PKC inhibitors for the treatment of septic cholestasis by CTM targeting

Abstract

The present invention relates to an inhibitor of the PKC signaling pathway for use in the treatment of septic cholestasis, wherein the inhibitor is targeted to 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. [Selected Figure] Figure 1

Description

[Technical field]

[0001] Cholestasis Cholestasis refers to the impairment of bile formation and flow and the subsequent retention of bilirubin and bile acids. Two forms of cholestasis are known: extrahepatic cholestasis, which is an obstructive type of cholestasis caused by mechanical blockage of the ductal system and displacement of the bile duct, which can arise, for example, from gallstones, tumors such as pancreatic cancer, bile duct cysts, bile duct strictures, or parasites, and intrahepatic cholestasis, where the cause of bile stasis is inside the liver (nonobstructive cholestasis). Intrahepatic cholestasis can occur due to genetic defects or can be acquired as a side effect of many drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), antihypertensives, antidiabetics, anticonvulsants, lipid-lowering drugs, anabolic steroids, psychotropic drugs, and various antibiotics. In addition, cholestasis can also occur as a result of viral or alcoholic hepatitis, hepatocellular carcinoma, granulomatous liver disease, or cirrhosis. However, the second most common cause is extrahepatic infection (sepsis). It may also occur during parenteral nutrition, pregnancy, and after liver transplantation. As a result of reduced bilirubin excretion, patients with cholestasis show symptoms of jaundice. Depending on the cause, cholestasis may be accompanied by digestive symptoms such as itching (pruritus), pale stools, dark urine, nausea, vomiting, and pain. Cholestatic liver disease is diagnosed by a marked elevation of serum alkaline phosphatase and bilirubin, although serum bilirubin may be normal until the later stages of the disease. Overt intrahepatic cholestasis is a rare but typical symptom of sepsis. In Germany, for example, about 30,000 people develop organ failure associated with sepsis, of which 3-6% develop a condition called septic cholestasis or sepsis-induced cholestasis, characterized by the additional symptom of jaundice. Mortality in the first 12 months after diagnosis is 92%, much higher than other sepsis-associated organ failure (Jaeger et al., Jaundice Increases the Rate if Complications and One-Year Mortality in Patients with Hypoxic Hepatitis, Hepatology 2012, 56(6), 2297).

[0002] sepsis Sepsis should be defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection by a pathogen, and is a major public health concern accounting for more than $20 billion of total hospital costs in the United States in 2011. Sepsis can be assumed to be the leading cause of mortality and critical illness worldwide. Moreover, patients who survive sepsis often have long-term physical, psychological and cognitive impairments with considerable health care and social impacts. The pathogens involved in the development of sepsis can have various origins and can result from bacterial, viral, fungal or protozoal infections. Sepsis is not only defined by systemic inflammation, but more importantly, the patient's dysregulated response to this infection, which causes organ dysfunction and accentuates the overall severity of this condition. Sepsis also involves pro- and anti-inflammatory responses, changes in cardiovascular, neurological, autonomic, hormonal, bioenergetic, metabolic processes and coagulation. Finally, organ failure or dysfunction is responsible for the high mortality associated with sepsis. Therefore, the so-called "Sequential Organ Failure Assessment" (SOFA) scoring scale has been developed to determine the severity of sepsis. They can be defined by clinical parameters such as Pao2 / Fio2, platelet count, bilirubin, creatinine, urine output, and the patient's mental status. It could be shown that early antibiotic eradication of the underlying infection is paramount for the patient's survival. Additional therapeutic considerations include avoidance of parenteral nutrition, avoidance of hepatotoxic drugs, monitoring of blood glucose levels and, if necessary, adequate provision, extracorporeal liver support (i.e., albumin dialysis). For example, immunocompromised patients after organ transplantation, cancer therapy or treatment for autoimmune diseases are not only at increased risk of infections with common pathogens, but also of opportunistic infections with less virulent microorganisms that are of little concern to patients with intact immune systems. It is therefore clear that this highly elevated risk of infection predisposes such individuals to an increased risk of sepsis and septic shock.

[0003] In sepsis, a dysregulated host response to systemic infection often leads to hepatocellular dysfunction of membrane transport processes with a continuous impairment of bile excretion (Zollner G., Trauner M.; Mechanism of cholestasis, Clin Liver Dis 2008; 12: 1-26). As a consequence, jaundice can be observed in septic patients as a result of intrahepatic (non-obstructive) cholestasis. The challenge is to distinguish in a timely manner between sepsis-related and other non-sepsis-related causes of cholestasis (see above). Usually, before the onset of septic cholestasis, symptoms of sepsis dominate the clinical picture. Uncontrolled infections can lead to a reduction in the function and expression of important hepatocellular transport proteins, resulting in reduced bilirubin excretion and jaundice (Zollner G., Trauner M., Ic). Septic cholestasis is accompanied by liver failure and has a mortality rate of over 92%.

[0004] Sepsis-induced cholestasis Sepsis-induced cholestasis is a special type of excretory dysfunction of the liver. Excretory dysfunction of the liver can have various causes: carcinoma, cysts in the bile ducts, inflammation of the liver (such as hepatitis), fibrosis or cirrhosis, fatty liver (alcoholic or non-alcoholic), side effects of certain drugs (e.g., anabolic drugs, antipsychotics, certain antibiotics), etc. In the case of septic cholestasis, the underlying systemic infection causes a disturbance of the entire immune system and induces secretory dysfunction of the liver. Thus, sepsis-induced cholestasis represents a complication of a systemic infection. Currently, the only effective treatment for septic cholestasis is the treatment of the underlying sepsis with antibiotic therapy of the infection, which should be started as soon as possible. The window of opportunity for successful intervention is short, and delays in diagnosing the infection and initiating antibiotic therapy significantly worsen the patient's prognosis and chances of survival (Fuchs M., Sanyal AJ.; Sepsis and cholestasis, Clin Liver Dis 2008; 12: 151 -72). To rapidly and efficiently eradicate bacterial infections, the maximum tolerated doses of a combination of different broad-spectrum antibiotics are usually used in treatment, because diagnosis of the causative pathogen (blood culture) is not possible within an acceptable time frame in most cases.

[0005] Antibiotic therapy of systemic infections is also associated with known problems and is certainly not a cure for organ failure resulting from a dysregulated response to infection. Moreover, some antibiotics may impede and block bile excretion and thus induce cholestasis (see above). The most prominent examples are amoxicillin and erythromycin. High doses of broad-spectrum antibiotic therapy may cause new emergence of pathogens with antibiotic resistance, hindering the success of future therapy. Moreover, it must be taken into account that antibiotic therapy is pointless or even counterproductive if the underlying infection is fungal, viral or protozoal, due to potential undesirable (toxic) side effects of some antibiotics, such as allergies, interactions with food and other medicines, and / or due to direct damage to the major organs, mainly the kidneys and liver, especially if organ function is impaired.

[0006] In addition to antibiotic therapy of sepsis, further therapeutic approaches for sepsis using kinase inhibitors have been described. USH1168H, filed in 1991, describes a method for treating septic shock, which reduces inflammation and improves tissue and organ perfusion, comprising injecting a PKC inhibitor selected from the group consisting of lipid analogues. This means systemic administration of the PKC inhibitor. US Patent No. 5,616,577, filed in 1996 (corresponding International Publication No. WO 93 / 16703), describes the treatment and prevention of conditions in which PKC inhibition is indicated. These conditions are described as cardiovascular and renal disorders, inflammation, central nervous system disorders, immunosuppression, and septic shock. US Patent Publication No. 2011 / 0130415 describes the treatment of various inflammatory diseases, including septic shock, by PKC inhibition.

[0007] Although the effect of kinase inhibitors on cholestasis, especially PKC inhibition, has been described (Anwer MS; Role of protein kinase C isoforms in bile formation and cholestasis, Hepatology 2014; 60(3): 1090-1097), treatment of sepsis with the use of kinase inhibitors can be counterproductive and cause life-threatening side effects for reasons outlined in the paragraph "Kinase inhibitors and sepsis" (see below). First, the role of kinase inhibitors on cholestasis will be described in more detail.

[0008] Kinase inhibitors and cholestasis In the case of intrahepatic cholestasis, the formation of bile itself from hepatocytes is impaired. Bile formation is a complex process involving many different transhepatic solute transporters, most notably the Na-taurocholate cotransporting polypeptide (NTCP) at the basolateral site, and the bile salt exporter (BSEP) and multidrug resistance-associated protein (MRP) at the apical hepatocyte membrane (Anwer MS, lc). The plasma membrane localization of these transporters is a highly dynamic process, which is regulated by posttranslational events, in particular by kinases such as protein kinase C (PKC), phosphoinositide 3-kinase (PI3K), AMP-activated protein kinase (AMPK) and mitogen-activated protein kinase (MAPK). Various experiments have shown that PKC inhibitors or PI3 kinase inhibitors are useful preclinical tools for the treatment of cholestasis. (Anwer MS, lc; Toledo et al., Arch Toxicol. 2017, 91 :2391-2403; and Li et al., Pharm. Res. 2017, 125, 105-113.) These kinase inhibitors profoundly affect cell proliferation and immune cell signaling and act as immunosuppressants.

[0009] Kinase inhibitors and sepsis As outlined above, sepsis is a severe and complex systemic immune response induced by an infection. The body relies on the immune system to combat this infection. It is known in the art that kinase inhibitors, such as PKC and PI3 kinase inhibitors, can suppress the immune response in cases of inflammation. It has therefore been suggested that excessive inflammatory responses can be treated with such compounds, also in cases of sepsis (see, for example, USH1168H, U.S. Pat. No. 5,558,969, WO 93 / 16703, supra). However, it must be recognized that, on the one hand, the underlying infection itself is certainly not treated by such proposed treatments, and, on the other hand, for conditions related to infections, it is certainly not desirable to suppress the body's immune system. As already mentioned, immunocompromised patients are at increased risk of infections with common pathogens, and it is clear that this highly developed risk of infection predisposes patients to an increased risk of sepsis and septic shock. Kinase inhibitors, such as PKC and PI3 kinase inhibitors, are also known to increase the incidence of various (additional) infections due to their immunosuppressive properties. For these reasons, manufacturers of commercially available kinase inhibitors for use in treating various pathogenic conditions, as well as certain types of cancer, expressly warn against systemic administration of these inhibitors in cases where they exhibit immunosuppressive effects and where infection or inflammation is present in the patient. See, for example, · EMEA Report EMEA / H / C / 753 of 24 May 2007 (Doc. Ref. EMEA / 150964 / 2007), page 13, on the PKC inhibitor ruboxistaurin (drug name Arxxant; indicated for diabetic retinopathy); Medscape, a medical professional site, states that the PI3K inhibitor copanlisib (drug name Aliqopa, indicated for relapsed follicular lymphoma) should be withheld in the event of infection; the increased risk of infection (even sepsis itself) is described in detail by Kim et al., BJC, 2018, 1 18, 462-470. · Medscape (lc) prescribes withholding treatment with the PI3K inhibitor idelalisib (drug name Zydelig; indicated for three classes of lymphoma) in case of infection, specifically mentioning sepsis; regulation: "withhold idelalisib until infection resolves." This is also stated in Zelenetz et al., Lancet Oncol. 2017, 18:297-311. They state a 5-fold higher risk of developing sepsis as an adverse effect while receiving idelalisib compared to placebo. Novartis Pharma GmbH (2017): Rydapt® 25 mg Weichkapseln, Fachinformation, Status September 2017 provides highlights of prescribing information for the PKC inhibitor midostaurin (drug name RYDAPT; indicated for AML). The right column on page 1 and Table 2 on page 5 show adverse reactions of midostaurin; the last paragraph on page 6 specifies that "Grade ≥ 3 (≥ 3) adverse reactions reported in ≥ 5% (≥ 5%) were fatigue, sepsis, gastrointestinal bleeding, pneumonia, diarrhea, febrile neutropenia... (Table 4)." Page 7 states: "Discontinuation of treatment due to adverse reactions occurred in 21% of patients. The most frequent adverse reactions leading to treatment discontinuation included infections....Serious adverse reactions were reported in 68% of patients, most commonly (>=20%) due to infections and gastrointestinal disorders," and "Treatment deaths unrelated to the underlying malignancy occurred in 16 patients (11%), most commonly from infections (sepsis or pneumonia), followed by cardiac events. Of the treatment deaths due to disease progression, four were due to infections." Page 8 outlines adverse reactions occurring in 9% or more (>=9%) of patients with sepsis.

[0010] From the above, it is clear that kinase inhibitors should not be administered systemically when sepsis is present. This is a teaching, and as a result, a person skilled in the art would not consider administering a kinase inhibitor to treat cholestasis during a systemic infection such as sepsis.

[0011] In summary: As outlined above, the currently known treatments for septic cholestasis (also known as sepsis-induced or sepsis-associated cholestasis) are treatments for the underlying systemic infection. In conditions with underlying systemic infection, suppressing the inflammatory response with systemically administered immunosuppressive kinase inhibitors is undesirable, as adverse effects of immunomodulation may lead to life-threatening conditions, especially when such drugs are administered systemically.

[0012] It is therefore an object of the present invention to provide an effective treatment of septic cholestasis by avoiding, or at least minimizing, adverse side effects.Furthermore, it is an object of the present invention to provide a direct treatment of septic cholestasis itself.

[0013] This objective has been achieved by the present invention and by treating septic cholestasis with compounds that reduce or inhibit the activity of protein kinase C (PKC), which ultimately regulates the transporters involved in bile formation. These compounds are targeted into the liver by a unique and selective delivery system.

[0014] In its first aspect, the present invention relates to an inhibitor of the PKC signaling pathway for use in the treatment of septic cholestasis, wherein the inhibitor is 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. According to the present invention, the inhibitor is preferably delivered into the parenchymal cells of the liver by the selective nanostructured delivery system of the present invention.

[0015] Bile formation is a complex process involving many different transhepatic solute transporters, most notably the sodium-taurocholate cotransporting polypeptide (NTCP), the bile salt exporter (BSEP), and the multidrug resistance-associated protein (MRP) (Anwer, lc). These transporters are located at basal or apical sites in the hepatocyte, respectively.

[0016] The plasma membrane localization of these transporters is a highly dynamic process, which is regulated by post-translational events, in particular by kinases such as protein kinase C (PKC), phosphoinositide 3-kinase (PI3K), AMP-activated protein kinase (AMPK) and mitogen-activated protein kinase (MAPK). Various experiments have shown that PKC or PI3K inhibitors are useful preclinical tools for the treatment of cholestasis (Anwer, lc; Toledo et al., Ic; Li et al., Ic). These kinase inhibitors significantly affect cell proliferation and immune cell signaling, acting as immunosuppressants.

[0017] According to the signaling pathway, the activity of PKC is regulated by diacylglycerol (DAG) and calcium (Ca 2+ ), which depends heavily on the concentration of regulatory molecules such as e.g. · DAG concentration is mediated by enzymes such as phospholipase C (PLC), which is highly regulated by the activation of various Gαq-coupled GPCRs, AKT and MAP kinases, growth factors, and cannabinoid receptors. · PLC activity is primarily regulated by PI3 kinase, which phosphorylates PIP2 to PIP3. Activated PLC cleaves PIP2 into IP3 and DAG. IP3 induces Ca release into the endoplasmic reticulum (ER), which then activates PKC. The molecule DAG itself also contributes to PKC activation. Therefore, PI3 kinase inhibitors, PLC inhibitors, DAG level reducers, or any agent that ultimately contributes to the reduction of PKC, are useful tools to treat septic cholestasis.

[0018] From a biochemical point of view, PI3 kinase generates the signaling substance diacylglycerol (DAG), which activates other protein kinases (e.g., PKC). Therefore, PI3 kinase inhibitors essentially reduce DAG levels and therefore inhibit downstream PKC. For this reason, other drugs that can reduce DAG levels are also useful in the treatment of septic cholestasis.

[0019] Thus, the term "inhibitors of the PKC signaling pathway" according to the present invention relates to any substance that affects the transmission and / or transduction of PKC-mediated signals in vivo and in cells. Such inhibitors include in particular inhibitors of kinases involved in the PKC signaling pathway.

[0020] The term "inhibitor of the PKC signaling pathway" further means any substance that directly or indirectly affects, preferably reduces or inhibits, the activity and / or expression and / or protein folding of PKC via upstream regulatory molecules of the PKC pathway. In a preferred embodiment, the activity and / or expression and / or protein folding of PKC itself, PI3 kinase, DAG, PLC, AMPK, MAPK, AKT is reduced by the "inhibitor of the PKC signaling pathway" of the present invention. The inhibitor of the PKC signaling pathway of the present invention can be understood as a PKC activity reducer. According to the present invention, the terms "inhibitor of the PKC signaling pathway", "PKC activity reducer" and "inhibitor of PKC activity", "inhibitor of PKC expression" and "inhibitor of PKC folding" are used synonymously.

[0021] Such "inhibitors of the PKC signaling pathway" can act to directly inhibit the above proteins / signaling molecules, for example, by: Direct inhibition of PKC with PKC inhibitors such as midostaurin, staurosporine, BIM-1 and other drugs; or Silencing protein biosynthesis of PKC or the respective protein / signaling molecule by siRNA, miRNA, shRNA, modified oligo analogues or antisense constructs; as well as by using other molecular biology methods known in the art, such as CRISPR / Cas, TALEN, zinc finger nucleases or antisense oligonucleotides.

[0022] Consequently, in a preferred embodiment, inhibitors of the PKC signaling pathway for use in the treatment of septic cholestasis directly or indirectly inhibit or reduce the activity of PKC or any PKC subtype.

[0023] Direct inhibition or reduction of the activity of PKC according to the present invention means that the activity is influenced by PKC inhibitors, including nucleic acid constructs that silence the respective genes, preferably via RNAi. This can be achieved by generally known methods, preferably using constructs such as siRNA, miRNA, shRNA, RNAse H, modified oligomers such as morpholino, etc. According to known biochemical approaches, the respective nucleic acid constructs are designed and directly linked to carbohydrate targeting moieties, either by covalent bonding or by encapsulation in nanocarriers carrying carbohydrate targeting moieties.

[0024] Indirect inhibition or reduction of the activity of PKC according to the present invention is preferably achieved by inhibition or reduction of pathways and / or signaling molecules required for PKC activity. PKC activity is promoted by various factors. PI3 kinase inhibitors, PLC inhibitors, DAG level reducers, or any agent that ultimately contributes to the reduction of PKC, are useful tools for treating septic cholestasis. Therefore, all kinds of agents (e.g., PI3 kinase inhibitors, PLC inhibitors) that can affect the PKC signaling pathway by reducing PKC activity are inhibitors according to the present invention and are useful tools for treating septic cholestasis. This can also be achieved by either small molecule inhibitors such as PI3 kinase inhibitors described herein, and / or PLC inhibitors such as U-73122, D609, manalide, edelfosin, or the respective nucleic acid constructs.

[0025] Direct and indirect inhibition or reduction of PKC activity according to the present invention should also be understood and include any effect on the expression of PKC genes, including PKC subtype genes, their transcription and / or translation and / or protein folding, resulting in less and / or no gene product and / or PKC protein. Such effects should be understood as decreasing (reducing) or preventing, blocking, switching off (inhibiting) PKC expression and thus PKC activity.

[0026] Currently, PKC inhibitors are primarily used in the treatment of cancer and autoimmune diseases. Currently, two PKC inhibitors are available as approved drugs: Rydapt® (midostaurin) and Arxxant® (ruboxistaurin).

[0027] Further known drugs are related to idelalisib and copanlisib. For both compounds, the information site for specialists (Medscape Reference: see above) states that due to their immunosuppressive properties, they are strictly contraindicated in case of severe infections, including sepsis, during which it is recommended to discontinue treatment.

[0028] To date, no specific treatment for septic cholestasis is available in the clinic, and treatment with any of the above-mentioned agents administered systemically would be too risky due to the underlying compromised immune state during sepsis and the therapeutic doses in the liver required to elicit a positive effect on septic cholestasis.

[0029] Thus, the present invention provides for the first time a specific treatment of septic cholestasis itself by avoiding or at least reducing the dangerous systemic immunosuppressive effects on the patient's body. This is achieved according to the present invention by selective targeting of therapeutic agents to the site of action, i.e., hepatocytes. The nanostructured delivery system of the present invention provides active hepatocyte targeting for treating septic cholestasis, which is achieved by carbohydrate-derived targeting moieties that are selectively recognized by special lectins present in liver tissue. According to the present invention, the systemic circulation and required therapeutic dose of these therapeutically active agents can also be significantly reduced compared to the systemic administration of PKC inhibitors in the treatment of pathological conditions.

[0030] According to the present invention, to treat septic cholestasis, inhibitors are administered, for example by injection, and selectively delivered to their site of action, i.e., the liver, by the nanostructured delivery system of the present invention. In this way, side effects (immunosuppression) associated with systemic treatment of infections with the above-mentioned kinase inhibitors are reduced and preferably avoided, without delivering the inhibitor to the desired site of action. Furthermore, the required dose of the inhibitor with the nanostructured delivery system is significantly reduced compared to the dose of the inhibitor without the liver-targeted nanostructured delivery system. The present invention provides for the first time a treatment of septic cholestasis itself.

[0031] To treat septic cholestasis as a condition with an underlying systemic infection, i.e. sepsis, it is not desirable to suppress the immune system. Therefore, the present invention describes a carbohydrate-driven selective delivery system for targeted transport of inhibitors of the PKC signaling pathway into the liver with negligible systemic immunosuppressive effects. The reason is that they act selectively in the liver, specifically modifying only bile excretion and restoring cholestasis. In this way, the agents of the present invention, i.e. inhibitors of the PKC signaling pathway / inhibitors of PKC activity, are administered at much lower doses compared to their non-targeted counterparts and transported only to the site of action, and are therefore suitable for the treatment of septic cholestasis. Thus, the present invention represents a highly effective method for treating septic cholestasis, with a significant reduction in the systemic adverse effects that typically occur when kinase inhibitors are administered in the treatment of frontline infections.

[0032] The term "agent" or "therapeutic agent" according to the present invention refers to an inhibitor of the PKC signaling pathway; furthermore, the term "agent" or "agents" is used synonymously with "anticholestatic agent" and "cholestatic agent" and the term "drug" or "drugs". Further, the terms "agent" and "drugs" are used synonymously according to the present invention.

[0033] When a nanostructured delivery system according to the present invention comprises at least one polymer, it is referred to herein as a "nanoparticle"; when it comprises at least one lipid, it is referred to herein as a "liposome". When a nanostructured delivery system according to the present invention comprises both a polymer and a lipid, it is also referred to herein as a "nanoparticle" or a "liposome". When a nanostructured delivery system according to the present invention comprises at least one polymer and at least one nucleic acid construct, it is also referred to herein as a "polyplex". According to the present invention, nanoparticles, liposomes, virus-like particles, as well as polyplexes, lipoplexes and peptoplexes relate to nanostructured delivery systems.

[0034] Nanoparticles can be constructed from multiple molecules. These nanoparticles can be composed of polymers, where these polymers are characterized by the fact that certain units (monomers) are the repeating units. The polymers are covalently bonded to each other by chemical reaction (polymerization) of these monomers. If some of these polymers are hydrophobic, they can form nanoscale structures (e.g. nanoparticles, micelles, vesicles) in an aqueous environment. Due to their hydrophobicity, lipids can also be used to form nanoparticles (micelles, liposomes).

[0035] When a nanostructured delivery system according to the present invention comprises at least one positively charged polymer that forms a complex with negatively charged genetic material, it is referred to herein as a "polyplex"; when it comprises at least one positively charged lipid and negatively charged genetic material, it is referred to herein as a "lipoplex"; and when it comprises at least one positively charged peptide and negatively charged genetic material, it is referred to herein as a "peptoplex."

[0036] According to the present invention, the terms "carbohydrate targeting moiety", "carbohydrate-based targeting moiety" and "carbohydrate-derived targeting moiety" have the same meaning and can be used synonymously. Carbohydrate targeting moiety (CTM) according to the present invention means a chemical structure that is recognized by a special surface molecule (e.g., lectin), preferably an ASGP receptor, and induces the internalization of the construct, drug, nanostructured delivery system, i.e. the nanostructured delivery system according to the present invention, into the cell, tissue or organ in which these surface molecules are expressed, preferably the liver. CTM can be either monovalent or multivalent, depending on the labeling density on the surface of the drug or drug construct or polymer. In the multivalent setting, there is a core molecule with at least one single unit (preferably a derivative of N-acetylgalactosamine (GalNAc), galactose mannose, and glucosamine). This can be either a repeat of the same unit or a mixture of different units. CTM with higher MW and multiple repeat units (e.g., pullulan or arabinogalactan) can form a nanostructured carrier system by itself, but can also be attached to a nanostructured carrier.

[0037] In a preferred embodiment, the carbohydrate targeting moiety is selected from the group consisting of N-acetylgalactosamine (GalNAc), galactose, lactose, mannose, glucosamine, asialofetuin, pullulan, arabinogalactan, glycyrrhizin, glycyrrhetinic acid and derivatives thereof. Preferably, the carbohydrate targeting moiety is recognized by the ASGPR recognition moiety.

[0038] Carbohydrate targeting moieties or carbohydrate liver or hepatocyte recognition moieties according to the present invention include classical monovalent ligands of ASGPR, such as galactose, glucosamine, N-acetylgalactosamine (GalNAc) oligosaccharide constructs, or multivalent constructs carrying these recognition units. Further preferred are fuconate, lactobionic acid, mannose, fibronectin, transferrin, asialofetuin, glycyrrhetinic acid, lithocholytaurin, steryl glycoside lipoproteins, or unclassified surface recognition moieties that can be addressed with specific ASGPR antibodies.

[0039] As a result, in a preferred embodiment of the present invention, the carbohydrate targeting moiety binds to a recognition unit located on the liver.

[0040] In a further preferred embodiment, the recognition or targeting unit is a receptor belonging to the family of lectins, preferably a lectin, more preferably the asialoglycoprotein receptor (ASGPR), also known as the Ashwell-Morell receptor.

[0041] The most prominent representative of hepatocyte-specific lectins is the asialoglycoprotein receptor (ASGPR). ASPGR is a liver-specific membrane-bound receptor involved in the endocytosis of carbohydrate-containing glycoproteins. This receptor acts like a "lock" for direct entry into hepatocytes, and recent studies have thoroughly investigated the properties and possible locks of this lock (Sanhueza CA et al., Efficient liver targeting by polyvalent display of a compact ligand for the asialoglycoprotein receptor; JACS, 2017; 139: 3528-3536). After binding of the appropriate ligand (lock), the entire receptor and ligand construct is internalized into hepatocytes, preferably by clathrin-mediated endocytosis.

[0042] X-ray crystallography has shown that ASGPR has a shallow binding cavity, which is best targeted by multivalent ligand constructs. Such multivalent structures are state of the art and can be set up in different ways and with different substructures (see Figure 2). Sanhueza et al., Ic, give an overview of possible configurations for multivalent ligands, but basically any molecule with a connection point (connection to drug / drug construct / polymer) and three further connection points for connecting the smallest unit of carbohydrate targeting units may be suitable (see Figure 1). Depending on the overall surface density of the targeting moieties, different configurations may be optimal. If the surface labeling of the nanoparticles is high enough, monovalent ASPGR ligands may also be suitable for proper targeting. On a case-by-case basis, the most efficient and synthetically feasible targeting should be investigated in a suitable model for tissue endocytosis, such as, for example, a chip-based microfluidic model (exemplified in Example 8).

[0043] In particularly preferred embodiments, the inhibitor of the PKC signaling pathway for use in the treatment of septic cholestasis is selected from the group consisting of PKC inhibitors, PI3 kinase inhibitors, MAPK inhibitors, PLC inhibitors, DAG level reducers, siRNAs, miRNAs, shRNAs, modified oligo analogs (e.g., morpholinos), antisense constructs and RNAse H.

[0044] The inhibitors siRNA, miRNA, shRNA, modified oligo analogs (e.g., morpholino), antisense constructs and RNAse H according to the present invention relate to oligonucleotide constructs capable of silencing the respective genes (e.g., silencing the PKC gene, PI3 kinase gene, MAPK gene, PLC gene) that can be constructed by gene silencing techniques well known in the art. These inhibitors can also be designated as PKC siRNA, PKC shRNA, PKC miRNA, PI3 kinase siRNA, PI3 kinase shRNA, PI3 kinase miRNA.

[0045] According to the present invention, inhibition of PKC activity can also be achieved using suitable gene editing methods such as CRISPR / Cas, TALEN, zinc finger nucleases.

[0046] In a preferred embodiment, the inhibitor of the PKC signaling pathway for use in the treatment of septic cholestasis is a PKC inhibitor selected from the group consisting of bisindolylmaleimides, staurosporine, midostaurin, UCN-01, sotrastaurin, enzastaurin, ruboxistaurin, tivantinib, enzastaurin, Go6983, K252a, ANA-12, lestaurtinib, stauprimide, CEP-701, Arcyriaflavin A, chelerythrine chloride, and bisindolylmaleimides I-XII, also known as BIM I-XII.

[0047] In a preferred embodiment, the inhibitor of the PKC signaling pathway for use in treating septic cholestasis is a PI3 kinase inhibitor selected from the group consisting of copanlisib, idelalisib, wortmannin derivatives, bryostain derivatives, taselisib, omipalisib, AS605240, GSK1059615, bupallisib, alpelisib, pictilisib, ceravilisib, dactolisib, dihydrosphingosine, calphostin C, and melittin. Preferred inhibitors of the present invention also include novel investigational compounds that exhibit PI3 kinase inhibitory effects.

[0048] The agents of the present invention, i.e., inhibitors of the PKC signaling pathway, may be directly attached to spacers or linkers comprising aliphatic, heteroaliphatic, aromatic, heteroaromatic, linear, branched or cyclic atomic aggregates, and / or carbohydrate targeting moieties, or may be attached to suitable carriers such as nanoparticles, liposomes or virus-like particles in which the agents are encapsulated or entrapped (see, e.g., Figures 1, 7a-c).

[0049] The carbohydrate targeting moieties of the present invention as selective liver targeting moieties can be attached to the agents of the present invention (i.e. directly to the inhibitor or to a suitable carrier) by conventional chemical coupling reactions well known in the art, preferably by activated carboxylic acid derivatives (e.g. anhydrides, acyl halides, active esters), which can then be coupled to amines by photoinduced thiol-ene click reaction, Michael addition (1,4-addition), cycloaddition reaction, Huisgen reaction (e.g. 1,3-cycloaddition of alkynes to azides), Diels-Alder reaction (e.g. trans-cyclooctene coupling to tetrazine derivatives), maleimide-thiol reaction, isocyanate-, isothiocyanate coupling, carbodiimide coupling, chloroacetamide coupling. Alternatively, reactive carbonyl compounds, preferably ketones, aldehyde acetals or hemiacetals with amines forming Schiff bases that can be reduced to the corresponding amines, can be used according to the present invention (see, e.g., FIG. 6).

[0050] The term "nanostructured delivery system" according to the present invention is characterized by 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 that delivers a therapeutic agent, i.e., an inhibitor of the present invention, into a target tissue, and includes contacting the target tissue with said nanostructured delivery system.

[0051] At least one carbohydrate targeting moiety as a targeting unit induces active and selective transport of the nanostructured delivery system into the target tissue.

[0052] The at least one carbohydrate targeting moiety interacts with the cell surface and accumulates the nanostructured delivery system on the cell surface, thereby further inducing uptake of the nanostructured delivery system into cells of the target tissue.

[0053] The term "nanostructured delivery system" according to the present invention also relates to polyplexes, which must be understood as complexes between negatively charged nucleic acid constructs linked to positively charged polymers. The terms "nanostructured carrier system" and "nanostructured delivery system" are used synonymously according to the present invention.

[0054] The nanostructured delivery system of the present invention comprises a combination of a nanostructured carrier and a carbohydrate targeting moiety. The nanostructured delivery system comprises at least one polymer and / or at least one lipid or virus-like particle, and can carry an active ingredient--according to the present invention, a PKC activity inhibitor or reducer (an inhibitor of the PKC signaling pathway) as a vehicle. These nanostructured systems can be detected and characterized by methods known in the art, such as DLS, AUC, AF4, DSC, ITC, XRD, SANS, SAXS, or specialized microscopy methods, such as SEM, STEM, or cryo-TEM, AFM. The shape can be preferably, but not limited to, either spherical, elliptical, rod-shaped, barrel-shaped, disc-shaped, or polyhedral. The size preferably varies from 1 nm to 800 nm.

[0055] In a preferred embodiment, at least one of the polymers, lipids, virus-like particles and / or active agents comprises functional groups that allow for chemical modification and attachment of carbohydrate targeting moieties (CTMs) (see, for example, Figures 4 and 6). The polymers can be organic or inorganic and immobilize the therapeutic agent as a vehicle. Inorganic particles can be functionalized by silanization with functionalizing silanes such as aminopropyltrimethylsilane (APTES), which introduces amine functional groups to the oxide.

[0056] In a preferred embodiment of the invention, the at least one polymer is selected from the group consisting of polyesters, polyacrylates, polystyrene derivatives, polyamides, polyurethanes, polyacrylonitriles, polytetrafluoroethylenes, silicones, silica particles, cerium oxide, aluminum oxide or apatite particles, polyethylene glycols, polyethylene oxides and polyoxazolines, and copolymers thereof, preferably of various compositions such as random, gradient, alternating, block, graft or star copolymers. More preferably, the at least one polymer is an organic, inorganic, hydrophobic, hydrophilic, amphiphilic, anionic and / or cationic polymer.

[0057] Even more preferably, the polymer is selected from the group consisting of PLGA, PLA, PCL, PGA, PDMAEMA, PMMA, PMAA, PEI, PEtOx, PEG, HPMA, APMA, PVP, hydrolyzed PVP, polysaccharides such as arabinogalactan, chitosan, pullulan, alginates, cellulose or starch derivatives, etc. Polymers according to the invention also include inorganic polymers capable of forming porous particles capable of trapping / encapsulating active principles, and which are preferably silica-, alumina-, titanium oxide-, cerium oxide-, carbon-, zeolite- or apatite-based.

[0058] In a preferred embodiment of the invention, the at least one lipid is selected from the group consisting of saturated and unsaturated fatty acids, cholesterol derivatives, phospholipids, sphingolipids, lipoproteins and glycolipids.

[0059] The at least one polymer and / or the at least one lipid according to the present invention is preferably a biocompatible polymer and / or lipid.

[0060] The nanostructured delivery system preferably comprises a virus-like particle, such as a protein or protein shell. Such virus-like particles preferably comprise a protein shell, preferably derived from, but not limited to, the following viruses: bacteriophage MS2, bacteriophage Qβ, enterobacteriaceae phage P22, cowpea mosaic virus (CPMV), cowpea chlorotic mottle virus (CCMV), hepatitis B virus carries (HBVc), adeno-associated virus (AAV). The proteins are obtained by transfecting the respective viral genetic material into a suitable expression system, such as Saccharomyces cerevisiae, by methods well known in the art.

[0061] Thus, in a preferred embodiment of the present invention, the at least one virus-like particle is derived from a virus selected from the group consisting of bacteriophage MS2, bacteriophage Qβ, enterobacteriaceae phage P22, cowpea mosaic virus (CPMV), cowpea chlorotic mottle virus (CCMV), hepatitis B virus carries (HBVc), and adeno-associated virus (AAV).

[0062] The present invention will now be explained in more detail with reference to figures, but these need not be understood to limit the scope of the invention. [Brief description of the drawings]

[0063] [Figure 1] Figure 1 shows a schematic representation of different constructs of the invention; monovalent and multivalent (currently trivalent) CTMs on ligands or ligand constructs (nanostructured carriers). Attachment is achieved via a spacer or linker moiety. [Diagram 2] Figure 2 shows examples of lectin binding moieties useful for hepatocyte targeting, where "R" represents a possible attachment point for a delivery system (polymer, virus-like particle, lipid, or gene construct). [Diagram 3]FIG. 3 shows a general synthetic approach for the synthesis / attachment of a lectin-binding carbohydrate moiety to a drug, drug construct, carrier polymer, virus-like particle or linker. [Figure 4] FIG. 4 shows exemplary methods for introducing and / or modifying functional groups for attachment of drugs / drug constructs, polymers, and targeting moieties. [Diagram 5] Figure 5 shows strategies for direct coupling of nucleic acid material to CTMs: Figure 5A: 3' end labeling strategy for primarily DNA-like constructs; Figure 5B: 5' end labeling strategy for DNA, RNA, or modified nucleotides. [Figure 6-1] FIG. 6 shows examples of attachment strategies between drugs or drug constructs comprising polymers and / or targeting moieties and / or linkers. [Figure 6-2] FIG. 6 shows examples of attachment strategies between drugs or drug constructs comprising polymers and / or targeting moieties and / or linkers. [Figure 7a] Figure 7 shows exemplary building blocks for generating / preparing a variety of different nanostructured delivery systems useful for the treatment of septic cholestasis. Figure 7a shows various potential compounds that reduce PKC activity (A); [Figure 7b] Figure 7b shows some carbohydrate targeting moieties (CTMs) (B); [Figure 7c] FIG. 7c shows an example of a targeted nanostructured delivery system (C). [Figure 8] Figure 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; essentially, this scheme can be adopted for other carbohydrate derivatives. The carboxy-terminated CTM can be attached to an 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 conventional peptide coupling known to those skilled in the art (e.g., EDC / NHS); as illustrated in Figure 11. [Figure 9] FIG. 9 shows a scheme for the synthesis of trivalent Gal-NAc constructs with a maleimide linker for coupling the construct to thiol groups as shown in FIG. [Figure 10] FIG. 10 shows a scheme for synthesizing amino-terminal trivalent GalNAc constructs. [Figure 11] FIG. 11 shows the coupling of an amino-terminal GalNac (CTM1) to the terminal carboxylic acid of PLGA. [Figure 12] Figure 12 shows the preparation of nanoparticles by emulsion, double emulsion and nanoprecipitation: Figure 12A: emulsion and double emulsion; Figure 12B: nanoprecipitation. [Figure 13] FIG. 13 shows the toxicity of targeted nanoparticles and free drugs (BIM-1, midostaurin and AS605240) in L929 mouse fibroblast cells. [Figure 14] FIG. 14 shows a Kaplan-Meier-Schatzer plot showing mouse survival in a peritoneal contamination and infection (PCI) model using two different stool batches. [Figure 15] Figure 15 shows Kaplan-Meier-Schatzer plots of survival rates of mice treated with PKC activity reducing compounds. These figures show the effect of drugs and targeted nanostructured particles on healthy animals (sham) and animals with PCI. [Figure 16] Figure 16 shows Kaplan-Meier-Schatzer plots of survival rates of mice treated with PKC activity reducing compounds. These figures show the effect of drugs and targeted nanostructured particles on healthy animals (sham) and animals with PCI. [Figure 17] Figure 17 shows Kaplan-Meier-Schatzer plots of survival rates of mice treated with PKC activity reducing compounds. These figures show the effect of drugs and targeted nanostructured particles on healthy animals (sham) and animals with PCI.

[0064] The carbohydrate moiety-induced endocytosis according to the present invention can be employed for tissue-specific transport of drugs. For this purpose, the drug of interest is linked to a linker / spacer that includes an ASGPR-specific recognition ligand or ligand construct. According to the present invention, an inhibitor of the PKC signaling pathway is linked to a polymer or nanostructured delivery system (i.e., polymer particles) directly or by using a spacer that includes an ASGRP-specific recognition ligand or ligand construct as shown in FIG. 1.

[0065] The ASPGR specific recognition ligand may be GalNAc or another liver specific lectin recognition ligand, as shown in Figure 2. These recognition ligands are involved in targeted delivery of drugs, drug constructs or carriers and cell / tissue / organ specificity. As outlined in Figure 2, such recognition ligands are preferably carbohydrate derivatives. They are useful for hepatocyte targeting. The molecules shown are preferred for the construction of CTMs, but larger molecules such as pullulan or arabinogalactan derivatives (shown in Figure 7b) can also be used.

[0066] Different approaches can be applied to attach ASPGR-specific carbohydrate-based recognition sites to drugs, drug constructs or carriers. Depending on the respective drug, drug construct or nanostructured delivery system of the invention and the functional groups present on the respective recognition ligand, the most appropriate method must be evaluated. In the case of carbohydrate derivatives, the appropriate leaving group (e.g. acetate) is preferably further activated by TMS-OTf or HBr and then displaced by various nucleophiles such as alcohols, amines, thiols or C-nucleophiles, as shown in Figure 3.

[0067] In cases where direct coupling is difficult due to lack of suitable attachment points, suitable functional groups are preferably introduced to link drugs / drug constructs or nanostructured delivery systems to carbohydrate targeting moieties according to commonly known functional group interconversion methods as shown in Figure 4. Figure 4 shows the interconversion of carboxylic acids to amines, alcohols to carboxylic acids, and alcohols to maleimides. Carboxylic acids are suitable for coupling to amines and vice versa, while maleimides can be attached to thiols.

[0068] To increase the distance between the targeting moiety and the drug / drug construct, polymer and / or delivery system, the carbohydrate targeting moiety (e.g., ASPGR recognition moiety) can be attached directly or via an additional spacer. An exemplary synthesis is shown in FIG. 11 for the preparation of Gal-NAc PLGA (CTM1-PLGA) useful as a nanocarrier. Further disclosure is given in Example 3. GalNac-labeled PLGA (FIG. 11 and Example 3) is useful for encapsulation of PKC inhibitors by nanoprecipitation, emulsion or double emulsion, preferably as described in Example 4.

[0069] Alternatively, the carbohydrate targeting moiety CTM (here GalNAc) can be attached 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 in a non-targeted carrier system accordingly. After preparation of the nanoparticles, the functional groups in the polymer are activated and attached to the CTM similar to coupling as shown in Figure 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 and a preferred coupling strategy that can be used according to the invention is shown in Figure 6.

[0070] This approach can be adopted for the carrier of the present invention, such as small molecules, nucleic acid constructs such as si-RNA, or organic or inorganic liposomes or nanoparticles.The methods used are known in the art and are described, for example, 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 Ahmed and Narain, Carbohydrate-based materials for targeted delivery of drugs and genes to the liver, Nanomedicine (Lond.) 2015, 10 (14), 2263-2288.

[0071] The carbohydrate targeting moiety (CTM) according to the present invention is preferably attached to the polymeric portion (polymer or virus-like particle) of the nanostructured delivery system, but can also be attached directly to the inhibitor of the PKC signaling pathway prior to formation of the nanostructured delivery system. For example, a carbohydrate targeting moiety containing a maleimide functional group can be attached to the 3' or 5' EndTAG TM The CTM is attached to the nucleic acid construct by known labeling methods such as. In either method, the preferred functional group on the CTM is maleimide, which can be generated as shown in FIG. 9. Two EndTAG coupling strategies for selectively linking nucleic acid constructs to carbohydrate targeting moieties are shown in FIG. 5. FIG. 5A shows a 3'-end labeling strategy for primarily DNA-like constructs; FIG. 5B shows a 5'-end labeling strategy for DNA, RNA, or modified nucleotides. The target nucleic acid construct can be used to form polyplexes with polymers (organic or inorganic) to generate nanostructured delivery systems.

[0072] Figure 6 shows an example of a conjugation strategy of a drug / drug construct with a polymer or targeting moiety according to the present invention. The carbohydrate-driven targeting moiety of the present invention, the selective liver targeting moiety, can be attached to the drug or drug construct of the present invention by a conventional chemical coupling reaction as mentioned above. For the coupling reaction, all reactions familiar to the skilled chemist can be applied. In a preferred embodiment, as shown in Figure 6, a reactive carbonyl compound, preferably a ketone, an aldehyde acetal or a hemiacetal can be used with an amine that forms a Schiff base that can be reduced to the corresponding amine.

[0073] The carbohydrate targeting moiety comprises a chemical moiety that is recognized by a specific recognition unit, preferably a lectin, on the surface of the target tissue, preferably the liver. Preferred lectins for recognition include ASGPR and GalNAc constructs as carbohydrate targeting moieties. In addition, galactose-terminal glycoproteins, arabinoglycans, pullulan, and sitosterol glycosides (also known as sitoG) are useful as lectin recognition constructs. Some representative carbohydrate targeting moieties are shown in Figure 7b.

[0074] Figures 7a-c show exemplary building blocks for preparing a variety of different nanostructured delivery systems according to the present invention, which are useful for the inventive treatment of septic cholestasis by reducing PKC activity. Figure 7a shows different PKC activity reducers that may be used in the building blocks. These PKC activity reducers, as well as small molecules and nucleic acid constructs, can be used according to the present invention. Figure 7b shows different preferred carbohydrate targeting moieties (CTMs) that can be used according to the present invention. Monovalent, trivalent and multivalent. "R" represents the point of attachment to the drug / drug construct or polymer. Possible chemical linkages are shown in Figure 6. The configuration of the trivalent construct is merely exemplary, the chains can also include PEG, amide, triazole or other moieties, and the length of the chains can vary between 2 and 30 atoms. Figure 7c shows different delivery systems carrying carbohydrate targeting moieties (shown as stars). Above are shown polymers (organic or inorganic), lipids, virus-like particles that can act as vehicles for targeted drug delivery. Basically, CTM can be linked to small molecules, nucleic acid constructs, and polyplexes between nucleic acid constructs and positively charged polymers. Such positively charged polymers can also be labeled with the carbohydrate targeting moieties (CTMs) of the present invention, and therefore can form targeted nanostructured delivery systems by themselves after ligation to nucleic acid constructs. Such targeted nanostructured delivery systems are preferably formed when CTMs are directly bound to the inhibitors of the present invention of PKC signaling pathway (preferably nucleic acid constructs), and these constructs form nanostructured delivery systems (with or without helper polymers).

[0075] To mimic septic cholestasis, systemic inflammation was induced using an established peritoneal contamination and infection (PCI) model. In this model, a human fecal suspension is applied intraperitoneally (i.p.) to rapidly induce sepsis accompanied by liver dysfunction. For each batch of human stool, the dose is carefully titrated to achieve a survival rate between 0% and 20% within 2 weeks.

[0076] Different doses were tested to find the appropriate dose of stool. Six hours after intraperitoneal (ip) application of stool, 8-12 week old C57 / BL6 or FVB / N mice were treated with nanoparticles or free drug, respectively. Figure 14 shows the survival of mice with the two different batches used in the Kaplan-Meier-Schatzer plot.

[0077] Figures 15, 16, and 17 show Kaplan-Meier-Schatzer plots of survival of mice treated with PKC activity reducing compounds. These figures show the effect of drugs and targeted nanostructured particles on healthy animals (sham) and animals with PCI. Figure 15 shows the effect of the PKC inhibitor BIM-1 as free drug and as cargo in a targeted nanoparticle formulation.

[0078] FIG. 16 shows the effect of the PI3-kinase inhibitor AS605240 as free drug and as cargo in a targeted nanoparticle formulation.

[0079] FIG. 17 shows the effect of the PKC inhibitor midostaurin as a free drug and as the cargo of a targeted nanoparticle formulation.

[0080] The present invention is further illustrated below on the basis of examples, but the present invention is not limited thereto. EXAMPLES

[0081] Example 1: Synthesis of precursors for the synthesis of CTM (here GalNAc construct) The fully acylated Gal-NAc 1 (1.0 mmol) was activated with TMS-OTf (0.7 mmol) in 5 mL of DCM containing 4 Å molecular sieves (375 mg) in the presence of CBZ-protected aminohexanol 2 (0.9 mmol) for 16 h at room temperature to give the chain-functionalized carbohydrate (3) in 85% yield after aqueous workup and recrystallization from EtOAc. This product 3 (0.85 mmol) was treated with (0.08 mmol) 25%-NaOMe solution in 5 mL of MeOH. After stirring with Amberlite resin (500 mg) for 1 h, filtration, and removal of the solvent, all acyl-protected hydroxyl groups were completely deprotected in quantitative yield. The CBZ-amine 4 (0.85 mmol) was deprotected by catalytic hydrogenation under atmospheric pressure hydrogen with 20% Pd / C (20 mg) in 5 mL of MeOH. After filtration and removal of the solvent, the desired product 5 was obtained quantitatively.

[0082] Example 2: Synthesis of trivalent Gal-NAc constructs A: Maleimide-functionalized trivalent Gal-NAc for direct coupling to nucleic acid constructs via introduced SH groups. (EndTAG® Labeling) As outlined in Figure 9, the amino triester (1 g) is dissolved in 10 mL of DMF and 5 equivalents of HBTU and DIEA are added at room temperature. Under nitrogen, 1 equivalent of 5-maleimidovaleric acid is added and stirred at room temperature for 24 hours. The reaction mixture is poured into 250 mL of 10% NaHCO3 and extracted three times with ethyl acetate. The combined organic phases are evaporated to dryness and dissolved in 25 mL of DCM with 1 M TFA and stirred for 24 hours. After evaporation of the solvent, the residue is dissolved in 300 mL of acetone and 3.5 equivalents of NaOMe are added. The precipitated compound is filtered off, dried and used without further purification. 1 g of the trisodium salt is dissolved in water, acidified to pH 2 and extracted three times with chloroform. The combined organic phases are evaporated to a final volume of 50 mL and 5 equivalents of Pfp-TFA and 20 equivalents of DIEA are added to the resulting tri-acid and the reaction mixture is stirred for 2 hours. After reaction, the mixture is poured into 500 mL of water, extracted 3 times with 200 mL of EtOAc, washed with brine, separated, dried over MgSO4, and evaporated to dryness. The resulting gum is crystallized from hexane / ethyl acetate to give a beige solid.

[0083] The Tris-Pfp ester is dissolved in THF, 5 equivalents of amino-GalNAc monomer is added, and stirred for 20 minutes. The reaction mixture is filtered and evaporated to dryness. The resulting oil is subjected to column chromatography using CHCl3 / MeOH 9:1 to obtain the final trivalent GalNAc maleimide construct (detected by KMnO4 or concentrated sulfuric acid). The synthetic scheme is shown in Figure 9.

[0084] Direct Coupling of Genetic-Based Inhibitors to Carbohydrate Targeting Moieties (EndTaq®) According to vectorlabs®, 1 μg of PKC-siRNA (custom made by JenaBioscience) is incubated in reaction buffer with T4 polynucleotide kinase and ATPγS for 30 minutes at 37°C. The reaction is purified using a ThermoFischer RNA purification kit and carefully stored as required for RNA. (Cold, sterile, 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 at 65°C for 30 minutes. The final construct is purified again under sterile conditions using a ThermoFischer RNA purification kit.

[0085] B: Amine-functionalized trivalent Gal-NAc for coupling to carboxylic acid derivatives (here PLGA) As outlined in Figure 10, aminotriester 10 (1.00 mmol) was dissolved in 10 mL of DCM and HBTU (5.00 mmol), DIEA (5.00 mmol) were added at room temperature. Under nitrogen, CBZ-5-aminovaleric acid 15 (5.00 mmol) was added and stirred at room temperature for 24 h. The reaction mixture was poured into 250 mL of 10% aqueous NaHCO3 and extracted three times with ethyl acetate. The combined organic phase was evaporated to dryness. The residue was dissolved in 25 mL of toluene containing 1 mL of phosphoric acid and stirred for 15 h. After aqueous workup, the combined organic phase was evaporated. Product 16 was dissolved in 20 mL of DMF and 5 equivalents of Pfp-TFA and 20 equivalents of DIEA were added to the solution. After stirring at room temperature for 16 h, the reaction mixture was quenched with saturated NH4Cl solution (10 mL) and extracted three times with DCM. After removal of the solvent, the residue was purified by flash column chromatography using n-hexane / EtOAc 3:1 as eluent to give Tris-Pfp ester 17 (Rf=0.27).

[0086] Tris-Pfp ester 17, 4 equivalents of EDC·HCl, and 0.1 equivalent of DMAP were dissolved in 15 mL of DCM. After 1 h reaction time, 3.5 equivalents of fully acylated carbohydrate 3 bearing an amine chain were added to the reaction mixture and stirred at room temperature for 12 h. The reaction mixture was quenched by adding 5 mL of water. After aqueous workup and solvent removal, the resulting oil was subjected to column chromatography using CHCl3 / MeOH 9:1 as eluent to give the fully protected trivalent GalNAc construct 18.

[0087] The product 18 (0.85 mmol) was treated with a solution of (0.08 mmol) 25%-NaOMe in 5 ml of MeOH. After stirring with Amberlite resin (500 mg) for 1 h, filtration and removal of the solvent, all acyl-protected hydroxyl groups were completely deprotected in quantitative yield. The crude product was carried to the next step without further purification. The CBZ group was cleaved by catalytic hydrogenation under atmospheric pressure hydrogen using 20% ​​Pd / C (20 mg) in 5 mL of MeOH. After filtration and removal of the solvent, the desired product 19 was purified by semi-prep C18 RP-HPLC using acetonitrile with 0.1% TFA as the eluent.

[0088] Example 3: Coupling of amino-functionalized Gal-NAc (CTM1) to PLGA as a delivery system. PLGA (Resomer RG 502 H, MW: 12.000, 100 mg, 8.33 μmol) was dissolved in 300 μL of DMSO. 60 μl (1 equiv, 8.33 μmol) of EDC·HCl solution (26.8 mg, dissolved in 1.00 mL of DMSO) and 60 μl (1 equiv, 8.33 μmol) of NHS solution (17.0 mg, in 1.00 mL of DMSO) were added to this solution in succession. After stirring for 3 h at room temperature, the solution was poured into a DMSO solution of CTM1 (5) (16 mg, 6.0 equiv, 49.97 μmol, in 300 μL of DMSO). The solution was stirred for 16 h. Triethylamine (10 μL, 16 equiv, 121.55 μmol) was added to this solution. After 3 hours, the solution was neutralized by adding glacial acetic acid (12 μL, 27 equiv., 223.81 μmol). After 5 min, the solution was poured into water (25 mL). The precipitate was washed several times with water and lyophilized. The CTM1-labeled PLGA was used for nanoprecipitation or emulsion procedures to encapsulate the appropriate drugs. In the case of nucleic acid derivatives, polyplexes consisting of PEI or any other basic polymer are formed. The encapsulation is then carried out via double emulsion in Gal-Nac PLGA. The coupling of CTM1 to PLGA is outlined in FIG. 11.

[0089] Example 4: Preparation of nanoparticles After functionalization of the polymer with carbohydrate targeting moieties (see Example 5), nanoparticles were produced by nanoprecipitation using polyvinyl alcohol (PVA) as a surfactant. The polymer and the PKC inhibitor BIM-1 or midostaurin or the PI3K inhibitor AS605240 were dissolved in DMSO, and the solution was slowly added dropwise to a vigorously stirred 0.3% aqueous solution of PVA. The nanoparticles formed contain 4 wt% BIM-1, 6 wt% midostaurin, or 10 wt% AS605240 encapsulated in Gal NAc-targeted (CTM1) PLGA. The solution was purified and concentrated by cross-flow filtration. The preparation method of the nanoparticles of the present invention by emulsion, double emulsion and nanoprecipitation is further exemplarily shown in FIG. 12.

[0090] To demonstrate cell / tissue targeting, neutral lipid orange (DYOMICS) is encapsulated in the same procedure instead of the PKC reducer. Evaluation and visualization of hepatocyte targeting is performed according to the intravital microscopy method described in WO 2015 / 035974, the disclosure of which is incorporated herein by reference in its entirety.

[0091] Example 5: Characterization of the nanoparticles of the invention Nanoparticles of Gal-Nac-PLGA were generated using certain parameters and reproduced according to the following protocol. - Size: Measurement of the size of various nanostructured delivery systems dissolved in deionized water by dynamic light scattering (eg Zetasizer (Malvern Instruments GmbH)) or electron microscopy. - Shape: Determination of shape by electron micrographs. - Charge: Measurement of different nanostructured delivery systems dissolved in deionized water using a Zetasizer (Malvern Instruments GmbH) by measuring the electrophoretic signal (zeta potential, surface charge). - Endotoxin: Endotoxin content was determined using a Charles River test kit based on the LAL chromogenic assay according to DE 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. - Hemolysis: measurement of the hemoglobin concentration of red blood cells incubated with particles in a physiological buffer for 1 hour. If there is damage to the red blood cell membrane, there will be an increase in the measurable hemoglobin concentration in the supernatant. - Agglutination: measurement of the absorption of red blood cells incubated with a polymer in a physiological buffer. Samples containing cell aggregates show lower absorption than homogeneously distributed non-agglutinated cells.

[0092] result: A: Non-targeted nanoparticles (PLGA / PVA) containing 2.5% encapsulated neutral lipid orange. B: CTM1-targeted nanoparticles from Example 4 containing 4% BIM-1 (PKC inhibitor) C: CTM1-targeted nanoparticles from Example 4 containing 10% AS605230 (PI3 kinase inhibitor) D: CTM1-targeted nanoparticles from Example 4 containing 7% midostaurin (PKC inhibitor)

[0093] Table 1: TIFF0007672972000001.tif30170

[0094] Example 6: Static macrophage assay and dynamic chip-based microfluidic model for hepatocyte targeting and interaction with macrophages A macrophage assay was used to investigate whether unwanted uptake and / or effects of nanoparticles by macrophages occurs. Interaction between NPs and macrophages can significantly reduce the efficacy of NPs. Furthermore, the interaction can lead to activation of macrophages, thereby potentially harming all host and surrounding tissues. Therefore, the interaction between NPs and macrophages must first be proven. Particle size, shape, coating, and surface charge are important determinants. Two assays were performed under static conditions.

[0095] A. Human peripheral blood mononuclear cell (PBMC) culture and macrophage differentiation. PBMC were freshly isolated immediately after drawing donor blood from healthy volunteers. Donors were informed about the purpose of the study and gave written informed consent. Blood sample volume was diluted in a 1:1 ratio with calcium- and magnesium-free PBS (Biochrom AG, Germany) containing 0.1% bovine serum albumin (BSA, Carl Roth, Germany) and 2 mM EDTA (Sigma-Aldrich, Germany; isolation buffer) and carefully placed on top of Biocoll separation solution (Biochrom AG, Germany). PBMC were obtained from density gradient centrifugation. Subsequently, cells were washed several times in isolation buffer and finally filtered through a 40 μm molecular mesh (BD Bioscience, Germany). For monocyte enrichment, wells (9,6 cm 2 ) per 10 7 PBMCs were plated in 6-well plates (or in smaller wells at equivalent cell density) in 2mL of X-VIVO15 (Lonza, Germany) supplemented with 10% autologous serum, 10ng / mL GM-CSF (PeproTech, Germany), 100 units / mL penicillin, and 100μg / mL streptomycin (Life Technologies, Germany). After 3 hours of incubation, cells were washed with plain X-VIVO 15 medium and fresh medium (as above) containing supplements was added. Macrophage (Mf) differentiation was carried out for 5 days, including the preparation time for nanoparticle experiments.

[0096] A1. Culture and differentiation of mouse macrophage cell line RAW264.7 RAW264.7 macrophages (CLS, Eppelheim, Germany) were cultured at 75 cm in a humidified 5% CO2 / 95% air atmosphere at 37 °C. 2The cells were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 100 units / mL penicillin, and 100 μg / mL streptomycin in 100 mL cell culture flasks. Medium changes were performed after 2–4 days (depending on the cell culture density). For the experiments, macrophages were detached by Accutase treatment, seeded, and cultured for 24 h, and then the particles, i.e., neutral lipid orange-loaded NPs, were incubated for individual periods in phenol red-free medium. After incubation, macrophages were harvested and / or lysed and then subjected to individual analyses (i.e., by a microplate reader equipped with a fluorescence detection system). Protein content was analyzed using a BCA assay (Thermo Fisher Scientific, USA).

[0097] To obtain more meaningful data compared to static single-cell cultures, several scalable co-culture models were used, which more closely resemble the in vivo situation than static single-cell cultures.

[0098] A2. Co-culture of endothelial cells and macrophages According to Rinkenauer AC 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, nanoparticles (NPs) were tested in a co-culture model of endothelial cells and macrophages under physiological shear stress conditions. Briefly, monocytes were cultured at 4 mg mL -1Monocytes were harvested 24 h after isolation by treatment with 1 mM 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. Monocytes and HUVECs were subsequently cultured in 10% autologous serum, 10 ng mL -1 of GM-CSF and 100UmL -1 Penicillin and 100 μg mL -1 Pool 1:3 in endothelial growth medium MV supplemented with streptomycin and incubate at 1.3x10 5 HUVECs cm-2 and 0.43x10 5 Monocytes cm 2 The cells were seeded in the diamond-chamber chip at a density of 0.7, 3.0, 6.0, and 10.0 dyn cm. The medium was changed daily. Mf differentiation was performed in the presence of GM-CSF under static culture conditions for 72 h. HUVECs were perfused using a peristaltic pump (Ismatec REGLO digital MS-CA-4 / 12-100, Germany). The shear stress in the diamond-chamber chip 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). -2 Shear stress of 200 μg mL -1 The nanoparticles were applied for 24 h after uptake of the nanoparticles for 60 min at a concentration of 0.1 mg / mL. Negatively charged nanoparticles containing Nile Red were dissolved in additive-free endothelial cell growth medium MV.

[0099] B. Dynamic42 Sinusoid-Chip based microfluidic model Cell specificity and targeting are determined in a chip-based microfluidically supported multi-cell culture system consisting of macrophages, hepatocytes, stellate cells, and endothelial cells. Cell culture and assembly of the Dynamic42 Sinusoid model was performed according to Rennert K. et al, A microfluidically perfused three-dimensional human liver model, Biomaterials 2015; 71 :1 19-131.

[0100] Preparation of HepaRG and endothelial cells for the Dynamic42 Sinusoid model 2.7x10 HepaRG cells 4 Cells / cm 2 Cells were seeded at a density of 1000 x 1000 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). Cells were cultured in a humidified cell incubator at 37°C with 5% CO2 for 14 days before differentiation. The medium was refreshed every 3-4 days. Cell differentiation was induced and cells were used for up to 4 weeks.

[0101] Endothelial cells: Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical veins. Donors were informed of the purpose of the study and gave written consent. HUVEC cells were cultured at 2.5 x 10 4 Cells / cm 2 and cultured in endothelial cell medium (ECM) (Promocell, Heidelberg, Germany) until passage 4.

[0102] Preparation of LX-2 stellate cells and macrophages for the Dynamic42 Sinusoid model LX-2 stellate cells (kindly provided by Scott L. Friedman, Division of Hepatology, Mount Sinai School of Medicine, New York, NY, USA) were cultured at 2.0 × 10 4 Cells / cm 2 Cells were seeded at a density of 1.0 × 10 cells / well 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 cultured at a density of 1.0 × 10 cells / well 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. 6 Cells / cm 2 After 3 h of incubation in a humidified cell incubator at 5% CO2 and 37°C, the cells were washed twice with X-VIVO15 medium. The attached monocytes were cultured in X-VIVO15 medium for 24 h and seeded into the hepatic sinusoids.

[0103] Assembly of Dvnamic42 Sinusoid The hepatic sinusoidal model was assembled by seeding alternating layers of blood vessels and hepatic cells. In each sterile biochip, 2.7x10 5 HUVECs / cm 2 (Total 3.0 10 5 cells) and 0.9x10 5 pieces / cm 2 of monocytes (total 1x10 5HUVECs / monocytes (2.7x10 cells) were mixed and seeded on top of the membrane of the upper chamber. HUVECs / monocytes were co-cultured for at least 3 days with daily medium changes in endothelial cell culture medium (ECM) supplemented with 10ng / ml epidermal growth factor, 90mg / ml heparin, 2.8mM hydrocortisone, endothelial cell growth supplement, 10ng / ml GM-CSF, 10ng / ml M-CSF to induce macrophage differentiation, 100U / ml penicillin / 100mg / ml streptomycin and 10% (v / v) autologous human serum (Life Technologies, Karlsruhe, Germany). Subsequently, 2.7x10 cells were co-cultured for at least 3 days with daily medium changes. 5 pieces / cm 2 of differentiated HepaRG (total 3x10 5 cells) and 0.9x10 4 pieces / cm 2 LX-2 (total 1x10 4 Cells) were seeded on the membrane opposite the HUVEC cells and cultured for 24 h in DMSO-free William's medium E (Biochrom, Berlin, Germany) hepatocyte growth medium containing 10% (v / v) FBS, 2 mM glutamine, and 100 U / ml penicillin / 100 mg / ml streptomycin, with 50 μM hydrocortisone and 5 μg / ml insulin, before use in experiments.

[0104] Table 2: Dimensions of the sinusoidal tip TIFF0007672972000002.tif57170

[0105] Table 3: Flow velocity within the sinusoidal chip TIFF0007672972000003.tif42170

[0106] The hepatic sinusoidal model was equilibrated after 7 days of static culture by perfusion at a flow rate of 50 μl / min for up to 72 hours. Subsequently, drug constructs and controls (at least in triplicate) were incubated in the hepatic sinusoidal model under variable dynamic conditions for individual periods. Afterwards, the hepatic sinusoids were fixed with paraformaldehyde and / or methanol and analyzed by immunofluorescence staining. The different cell layers were examined by fluorescence microscopy to analyze the enrichment of the constructs in or on the different cell types. Furthermore, it is possible to separately dissolve the vascular cell layer and the hepatic cell layer and measure the cell-specific uptake of nanoparticles by a microplate reader equipped with a fluorescence detection system.

[0107] Example 7: Determination of Cytotoxicity Cytotoxicity tests were performed with L929 mouse fibroblasts and HepG2 cells (human hepatoma cell line) as recommended by IS010993-5. Cells were seeded at 104 cells per well in 96-well plates in Dulbecco's modified Eagle's medium (DMEM, Lonza, Basel) supplemented with 10% fetal calf serum (FCS), 100 U / mL penicillin, 100 mg / mL streptomycin and incubated for 24 h at 37 °C in a humidified 5% (v / v) CO2 atmosphere. Test substances (polymers) at the indicated concentrations (0.5 μg / mL to 50 μg / mL) were added to the cells and the plates were incubated for another 24 h. Control cells were incubated in fresh medium. Afterwards, the medium was replaced by a mixture of fresh medium and Alamar Blue solution (PrestoBlue for mouse fibroblasts) (Life technologies, Darmstadt, Germany) prepared according to the manufacturer's instructions. After a further incubation at 37°C for 4 h (30 min for PrestoBlue), the fluorescence was measured at Ex 570 / Em 610 nm (560 / 590 for PrestoBlue) using untreated cells on the same well plate as a negative control. The negative control was normalized as 0% of metabolic inhibition and taken as 100% viability. Cell viability below 70% was considered to indicate cytotoxicity. Data are expressed as mean ± SD of triplicate determinations. After 24 h, Figure 13 shows more or less similar toxicity for all drugs, regardless of the formulation. This observation reflects the limited stability of the nanoparticles in this experimental setting. Nearly identical results were obtained using HeGP2 cells (data not shown).

[0108] Example 8: Survival in a Cholestatic Model Under Septic Conditions "Peritoneal Contamination and Infection (PCI)" Experimental setup: Sepsis with systemic infection / organ failure was induced in male C57 / BL6 mice by using the PCI model. For this purpose, human fecal suspensions (2.5 μl / g BW for fecal batch 1 and 6 μl / g for fecal batch 2, respectively) were injected intraperitoneally (without anesthesia) in weight-adapted doses, thus inducing peritonitis with subsequent systemic infection. To avoid animal burden and mortality, the broad-spectrum antibiotic meropenem was administered subcutaneously (2.5 μg / g BW) twice a day starting 6 h after infection. Animals were closely monitored and scored every 6 h for signs of infection to ensure timeliness. For fecal batch 1, 70% of mice died within the first 2 days at the dose of 2.5 μg / g, and the remaining 30% died by day 7 (Figure 14, left panel). At the evaluated dose of 6 μl / g BW stool and additional antibiotic therapy, all mice died within 3 days, as shown in the Kaplan-Meier-Schatzer plot (FIG. 14, right panel). The experimental data are partly based on the batch 1 experiment and partly based on the batch 2 experiment. Details are given in FIG. 14.

[0109] For dose determination, three drug concentrations per formulation were tested in small groups and changes in survival rates were documented. Free drug was used for dose evaluation (data not shown) and 1 / 8 of the effective dose was used in the targeted nanoparticles. The PI3K inhibitor AS605240 and the PKC inhibitor BIM-1 alone were active at 4 mg / kg body weight. In nanoparticles we used 0.5 mg / kg, with more pronounced effects in all cases. For midostaurin, 6 mg / kg of free drug and 0.75 mg / kg were used in the nanoparticle formulation.

[0110] Six hours after infection (PCI model), treatment is performed with different drugs capable of reducing the activity of PKC (BIM-1 and midostaurin as PKC inhibitors and AS605240 as PI3 kinase inhibitor) or with control preparations (once a day, intraperitoneally or intravenously) and a combination of dose and antibiotic therapy (twice a day, subcutaneously). Treatment with drugs is scheduled for 5 days. The dose / antibiotic therapy is performed for 7 days (2 days longer than drug therapy). Observations during the first 5 days are performed 24 hours a day, with 3-hour intervals. After that, observation of the animals continues until the 14th day (twice a day).

[0111] 9a) CTM1-targeted PLGA nanoparticles with BIM-1 as a typical PKC inhibitor as cargo: We prepared nanoparticles as described in Example 5 using the synthesized PTM1-PLGA, PVA as a surfactant, and BIM-1 at the following concentrations / loading efficiencies: PTM-PLGA: 56% PVA: 40% BIM-1: 4% Size / Zeta potential: 72nm / -0.2 The particle suspension was diluted with 45% glucose solution to give a final glucose concentration of 5%. Ten mice were treated with the targeted nanoparticles and evaluated with two sham mice to test the tolerability of the nanoparticles in healthy mice. The results are shown in Figure 15 and show that survival increased from 10% to 60% within 7 days.

[0112] 9b) PTM-targeted PLGA nanoparticles with AS605240 as an experimental Pi3K inhibitor as cargo: Particles were prepared similarly to Example 5 with slightly modified parameters: we prepared nanoparticles as described above using the synthesized PTM-PLGA, PVA as surfactant, and AS605240 at the following concentrations / loading efficiencies: PTM-PLGA: 58% PVA: 32% AS605240:10% Size / Zeta potential: 93nm / -2

[0113] The particle suspension was diluted with 45% glucose solution to give a final glucose concentration of 5%.

[0114] Six mice were treated with the targeted nanoparticles and evaluated with two sham mice to test the tolerability of the nanoparticles in healthy mice. The results are shown in Figure 16 and show that survival increased from 0% to 40% within 7 days.

[0115] 9c) PTM1-targeted PLGA nanoparticles using midostaurin as an approved kinase inhibitor with significant PKC inhibition as cargo: Particles were prepared similarly to Example 5 with slightly modified parameters: we prepared nanoparticles as described above using the synthesized PTM-PLGA, PVA as surfactant, and midostaurin at the following concentration / loading efficiency: PTM-PLGA: 41% PVA: 52% Midostaurin: 7% Size / Zeta potential: 185nm / -1

[0116] The particle suspension was diluted with 45% glucose solution to give a final glucose concentration of 5%.

[0117] Five mice were treated with the targeted nanoparticles and evaluated with two sham mice to test the tolerability of the nanoparticles in healthy mice.

[0118] The results are shown in Figure 17 and demonstrate that survival increased from 10% to 60% within 7 days.

Claims

1. A selective nanostructured delivery system for targeting an inhibitor of the PKC signaling pathway into the liver for use in the treatment of septic cholestasis, the selective nanostructured delivery system comprising 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, the at least one carbohydrate targeting moiety being chemically bound to the polymer or lipid nanoparticle or liposome or to the virus-like particle, the inhibitor of the PKC signaling pathway being encapsulated or entrapped in the selective nanostructured delivery system, the carbohydrate targeting moiety being selected from the group consisting of N-acetylgalactosamine (GalNAc), galactose, lactose, mannose, glucosamine, asialofetuin, pullulan, arabinogalactan, glycyrrhizin, and glycyrrhetinic acid.

2. The selective nanostructured delivery system of claim 1 , wherein the carbohydrate targeting moiety binds to a recognition unit located on the liver.

3. The selective nanostructured delivery system of claim 2, wherein the recognition unit is a receptor, preferably a lectin, more preferably the asialoglycoprotein receptor (ASGPR) or also known as the Ashwell-Morell receptor.

4. The selective nanostructure delivery system of any one of claims 1 to 3, wherein the inhibitor is selected from the group consisting of a PKC inhibitor, a PI3 kinase inhibitor, a MAPK inhibitor, a PLC inhibitor, a DAG level reducer, and an inhibitor comprising siRNA, shRNA, miRNA, morpholino, an antisense construct, or RNase H.

5. 5. The selective nanostructured delivery system of claim 4, wherein the inhibitor is a PKC inhibitor selected from the group consisting of bisindolylmaleimides, staurosporine, midostaurin, UCN-01, sotrastaurin, enzastaurin, ruboxistaurin, tivantinib, enzastaurin, Go6983, K252a, ANA-12, lestaurtinib, stauprimide, CEP-701, Arcyriaflavin a, or inhibitors including bisindolylmaleimides I-XII, also known as BIM I-XII.

6. 5. The selective nanostructured delivery system of claim 4, wherein the inhibitor is a PI3 kinase inhibitor selected from the group consisting of inhibitors including copanlisib, idelalisib, wortmannin, bryostain, taselisib, omipalisib, AS605240, GSK1059615, bupallisib, alpelisib, pictilisib, ceravilisib, dactolisib, dihydrosphingosine, calphostin C, or melittin.

7. The selective nanostructured delivery system of claim 1 , wherein the inhibitor directly or indirectly inhibits or reduces the activity of PKC or a PKC subtype.

8. The selective nanostructured delivery system of any one of claims 1 to 7, wherein at least one polymer is selected from the group consisting of polyesters, polyacrylates, polystyrenes, polyamides, polyurethanes, polyacrylonitriles, polytetrafluoroethylenes, silicones, silica particles, cerium oxide, aluminum oxide or apatite particles, polyethylene glycols, polyethylene oxides and polyoxazolines, and copolymers thereof, preferably copolymers of various compositions such as random, gradient, alternating, block, graft or star copolymers.

9. 9. The selective nanostructured delivery system of claim 8, wherein at least one polymer is an organic, inorganic, hydrophobic, hydrophilic, amphiphilic, anionic and / or cationic polymer.

10. 10. The selective nanostructured delivery system of claim 8 or 9, wherein at least one polymer is selected from the group consisting of PLGA, PLA, PCL, PGA, PDMAEMA, PMMA, PMAA, PEI, PEtOx, PEG, HPMA, APMA, PVP, hydrolyzed PVP, and polysaccharides.

11. The selective nanostructured delivery system of any one of claims 1 to 10, wherein at least one lipid is selected from the group consisting of saturated and unsaturated fatty acids, cholesterol, phospholipids, sphingolipids, lipoproteins and glycolipids.

12. 12. The selective nanostructured delivery system of any one of claims 1 to 11, wherein at least one virus-like particle is derived from a virus selected from the group consisting of bacteriophage MS2, bacteriophage Qβ, enterobacteriaceae phage P22, cowpea mosaic virus (CPMV), cowpea chlorotic mottle virus (CCMV), hepatitis B virus carrier (HBVc), and adeno-associated virus (AAV).

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