, nucleic acid constructs, like si-RNA, or inventive carriers, such as liposomes or nanoparticles, either organic or inorganic. Methods to be used are known in the art and described, for examples, in Huang, Mol. Ther. Nucl. Acids, 2017, Preclinical and Clinical Advances of GalNAc-decorated Nucleic Acid Therapeutics Molecular Therapy. Nucleic Acids Vol. 6, 2017, p.116 or in Ahmed and Narain, Carbohydrate-based materials for targeted delivery of drugs and genes to the liver, Nanomedicine (Lond.) 2015, 10 (14), 2263-2288.

[0099] The carbohydrate targeting moiety (CTM) according to the invention is preferably attached to the polymeric moiety (polymer or virus like particle) of the nanostructured delivery system, but can also be directly attached to the inhibitor of the PKC signaling pathway before the formation of the nanostructured delivery system. For example, a carbohydrate targeting moiety comprising a maleimide functional group is attached to the nucleic acid construct by known labelling methods like 3′ or 5′EndTAG™ For both methods, the preferable functional group at the CTM is the maleimide, which can be generated as shown in FIG. 9. The two EndTAG coupling strategies to selectively couple nucleic acid constructs to the carbohydrate targeting moiety are shown in FIG. 5. FIG. 5A shows 3′EndLabeling strategy mainly for DNA-like constructs; FIG. 5B shows 5′EndLabeling strategy for DNA, RNA or modified nucleotides. The targeted nucleic acid constructs can be used to form polyplexes with polymers (organic or inorganic) in order to generate a nanostructured delivery system.

[0100] FIG. 6 demonstrates examples for binding strategies for agent/agent constructs with the polymer or targeting moiety according to the invention. The inventive carbohydrate driven targeting moiety selective liver targeting moiety can be attached to the agent or agent construct of the invention by regular chemical coupling reactions as referred to above. For the coupling reaction, all the reactions, well known for a skilled chemist can be applied. In a preferred embodiment, reactive carbonyl compounds, preferably ketones, aldehydes acetals or hemiacetals with amines to form a Schiff-base, which can be reduced to a corresponding amine are used as shown in FIG. 6.

[0101] The carbohydrate targeting moiety comprises a chemical moiety, which is recognized by certain recognition unit, preferably a lectin, on the surface of the target tissue, preferably the liver. A preferred lectin for the recognition comprises the ASGPR and GalNAc constructs as carbohydrate targeting moiety. Furthermore, galactose-terminated glycoproteins, arabinoglycans, pullulans and sitosterol glycosides (aka sitoG) are useful as lectin recognition constructs. In FIG. 7b some representative carbohydrate targeting moieties are shown.

[0102] FIG. 7a-c shows an exemplary building block to prepare various different nanostructured delivery systems according to the invention, which are useful for the inventive treatment of septic cholestasis by reducing the PKC activity. FIG. 7a shows different PKC-activity reducing agents, which might be used in the building block. These PKC-activity reducing agents as well as small molecules and also nucleic acid constructs can be used according to the invention. FIG. 7b shows different preferred carbohydrate targeting moieties (CTM), which can be used according to the invention are shown. Monovalent, trivalent and multivalent. “R” represents the connection points to the agent/agent construct or polymer. Possible chemical bindings are shown in FIG. 6. The setup of the trivalent construct is only exemplary and the chains might also contain PEGs, amides, triazols or other moieties and the length of the chains can vary between 2 and 30 atoms. FIG. 7c shows different delivery systems that carry the carbohydrate targeting moiety (shown as asterisks). On top, a polymer (organic or inorganic), lipid, virus-like particle is shown, which can act as a vehicle for the targeted drug delivery. Basically, the CTM can be linked to small molecules, nucleic acid constructs and also polyplexes between nucleic acid construct and a positively charged polymer. Such positively charged polymers can also be labelled with a carbohydrate targeting moiety (CTM) of the invention and hence also form after ligation to nucleic acid constructs, a targeted nanostructured delivery system by themselves. Such targeted nanostructured delivery systems are preferably formed, if the CTM is directly bound to the inventive inhibitors of the PKC signaling pathway (preferably nucleic acid constructs) and these constructs form a nanostructured delivery system (with or without helper polymer).

[0103] In order to mimic the septic cholestasis, a systemic inflammation was induced using the well-established peritoneal contamination and infection (PCI) model. In this model, a human faeces suspension is applied intraperitoneally (ip.) and rapidly triggers sepsis with liver dysfunction. For each batch of human stool, the dose is titrated carefully for a survival between 0% and 20% within two weeks.

[0104] To find the adequate dose of stool, different doses were tested. 6 h after i.p. application of the stool, 8-12 weeks old C57/BL6 mice or FVB/N mice were treated with the nanoparticles or the free drug respectively. FIG. 14 shows the survival of mice with two different batches that were used in a Kaplan-Meier-Schatzer plot.

[0105] FIGS. 15, 16 and 17 show the survival rates of mice treated with PKC activity lowering compounds in Kaplan-Meier-Schatzer plots. The figures show the effects of the drug and the targeted nanostructured particles on healthy animals (sham) and on animals with PCI. FIG. 15 shows the effect of the PKC inhibitor BIM-1 as free drug and as cargo of a targeted nanoparticle formulation.

[0106] FIG. 16 shows the effect of the PI3-kinase inhibitor AS605240 as free drug and as cargo of a targeted nanoparticle formulation.

[0107] FIG. 17 shows the effect of the PKC inhibitor midostaurin as free drug and as cargo of a targeted nanoparticle formulation.

[0108] The invention is further demonstrated below on the basis of Examples, although it is not limited thereto.

EXAMPLES

Example 1

Synthesis of Precursors for the Synthesis of CTM (Here GalNAc Constructs)

[0109] The fully acylated Gal-NAc 1 (1.0 mmol) was activated with TMS-OTf (0.7 mmol) in 5 mL DCM with 4 Å molecular sieves (375 mg) for 16 h at rt in the presence of the CBZ-protected aminohexanol 2 (0.9 mmol), after aqueous workup and recrystallization with EtOAc, the chain-functionalized carbohydrate (3) was obtained with 85% yield. This product 3 (0.85 mmol) was treated with (0.08 mmol) 25%-NaOMe-solution in 5 mL MeOH. After stirring with Amberlite-resin (500 mg) for 1 hour, filtration and solvent removal, all the acyl protected hydroxyl-groups were fully deprodected in quantitative yield. The CBZ-amine 4 (0.85 mmol) was deprotected by catalytic hydrogenation under atmospheric hydrogen with 20% Pd/C (20 mg) in 5 mL MeOH. After filtration and solvent removal, the desired product 5 was obtained, quantitatively.

Example 2

Synthesis of Trivalent Gal-NAc Constructs

[0110] A: Maleimide Functionalized Trivalent Gal-NAc for Direct Coupling to Nucleic Acid Constructs via Introduced SH Groups. (EndTAG® Labeling)

[0111] As outlined in FIG. 9, aminotriester (1 g) is dissolved in 10 mL of DMF, 5 eq. of HBTU and DIEA are added at rt. Under nitrogen, 1 eq. 5-maleimido valeric acid is added and stirred for 24 h at rt. The reaction mixture is poured into 250 mL of 10% NaHCO.sub.3 and extracted three times with ethyl acetate. The combined organic phases are evaporated to dryness and dissolved and stirred for 24 h in 25 mL DCM, containing 1M TFA. After evaporation of the solvents, the residue is dissolved in 300 mL of acetone and 3.5 eq of NaOMe are added. The precipitating compound is filtered off, dried and used without further purification. 1 g of the tri-sodium salt is dissolved in water, acidified to pH2 and extracted 3 times with chloroform. The pooled organic phases are evaporated to a final volume of 50 mL and to the resulting tri-acid 5 eq. of Pfp-TFA and 20 eq. of DIEA are added and the reaction mixture is stirred for 2 h. After the reaction, the mixture is poured into 500 mL of water, extracted 3 times with 200 mL of EtOAc, washed with brine, separated, dried over MgSO.sub.4 and evaporated to dryness. The resulting gum is crystallized from hexane/ethyl acetate to give a beige solid.

[0112] Tris-Pfp ester is dissolved in THF and 5 eq. of the amino-GalNac monomer are added and stirred for 20 min. The reaction mixture is filtered off and evaporated to dryness. The resulting oil is subjected to column chromatography using CHCl.sub.3/MeOH 9:1 to yield the final trivalent GalNAc maleimide construct (detection with KMnO.sub.4 or conc. H.sub.2SO.sub.4). The synthesis scheme is shown in FIG. 9.

[0113] Direct Coupling of Genetic Material-Based Inhibitors to a Carbohydrate Targeting Moiety (EndTag®)

[0114] According to vectorlabs ®, 1 μg PKC-siRNA (custom made by JenaBioscience) is incubated with T4 polynucleotide kinase and ATPγS in reaction buffer for 30 min at 37° C. The reaction is purified with a ThermoFischer RNA purification kit and stored carefully, as it is necessary for RNA. (low temperature, sterile and RNAse free!). The activated siRNA is then suspended in 50 μL of PBS buffer and 1 μg of trivalent Gal-NAc maleimide is added and shaken for 30 min at 65° C. The final construct is purified again under sterile conditions with a ThermoFischer RNA purification kit.

[0115] B: Amine Functionalized Trivalent Gal-NAc for Coupling to Carboxylic Acid Derivatives (Here PLGA)

[0116] As outlined in FIG. 10, aminotriester 10 (1.00 mmol) was dissolved in 10 mL DCM and HBTU (5.00 mmol), DIEA (5.00 mmol) were added at rt. Under nitrogen, CBZ-5-amino valeric acid 15 (5.00 mmol) was added and stirred for 24 h at rt. The reaction mixture was poured into of 10% NaHCO.sub.3— aqueous solution 250 mL and extracted three times with ethyl acetate. The combined organic phases were evaporated to dryness. The residue was dissolved in 25 mL toluene, containing 1 mL phosphoric acid and stirred for 15 h, after aqueous workup, the combined organic phases, were evaporated. The product 16 was dissolved in 20 mL DMF, 5 eq. of Pfp-TFA and 20 eq. of DIEA were added to the solution. After 16 h stirring at rt the reaction mixture was quenched by a sat. NH.sub.4Cl-solution (10 mL) and extracted three times with DCM. After solvent removal, the residue was purified by flash column chromatography using n-Hexane/EtOAc 3:1 as eluent, to yield the Tris-Pfp ester 17 (Rf=0.27).

[0117] Tris-Pfp ester 17, 4 eq. of EDC.HCl, 0.1 eq. DMAP were dissolved in 15 mL DCM. After 1 h reaction time 3.5 eq. of the fully acylated carbohydrate with the amine-chain 3 was added to the reaction mixture and stirred for 12 h at rt. The reaction mixture was quenched by addition of 5 mL water. After aqueous workup and solvent removal, the resulting oil was subjected to column chromatography using CHCl.sub.3/MeOH 9:1 as eluent to yield the fully protected trivalent GalNAc construct 18.

[0118] This product 18 (0.85 mmol) was treated with (0.08 mmol) 25%-NaOMe-solution in 5 mL MeOH. After stirring with Amberlite-resin (500 mg) for 1 hour, filtration and solvent removal, all the acyl protected hydroxyl-groups were fully deprotected in quantitative yield. The crude product was subjected to the next step without further purification. The CBZ-group was cleaved off by catalytic hydrogenation under atmospheric hydrogen with 20% Pd/C (20 mg) in 5 mL MeOH. After filtration and solvent removal, the desired product 19 was purified by semiprep. C18 RP-HPLC with acetonitrile, containing 0.1% of TFA as eluent.

Example 3

Coupling of an Amino-Functionalized Gal-NAc (CTM1) to PLGA as a Delivery System

[0119] PLGA (Resomer RG 502 H, MW: 12.000, 100 mg, 8.33 μmol) was dissolved in 300 μL DMSO. 60 μl (1. eq., 8.33 μmol) of an EDC.HCl-solution (26.8 mg, dissolved 1.00 mL DMSO) and 60 μL (1. eq., 8.33 μmol) of an NHS-solution (17.0 mg, in 1.00 mL DMSO) were added to this solution, successively. After stirring for 3 h at rt. The solution was poured into a DMSO solution of CTM1 (5) (16 mg, 6.0 eq., 49.97 μmol, in 300 μL DMSO). The solution was stirred for 16 h. Triethylamine (10 μL, 16 eq., 121.55 μmol) was added to this solution. After 3 h glacial acetic acid (12 μL, 27 eq., 223.81 μmol) was added to neutralize the solution. After 5 min. the solution was poured into water (25 mL). The precipitate was washed serval times with water and lyophilized. The CTM1-labelled PLGA was used for a nanoprecipitation procedure or an emulsion procedure for the encapsulation of an appropriate agent. In case of nucleic acid derivatives, a polyplex consisting of PEI or any other basic polymers is formed. Then the encapsulation is performed via double emulsion into the Gal-NAc PLGA. The coupling of CTM1 to PLGA is outlined in FIG. 11.

Example 4

Preparation of Nanoparticles

[0120] After functionalization of the polymer with the carbohydrate targeting moiety (see Example 5), nanoparticles were produced by nanoprecipitation using polyvinylalcohol (PVA) as surfactant. The polymer and the PKC inhibitor BIM-1 or midostaurin or the PI3K inhibitor AS 605240 were dissolved in DMSO and the solution was slowly dropped into a vigorously stirred aqueous 0.3% PVA solution. The formed nanoparticles contain 4wt % of BIM-1, 6wt % of midostaurin or 10 wt % of AS 605240, encapsulated in the GalNAc-targeted (CTM1) PLGA. The solution was purified and concentrated by cross-flow filtration. Methods for the preparation of inventive nanoparticles by emulsion, double emulsion and nanoprecipitation is further exemplary illustrated in FIG. 12.

[0121] To proof the cell/tissue targeting, neutral-lipid orange (DYOMICS) is encapsulated instead of the PKC reducing agent, with an identical procedure. The evaluation and visualization of the hepatocyte targeting is performed according to the intravital microscopic methods of WO2015/035974, the disclosure of which is herewith fully referred to and incorporated.

Example 5

Characterization of Inventive Nanoparticles

[0122] Nanoparticles of Gal-NAc-PLGA were produced with constant parameters and reproduced according to the protocol as follows: [0123] Size: Measurement of the size of the various nanostructured delivery systems dissolved in deionized water by dynamic light scatter (for example, Zetasizer (Malvern Instruments GmbH)) or by electron micrographs. [0124] Shape: Determination of shape by electron micrographs. [0125] Charge: Measurement of the various nanostructured delivery systems dissolved in deionized water using a Zetasizer (Malvern Instruments GmbH) by determining the electrophoretic signal (zeta potential, surface charge). [0126] Endotoxins: Endotoxin content was determined with a Charles River test kit basing on the LAL chromogenic assay according to D. E. Guilfoyle, et al., Evaluation of a chromogenic procedure for use with the Limulus lysate assay of bacterial endotoxins drug products, J Parenter Sci Technol, 1985, 39(6): pp. 233-6. [0127] Hemolysis: Measurement of the hemoglobin concentration of erythrocytes which were incubated with the particles in physiological buffer for one hour. The measurable hemoglobin concentration in the supernatant increases when there is damage to the erythrocyte membrane. [0128] Aggregation: Measurement of the absorption of erythrocytes incubated with the polymers in physiological buffer. Samples with cell aggregates show a lower absorption than homogeneously distributed non-aggregated cells.

[0129] Results:

[0130] A: Untargeted nanoparticles (PLGA/PVA) with 2.5% encapsulated Neutral-lipid orange

[0131] B: CTM1-targeted nanoparticles from example 4 with 4% BIM-1 (PKC inhibitor)

[0132] C: CTM1-targeted nanoparticles from example 4 with 10% AS605230 (P13 kinase inhibitor)

[0133] D: CTM1-targeted nanoparticles from example 4 with 7% midostaurin (PKC inhibitor)

TABLE-US-00001 TABLE 1 A B C D Size [nm] 80 72 93 185 PDI 0.13 0.14 0.21 0.18 Zeta potential −12 −0.2 −2 −1

Example 6

Static Macrophage Assay and Dynamic Chip Based Microfluidic Model for Hepatocyte Targeting and Interaction with Macrophages

[0134] The Macrophage assay was used to investigate if any unwanted uptake and/or effect of nanoparticles by macrophages occur. Interactions between NPs and macrophages can seriously reduce the efficacy of NPs. In addition, interaction can result in activation of macrophages, thereby harming the surrounded tissue, after all the host. Therefore, the interaction between NPs and macrophages should be proven first. Particle size, shape and coating and surface charge are critical determinants. Two assays were performed under static conditions:

[0135] A. Human Peripheral Blood Mononuclear Cell (PBMC) Culture and Macrophage Differentiation

[0136] PBMCs were freshly isolated immediately after collecting donor blood from healthy volunteers. The donors were informed about the aim of the study and gave written informed consent. Blood sample volume was diluted in a ratio 1:1 with PBS without calcium and magnesium (Biochrom AG, Germany) containing 0.1% bovine serum albumin (BSA, Carl Roth, Germany) and 2 mM EDTA (Sigma-Aldrich, Germany; isolation buffer) and carefully laid on top of Biocoll separating solution (Biochrom AG, Germany). PBMCs were obtained from density gradient centrifugation. The cells were washed subsequently in isolation buffer for several times and were finally strained by a 40 μm molecular mesh (BD Bioscience, Germany). For monocyte enrichment 10.sup.7 PBMCs per well (9.6 cm.sup.2) were plated on a six well plate (or in smaller wells with comparable cell density) in 2 mL X-VIVO 15 (Lonza, Germany) supplemented with 10% autologous serum, 10 ng/mL GM-CSF (PeproTech, Germany), 100 units/mL penicillin, and 100 μg/mL streptomycin (Life Technologies, Germany). The cells were washed with plain X-VIVO 15 medium after 3 h of incubation and fresh medium with supplements (stated above) was added. Including the preparation time for nanoparticle experiments, macrophage (Mϕ) differentiation was performed for five days.

[0137] A1. Murine Macrophage Cell Line RAW264.7 Culture and Differentiation

[0138] RAW 264.7 macrophages (CLS, Eppelheim, Germany) were cultivated in 75 cm.sup.2 cell culture flasks in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum and 100 units/mL penicillin, and 100 μg/mL streptomycin at 37° C. in humidified 5% CO.sub.2/95% air atmosphere. Media exchange was performed after 2-4 days (depending on cell confluency). For experiments macrophages were detached by Accutase treatment and were seeded, cultured for 24 hours and then incubated with particles (i.e. NPs with loaded neutral lipid orange in phenol-red free medium for individual time periods. After incubation macrophages were harvested and/or lysed followed by individual analysis (i.e. by a microplate reader with fluorescence detection system). Protein contents were analyzed using BCA Assay (Thermo Fisher Scientific, USA)

[0139] To achieve more meaningful data compared to static mono-cell culture, several scalable co-culture-models were used. They resemble the in vivo situation better than static mono-cell cultures:

[0140] A2. Co-Culture of Endothelial Cells and Macrophages

[0141] According to Rinkenauer A C et al., Comparison of the uptake of methacrylate-based nanoparticles in static and dynamic in vitro systems as well as in vivo, J Control Release. 2015; 216:158-68, Nanoparticle (NP) were tested in co-culture model of endothelial cells and macrophages under physiologic shear stress conditions. Briefly, monocytes were harvested 24 h after isolation by treatment with 4 mg mL.sup.−1 lidocaine (Sigma-Aldrich, Germany) and 5 mM EDTA. Confluent HUVECs were detached using trypsin. Monocytes were stained with 1 μM CellTracker green CMFDA (Life Technologies, Karlsruhe, Germany) for 45 min in serum-free X-VIVO 15. Subsequently, monocytes and HUVECs were pooled 1:3 in Endothelial Growth Medium MV supplemented with 10% autologous serum, 10 ng mL.sup.−1 GM-CSF and 100 UmL.sup.−1 penicillin and 100 μgmL.sup.−1 streptomycin and seeded at a density of 1.3×10.sup.5 HUVECs cm-2 and 0.43×10.sup.5 monocytes cm.sup.2 into rhombic chamber chips. Medium was changed on a daily basis. Mϕ differentiation was performed in presence of GM-CSF for 72 h under static culture conditions. HUVEC were perfused using peristaltic pumps (Ismatec REGLO digital MS-CA-4/12-100, Germany). Shear stress within rhombic chamber chips was calculated as previously described (Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions. Raasch et al,; Biofabrication, 2015, 7(1):015013). Shear stress of 0.7, 3.0, 6.0 and 10.0 dyn cm.sup.−2 was applied for 24 h following 60 min nanoparticle uptake at a concentration of 200 μg mL.sup.−1. Negative charged nanoparticles containing nile red were solved in Endothelial Cell Growth Medium MV without additives

[0142] B. Dynamic42 Sinusoid—Chip Based Microfluidic Model

[0143] Cell specificity and targeting is determined in a chip based microfluidically supported multi-cell culture system consisting of macrophages, hepatocytes, stellate cell and, endothelial cells. According to Rennert K. et al, A microfluidically perfused three-dimensional human liver model, Biomaterials 2015; 71:119-131, the cell culture and assembling of the Dynamic42 Sinusoid—model was performed:

[0144] HepaRG and Endothelial Cell preparation for Dynamic42 Sinusoid Model

[0145] HepaRG cells were seeded at a density of 2.7×10.sup.4 cells/cm.sup.2 and cultured in William's Medium E (Biochrom, Berlin, Germany) containing 10% (v/v) FCS (Life Technologies, Darmstadt, Germany), 5 μg/ml insulin (Sigma Aldrich, Steinheim, Germany), 2 mM glutamine (GIBCO, Darmstadt, Germany), 50 μM hydrocortisone-hemisuccinate (Sigma-Aldrich) and 100 U/ml Penicillin/100 mg/ml Streptomycin mixture (Pen/Strep) (GIBCO). The cells were cultured in a humidified cell incubator at 5% CO.sub.2 and 37° C. for 14 days before differentiation. Medium was renewed every 3-4 days. Cell differentiation was induced and cells were used up to 4 weeks.

[0146] Endothelial cells: Human umbilical cord vein endothelial cells (HUVECs) were isolated from human umbilical cord veins. Donors were informed about the aim of the study and gave written consent. HUVEC cells were seeded at a density of 2.5 10.sup.4 cells/cm.sup.2 and cultured in Endothelial Cell Medium (ECM) (Promocell, Heidelberg, Germany) up to passage 4.

[0147] LX-2 Stellate Cell and Macrophage Preparation for Dynamic42 Sinusoid Model

[0148] LX-2 stellate cells (kindly provided by Scott L. Friedman, Division of Liver Diseases, Mount Sinai School of Medicine, New York City, N.Y., USA) were seeded at a density of 2.0×10.sup.4 cells/cm.sup.2 and cultured in Dulbecco's Minimum Essential Medium (DMEM) (Biochrom) supplemented with 10% (v/v) FCS, 1 mM sodium pyruvate (GIBCO) and Pen/Strep. Peripheral Blood Mononuclear Cells (PBMCs) were isolated by Ficoll density gradient centrifugation and seeded at a density of 1.0×10.sup.6 cells/cm.sup.2 in X-VIVO 15 medium (Lonza, Cologne, Germany) supplemented with 10% (v/v) autologous human serum, 10 ng/ml human granulocyte macrophage colony-stimulating factor (GM-CSF) (PeproTech, Hamburg, Germany) and Pen/Strep. After 3 h incubation in a humidified cell incubator at 5% CO.sub.2 and 37° C. the cells were washed twice with X-VIVO 15 medium. Adherent monocytes were cultivated for 24 h in X-VIVO 15 medium and seeded into the liver sinusoid.

[0149] Assembly of the Dynamic42 Sinusoid

[0150] Liver sinusoid models were assembled by staggered seeding of vascular and hepatic cell layers. In each sterilized biochip 2.7×10.sup.5 HUVEC's/cm.sup.2 (in total 3.0 10.sup.5 cells) and 0.9×10.sup.5/cm.sup.2 Monocytes (in total 1×10.sup.5 cells) were mixed and seeded on top of the membrane in the upper chamber. HUVEC/monocytes were co-cultured for at least 3 days with a daily medium exchange in endothelial cell culture medium (ECM) supplemented with 10 ng/ml epidermal growth factor, 90 mg/ml heparin, 2.8 mM hydrocortisone, endothelial cell growth supplement, 10 ng/ml GM-CSF, 10 ng/ml M-CSF to induce macrophage differentiation, 100 U/ml penicillin/100 mg/ml streptomycin and 10% (v/v) autologous human serum (Life Technologies, Karlsruhe, Germany). Subsequently, 2.7×10.sup.5/cm.sup.2 differentiated HepaRG (in total 3×10.sup.5 cells) and 0.9×10.sup.4/cm.sup.2 LX-2 (in total 1×10.sup.4 cells) were seeded on the membrane at the opposite side of HUVEC cells and cultured for 24 h in DMSO-free William's Medium E (Biochrom, Berlin, Germany) hepatocyte growth medium containing 50 μM hydrocortisone, 10% (v/v) FBS containing, 5 μg/ml insulin, 2 mM glutamine and 100 U/ml penicillin/100 mg/ml streptomycin prior to experimental use.

TABLE-US-00002 TABLE 2 Dimensions of the sinusoid chip length/width/height (mm) chip body 75.5/22.5/1.5 upper channel 15.0/2/0.45 lower channel 16.8/2/0.40 membrane (8 μm pore diameter) 13/8.5/0.02 distance (mm) membrane to upper sealing foil 0.7 membrane to lower sealing foil 0.8

TABLE-US-00003 TABLE 3 Flow rates within the sinusoid chip flow rate (μl/min) shear stress ((dyn * s)/cm.sup.2) upper channel 50 0.7 lower channel 1 0.01 (as indicated in 3 0.03 corresponding 10 0.12 experiments)

[0151] Liver sinusoid models were equilibrated after 7 days in static culture by perfusion with a flow rate 50 μl/min for up to 72 hours. Subsequently, drug constructs and controls (at least triplicates) were incubated for individual time periods in the liver sinusoid model under variable dynamic conditions. Afterwards liver sinusoids were fixed by paraformaldehyde or methanol or both and analyzed by immunofluorescence staining. The different cell layers were examined with a fluorescence microscope to analyze the enrichment of the constructs in or on different cell types. In addition, it is possible to lyse the vascular and hepatic cell layer separately and to measure the cell-specific uptaken nanoparticle by a microplate reader with fluorescence detection system.

Example 7

Determination of the Cytotoxicity

[0152] Cytotoxicity studies were performed with L929 mouse fibroblast cells and with HepG2 cells (human liver cancer cell line), as recommended by ISO10993-5. Cells were seeded at 104 cells per well in a 96-well plate in Dulbecco's modified eagle's medium (DMEM, Lonza, Basel) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 mg/mL streptomycin and incubated for 24 h at 37° C. in a humidified 5% (v/v) CO.sub.2 atmosphere. The testing substances (polymers) at indicated concentrations (from 0.5 μg/mL to 50 μg/mL) were added to the cells and the plates were incubated for further 24 h. Control cells were incubated with fresh culture medium. Subsequently, the medium was replaced by a mixture of fresh culture medium and Alamar-Blue solution (PrestoBlue for mouse fibroblasts) (Life technologies, Darmstadt, Germany), prepared according to the manufacturer's instructions. After a further incubation of 4 h at 37° C. (30 min for PrestoBlue), the fluorescence was measured at Ex 570/Em 610 nm (560/590 for PrestoBlue), with untreated cells on the same well plate serving as negative controls. The negative control was standardized as 0% of metabolism inhibition and referred as 100% viability. Cell viability below 70% was considered indicative of cytotoxicity. Data are expressed as mean±S.D. of three determinations. After 24 h FIG. 13 shows for all drugs more or less similar toxicities, regardless of the formulation. This observation reflects a limited stability of the nanoparticles in this experimental setup. Nearly identical results were obtained using HeGP2 cells (data not shown).

Example 8

Survival-Rate in Cholestasis Model Under Septic Conditions “Peritoneal Contamination and Infection (PCI)”

[0153] Experimental setup: systemic infection/sepsis with organ failure was induced in male C57/BL6 mice by using the PCI model. For this purpose, a human fecal suspension (2.5 μl/g BW for stool batch1 and 6 μl/g for stool batch 2, respectively) was injected intraperitoneally (without anesthesia) with weight adaptation, thus triggering peritonitis with subsequent systemic infection. In order to avoid the burden on the animals and a dying, 6 hours after infection twice a day, the broad-spectrum antibiotic Meropenem is administered subcutaneously (2.5 μg/g body weight). The animals were closely monitored and scored every 6 hours for signs of infection in order to timely. With stool batch 1, a dose of 2.5 μg/g produced a 70% of the mice died within the first two days and the remaining 30% died until day 7 (FIG. 14, left panel). With the evaluated dose of 6 μl/g BW stool and additional antibiotic therapy, all mice died within three days, as shown in the Kaplan-Meier-Schatzer diagram (FIG. 14, right panel). Experimental data partly rely on experiments with batch 1 and partly with batch 2. Details are stated in the FIG. 14.

[0154] For dose determination, three drug concentrations per formulation were tested in small groups and changes in survival are documented. The free drugs were used for dose evaluation (data not shown) and ⅛ of the effective dose was used in the targeted nanoparticles. The PI3K inhibitor AS605240 and the PKC inhibitors BIM-1 alone were active at 4 mg/kg body weight. In the nanoparticle, we used 0.5 mg/kg and obtained in all cases an even more pronounced effect. For midostaurin, 6 mg/kg of the free drug and 0.75 mg/kg were used in the nanoparticle formulation.

[0155] Six hours after infection (PCI model), the therapy is carried out with different drug, capable to reduce the activity of PKC (BIM-1 and midostaurin as PKC inhibitors and AS605240 as PI3 kinase inhibitor) or control formulations (once daily, i.p. or i.v.) and also the combined volume and antibiotic therapy (twice daily, s.c.). The therapy with the drug is scheduled for 5 days. The volume/antibiotic therapy takes place over 7 days (2 days longer than the drug therapy). The observation in the first 5 days is performed in a 3-hour interval for 24 hours a day. This is followed by observation of the animals until day 14 (twice a day).

[0156] 9a) CTM1 Targeted PLGA-Nanoparticles with BIM-1 as Archetypical PKC Inhibitor as Cargo:

[0157] We prepared nanoparticles as described in example 5 with the synthesized PTM1-PLGA, PVA as surfactant and BIM-1 with the following concentrations/loading efficiency.

[0158] PTM-PLGA: 56%

[0159] PVA: 40%

[0160] BIM-1: 4%

[0161] Size/zeta potential: 72 nm/−0.2

[0162] The particle suspension was diluted with a 45% glucose solution to a final glucose concentration of 5%.

[0163] Ten mice were treated with the targeted nanoparticles and to test the tolerability of the nanoparticles in healthy mice was evaluated with two sham mice. The results are illustrated in FIG. 15 and demonstrate within 7 days an increased survival from 10% to 60%.

[0164] 9b) PTM Targeted PLGA-Nanoparticles with AS605240 as Experimental Pi3K Inhibitor as Cargo:

[0165] The particles were prepared analogously to example 5 with slightly modified parameters: We prepared nanoparticles as described above with the synthesized PTM-PLGA, PVA as surfactant and AS605240 with the following concentrations/loading efficiency.

[0166] PTM-PLGA: 58%

[0167] PVA: 32%

[0168] AS605240: 10%

[0169] Size/zeta potential: 93 nm/−2

[0170] The particle suspension was diluted with a 45% glucose solution to a final glucose concentration of 5%.

[0171] Six mice were treated with the targeted nanoparticles and to test the tolerability of the nanoparticles in healthy mice was evaluated with two sham mice. The results are illustrated in FIG. 16 and demonstrate within 7 days an increased survival from 0% to 40%.

[0172] 9c) PTM1-Targeted PLGA-Nanoparticles with Midostaurin as Approved Kinase Inhibitor with Marked PKC Inhibition as Cargo:

[0173] The particles were prepared analogously to example 5 with slightly modified parameters: We prepared nanoparticles as described above with the synthesized PTM-PLGA, PVA as surfactant and midostaurin with the following concentrations/loading efficiency.

[0174] PTM-PLGA: 41%

[0175] PVA: 52%

[0176] midostaurin: 7%

[0177] Size/zeta potential: 185 nm/−1

[0178] The particle suspension was diluted with a 45% glucose solution to a final glucose concentration of 5%.

[0179] Five mice were treated with the targeted nanoparticles and to test the tolerability of the nanoparticles in healthy mice was evaluated with two sham mice.

[0180] The results are illustrated in FIG. 17 and demonstrate within 7 days an increased survival from 10% to 60%.