PKC inhibitors for the treatment of septic cholestasis by ctm targeting

Abstract

To provide a selective nanostructure delivery system for targeting an inhibitor of the PKC signaling pathway into the liver for use in the treatment of septic cholestasis.SOLUTION: The selective nanostructure delivery system comprises a carbohydrate targeting moiety and a PKC signaling pathway, wherein the inhibitor of the PKC signaling pathway is covalently linked, directly or through a linker or spacer moiety, to the carbohydrate targeting moiety, wherein the carbohydrate targeting moiety is selected from the group consisting of N-acetylgalactosamine (GalNAc), galactose, lactose, mannose, glucosamine, asialofetuin, pullulan, arabinogalactan, arabinoglycan, fuconic acid, lactobionic acid, transferrin, sitosterol glycoside (sitoG), glycyrrhizin, and glycyrrhetinic acid.SELECTED DRAWING: 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 caused by mechanical obstruction of the ductal system and displacement of the bile duct, which can result from, for example, gallstones, tumors such as pancreatic cancer, bile duct cysts, bile duct strictures, or parasites; and intrahepatic cholestasis (non-obstructive cholestasis), in which the cause of bile stasis is internal to the liver. Intrahepatic cholestasis can occur due to genetic defects or can develop laterally as a side effect of many medications, such as nonsteroidal anti-inflammatory drugs (NSAIDs), antihypertensives, antidiabetics, anticonvulsants, lipid-lowering drugs, anabolic steroids, psychotropic drugs, and various antibiotics. 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 can also occur during parenteral nutrition, pregnancy, and after liver transplantation. As a result of reduced bilirubin excretion, patients with cholestasis exhibit symptoms of jaundice. Depending on the cause, cholestasis may be accompanied by gastrointestinal symptoms such as itching (pruritus), pale stools, dark urine, nausea, vomiting, and pain. Cholestatic liver disease is diagnosed by marked elevations of serum alkaline phosphatase and bilirubin, although serum bilirubin may remain normal until the late stages of the disease. Although frank intrahepatic cholestasis is rare, it is a typical symptom of sepsis. In Germany, for example, approximately 30,000 people develop sepsis-related organ failure, and 3–6% of these patients develop a condition called septic cholestasis or sepsis-induced cholestasis, characterized by the additional symptom of jaundice. The mortality rate in the first 12 months after diagnosis is 92%, much higher than other sepsis-related organ failures (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, defined as life-threatening organ dysfunction caused by a dysregulated host response to pathogenic infection, is a major public health concern, accounting for more than $20 billion in total hospital costs in the United States in 2011. Sepsis can be considered a leading cause of mortality and critical illness worldwide. Furthermore, patients who survive sepsis often suffer long-term physical, psychological, and cognitive impairments with significant health care and societal impacts. The pathogens involved in the development of sepsis can be of various origins and can result from bacterial, viral, fungal, or protozoan infections. Sepsis is not only defined by systemic inflammation, but more importantly, it is the patient's dysregulated response to this infection, which causes organ dysfunction and accentuates the overall severity of the condition. Sepsis also involves alterations in pro- and anti-inflammatory responses, cardiovascular, neurological, autonomic, hormonal, bioenergetic, metabolic processes, and coagulation. Finally, organ failure or dysfunction contributes to the high mortality rate associated with sepsis. Therefore, a scoring system called the "Sequential Organ Failure Assessment" (SOFA) has been developed to assess 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. Early antibiotic eradication of the underlying infection has been shown to be paramount for patient survival. Additional therapeutic considerations include avoidance of parenteral nutrition, avoidance of hepatotoxic drugs, monitoring blood glucose levels and, if necessary, adequate provision, and extracorporeal liver support (i.e., albumin dialysis). For example, immunocompromised patients after organ transplantation, cancer treatment, or treatment for autoimmune diseases are at increased risk not only for infections caused by common pathogens, but also for opportunistic infections caused by less virulent microorganisms that are of little concern to patients with intact immune systems. Therefore, it is 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 subsequent impairment of bile excretion (Zollner G., Trauner M.; Mechanism of cholestasis, Clin Liver Dis 2008;12:1-26). Consequently, jaundice can be observed in septic patients as a result of intrahepatic (non-obstructive) cholestasis. The challenge is to timely distinguish between sepsis-related and non-sepsis-related causes of cholestasis (see above). Usually, symptoms of sepsis dominate the clinical picture before the onset of septic cholestasis. Uncontrolled infection can lead to reduced function and expression of important hepatocellular transport proteins, resulting in reduced bilirubin excretion and jaundice (Zollner G., Trauner M., Ic). Septic cholestasis is associated with 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. Hepatic excretory dysfunction can have a variety of causes: carcinoma, cysts in the bile duct, liver inflammation (e.g., hepatitis), fibrosis or cirrhosis, fatty liver (alcoholic or non-alcoholic), and side effects of certain medications (e.g., anabolic drugs, antipsychotics, and certain antibiotics). In the case of septic cholestasis, an underlying systemic infection causes a disturbance of the entire immune system, inducing liver secretory dysfunction. Therefore, sepsis-induced cholestasis represents a complication of systemic infection. Currently, the only effective treatment for septic cholestasis is treatment of the underlying sepsis with antibiotic therapy for the infection, which should be initiated 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, treatment typically involves the use of maximum tolerated doses of combinations of different broad-spectrum antibiotics, because diagnosis of the causative pathogen (blood culture) is often not possible within an acceptable time frame.

[0005] Antibiotic therapy for systemic infections is associated with known problems and is certainly not a cure for organ failure resulting from a dysregulated response to infection. Furthermore, some antibiotics may interfere with or block bile excretion, thus inducing cholestasis (see above). The most prominent examples are amoxicillin and erythromycin. High-dose, broad-spectrum antibiotic therapy can lead to the emergence of new antibiotic-resistant pathogens, hindering the success of future therapy. Furthermore, it must be considered that antibiotic therapy is pointless or even counterproductive when 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 medications, and / or direct damage to major organs, primarily the kidneys and liver, especially when organ function is impaired.

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

[0007] Although the effects of kinase inhibitors, particularly PKC inhibition, on cholestasis have been described (Anwer MS; Role of protein kinase C isoforms in bile formation and cholestasis, Hepatology 2014; 60(3): 1090-1097), treating sepsis with kinase inhibitors can be counterproductive and can 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 intrahepatic cholestasis, bile formation from hepatocytes is impaired. Bile formation is a complex process involving many different transhepatic solute transporters, most notably the sodium-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, regulated by posttranslational events, particularly by kinases such as protein kinase C (PKC), phosphoinositide 3-kinase (PI3K), AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase (MAPK). Various studies 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 significantly affect cell proliferation and immune cell signaling, acting as immunosuppressants.

[0009] Kinase inhibitors and sepsis As outlined above, sepsis is a severe and complex systemic immune response triggered 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 immune responses in cases of inflammation. Accordingly, it has been suggested that excessive inflammatory responses can be treated with such compounds in cases of sepsis (see, e.g., USH1168H, U.S. Pat. No. 5,558,969, and WO 93 / 16703, supra). However, it must be recognized that, on the one hand, the underlying infection itself is not treated by such proposed treatments, and, on the other hand, suppressing the body's immune system is certainly undesirable for conditions associated with infections. As already mentioned, immunocompromised patients are at increased risk for infections caused by 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, explicitly 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 (Doc.Ref.EMEA / 150964 / 2007) of 24 May 2007, page 13, concerning the PKC inhibitor ruboxistaurin (drug name Arxxant; indicated for diabetic retinopathy); Medscape, a website for medical professionals, notes that the PI3K inhibitor copanlisib (drug name Aliqopa, indicated for recurrent follicular lymphoma) should be withheld in the event of an 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) recommends withholding treatment with the PI3K inhibitor idelalisib (drug name: Zydelig; indicated for three classes of lymphoma) in the event of infection, specifically sepsis; the regulation states: "Interrupt idelalisib until the infection resolves." This is also stated in Zelenetz et al., Lancet Oncol. 2017, 18:297-311. They note a five-fold increased risk of developing sepsis as an adverse effect during idelalisib treatment compared with placebo. Novartis Pharma GmbH (2017): Rydapt® 25 mg Weichkapseln, Fachinformation (professional information), status September 2017, provides prescribing information highlights for the PKC inhibitor midostaurin (drug name RYDAPT; indicated for AML [acute myeloid leukemia]). The right column on page 1 and Table 2 on page 5 list adverse reactions of midostaurin; the last paragraph on page 6 states that "Grade 3 or higher (≥3) adverse reactions reported in ≥5% (≥5%) of patients were fatigue, sepsis, gastrointestinal bleeding, pneumonia, diarrhea, and febrile neutropenia... (Table 4)." Page 7 states: "Treatment discontinuation due to adverse reactions occurred in 21% of patients. The most common 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 foregoing, it is clear that kinase inhibitors should not be administered systemically when sepsis is present. This is a teaching, and as a result, one 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, 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 because adverse effects of immunomodulation, especially when such drugs are administered systemically, can lead to life-threatening conditions.

[0012] It is therefore an object of the present invention to provide an effective treatment for 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 for 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 transporters involved in bile formation. These compounds are targeted into the liver by a unique and selective delivery system.

[0014] In a 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 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. 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, 1c). 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, regulated by post-translational events, particularly by protein kinases such as protein kinase C (PKC), phosphoinositide 3-kinase (PI3K), AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase (MAPK). Various studies have shown that PKC or PI3K inhibitors are useful preclinical tools for the treatment of cholestasis (Anwer, lc; Toledo et al., lc; Li et al., lc). 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+ ) is highly dependent 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 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 for treating septic cholestasis.

[0018] From a biochemical perspective, 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 thus inhibit downstream PKC. For this reason, other drugs that can reduce DAG levels may also be useful in treating septic cholestasis.

[0019] Thus, the term "inhibitor 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 intracellularly. Such inhibitors include in particular inhibitors of kinases involved in the PKC signaling pathway.

[0020] The term "inhibitor of the PKC signaling pathway" further refers to 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, or AKT are 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 the 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 PKC activity according to the present invention means influencing its activity with PKC inhibitors, including nucleic acid constructs that silence the respective genes, preferably via RNAi. This can be achieved by commonly known methods, preferably using constructs such as siRNA, miRNA, shRNA, RNAse H, modified oligomers such as morpholinos, etc. According to known biochemical approaches, the respective nucleic acid constructs are designed and directly linked to carbohydrate targeting moieties by covalent bonding or by encapsulation in nanocarriers bearing the carbohydrate targeting moieties.

[0024] Indirect inhibition or reduction of PKC activity according to the present invention is preferably achieved by inhibiting or reducing 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 activity are useful tools for treating septic cholestasis. Therefore, all types 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 the treatment of septic cholestasis. This can also be achieved by small molecule inhibitors such as the 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 an effect 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. Two PKC inhibitors are currently available as approved drugs: Rydapt® (midostaurin) and Arxxant® (ruboxistaurin).

[0027] Further known drugs are idelalisib and copanlisib, both of which, according to a specialist information site (Medscape Reference: see above), are strictly contraindicated in cases of severe infections, including sepsis, due to their immunosuppressive properties, and it is recommended to discontinue treatment during these times.

[0028] To date, no specific treatment for septic cholestasis is available in the clinic, and treatment with any of the above systemically administered agents 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 for septic cholestasis itself, 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 the treatment of septic cholestasis, achieved by carbohydrate-derived targeting moieties that are selectively recognized by specialized 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, the 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 for septic cholestasis itself.

[0031] To treat septic cholestasis as a condition involving an underlying systemic infection, i.e., sepsis, it is undesirable to suppress the immune system. Therefore, the present invention describes a carbohydrate-driven selective delivery system for targeted delivery of PKC signaling pathway inhibitors into the liver with negligible systemic immunosuppressive effects. This is because they act selectively in the liver, specifically modifying biliary excretion and reversing cholestasis. In this way, the agents of the present invention, i.e., PKC signaling pathway inhibitors / PKC activity inhibitors, can be administered at much lower doses than their non-targeted counterparts and delivered only to the site of action, making them suitable for the treatment of septic cholestasis. Therefore, the present invention represents a highly effective method for treating septic cholestasis, significantly reducing the systemic adverse effects that commonly occur when kinase inhibitors are administered to treat advanced infections.

[0032] The term "drug" or "therapeutic agent" according to the present invention refers to an inhibitor of the PKC signaling pathway; furthermore, the term "drug" or "agents" is used synonymously with "anticholestatic agent" and "cholestatic agent" and the term "drug" or "drugs". Furthermore, the terms "drug" and "drug" 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, characterized by the fact that certain units (monomers) are repeating units. Polymers are covalently bonded to each other through chemical reactions (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. A carbohydrate targeting moiety (CTM) according to the present invention refers to a chemical structure that is recognized by a specific surface molecule (e.g., a lectin), preferably an ASGP receptor, and induces internalization of the construct, drug, or nanostructured delivery system, i.e., the nanostructured delivery system according to the present invention, into cells, tissues, or organs in which these surface molecules are expressed, preferably the liver. CTMs can be either monovalent or multivalent, depending on the labeling density on the surface of the drug or drug construct or polymer. In a multivalent configuration, 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. CTMs with higher MW and multiple repeating units (e.g., pullulan or arabinogalactan) can form nanostructured carrier systems by themselves, but can also be attached to nanostructured carriers.

[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 by specific ASGPR antibodies.

[0039] Consequently, 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 via clathrin-mediated endocytosis.

[0042] X-ray crystallography has shown that ASGPR possesses a shallow binding cavity, which is best targeted by multivalent ligand constructs. Such multivalent structures are state-of-the-art and can be configured in different ways and with different substructures (see Figure 2). Sanhueza et al., Ic, outlines possible configurations for multivalent ligands, but essentially, any molecule with a connection point (to a drug / drug construct / polymer) and three additional connection points for the smallest unit of carbohydrate targeting may be suitable (see Figure 1). Depending on the overall surface density of the targeting moiety, different configurations may be optimal. If the surface labeling of the nanoparticles is sufficiently high, monovalent ASGPR 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 an appropriate model for tissue endocytosis, such as 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 a PKC inhibitor, a PI3 kinase inhibitor, a MAPK inhibitor, a PLC inhibitor, a DAG level reducer, an siRNA, an miRNA, an shRNA, a modified oligo analog (e.g., morpholino), an antisense construct, and RNAse H.

[0044] The inhibitors siRNA, miRNA, shRNA, modified oligo analogs (e.g., morpholinos), antisense constructs, and RNAse H according to the present invention relate to oligonucleotide constructs capable of silencing the respective genes (e.g., silencing the expression of PKC genes, PI3 kinase genes, MAPK genes, and PLC genes) 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, and 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, bupalisib, alpelisib, pictilisib, cerabilisib, 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, can be directly attached to spacers or linkers comprising aliphatic, heteroaliphatic, aromatic, heteroaromatic, linear, branched, or cyclic atomic aggregates, and / or carbohydrate targeting moieties, or can 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 reactions, Michael additions (1,4-additions), cycloaddition reactions, Huisgen reactions (e.g., 1,3-cycloadditions of alkynes to azides), Diels-Alder reactions (e.g., trans-cyclooctene coupling to tetrazine derivatives), maleimide-thiol reactions, isocyanate-, isothiocyanate-, carbodiimide-, or chloroacetamide-coupling. Alternatively, reactive carbonyl compounds, preferably ketones, aldehyde acetals, or hemiacetals, can be used in accordance with the present invention, forming Schiff bases that can be reduced to the corresponding amines (see, e.g., Figure 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 involves 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—in this 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, cryo-TEM, and AFM. Their shape can be, but is not limited to, spherical, ellipsoidal, rod-shaped, barrel-shaped, disc-shaped, or polyhedral. Their size preferably varies from 1 nm to 800 nm.

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

[0056] In a preferred embodiment of the present invention, the at least one polymer is selected from the group consisting of polyesters, polyacrylates, polystyrene derivatives, polyamides, polyurethanes, polyacrylonitrile, polytetrafluoroethylene, 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. 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 entrapping / encapsulating active ingredients, 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, e.g., a protein or protein shell. Such virus-like particles preferably comprise a protein shell 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 (HBVc), and adeno-associated virus (AAV). Proteins can be obtained by transfecting the respective viral genetic material into a suitable expression system, such as Saccharomyces cerevisiae, using methods well known in the art.

[0061] Accordingly, 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, which, however, need not be understood to limit the scope of the invention. [Brief explanation 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. [Figure 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). [Figure 3]FIG. 3 shows a general synthetic approach for the synthesis / attachment of lectin-binding carbohydrate moieties to drugs, drug constructs, carrier polymers, virus-like particles or linkers. [Figure 4] FIG. 4 shows exemplary methods for introducing and / or modifying functional groups for attachment of drugs / drug constructs, polymers, and targeting moieties. [Figure 5] Figure 5 shows strategies for direct coupling of nucleic acid materials 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] Figure 7c shows an example of a targeted nanostructured delivery system (C). [Figure 8] Figure 8 shows synthetic routes to carboxy- and amine-functionalized GalNAc (CTM1) derivatives. For the design of monomeric carbohydrate-based targeting units, a 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, while the amine-terminated CTM (CTM1) can be coupled to any carrier, polymer, lipid, protein, or drug via conventional peptide coupling (e.g., EDC / NHS) known to those skilled in the art; 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 amino-terminal GalNac (CTM1) to the terminal carboxylic acid of PLGA. [Figure 12] Figure 12 shows the preparation methods 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 in mice treated with PKC activity-reducing compounds. These figures show the effects 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 in mice treated with PKC activity-reducing compounds. These figures show the effects 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 in mice treated with PKC activity-reducing compounds. These figures show the effects of drugs and targeted nanostructured particles on healthy animals (sham) and animals with PCI.

[0064] The carbohydrate moiety-induced endocytosis of the present invention can be employed for tissue-specific drug delivery. For this purpose, the drug of interest is conjugated to a linker / spacer containing an ASGRP-specific recognition ligand or ligand construct. According to the present invention, an inhibitor of the PKC signaling pathway is conjugated to a polymer or nanostructured delivery system (i.e., polymer particles) directly or using a spacer containing an ASGRP-specific recognition ligand or ligand construct, as shown in Figure 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 and cell / tissue / organ specificity of drugs, drug constructs, or carriers. 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 conjugating ASPGR-specific carbohydrate-based recognition sites to drugs, drug constructs, or carriers. The most appropriate method must be evaluated depending on the functional groups present in the respective drug, drug construct, or nanostructured delivery system of the present invention and the respective recognition ligand. In the case of carbohydrate derivatives, a suitable leaving group (e.g., acetate) is preferably further activated with TMS-OTf or HBr and then displaced by various nucleophiles, such as alcohols, amines, thiols, or C-nucleophiles, as shown in Figure 3.

[0067] When direct coupling is difficult due to a lack of suitable attachment points, suitable functional groups are preferably introduced according to commonly known functional group interconversion methods to link drugs / drug constructs or nanostructured delivery systems to carbohydrate targeting moieties, 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, a carbohydrate targeting moiety (e.g., an ASPGR recognition moiety) can be attached directly or via an additional spacer. An exemplary synthesis is shown in Figure 11 for the preparation of Gal-NAc PLGA (CTM1-PLGA), useful as a nanocarrier. Further disclosure is provided in Example 3. GalNac-labeled PLGA (Figure 11 and Example 3) is useful for encapsulating PKC inhibitors, preferably by nanoprecipitation, emulsion, or double emulsion, as described in Example 4.

[0069] Alternatively, a carbohydrate targeting moiety, CTM (here, GalNAc), can be attached to the final particle (nanostructured delivery system) after encapsulation of the inhibitor of the present 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 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 in accordance with the present invention is shown in Figure 6.

[0070] This approach can be used 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 method used is known in the art and is 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 (London) 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 (1) or (2). In either method, the preferred functional group on the CTM is maleimide, which can be generated as shown in Figure 9. Two EndTAG coupling strategies for selectively attaching nucleic acid constructs to carbohydrate targeting moieties are shown in Figure 5. Figure 5A shows a 3'-end labeling strategy primarily for DNA-like constructs; Figure 5B shows a 5'-end labeling strategy for DNA, RNA, or modified nucleotides. The targeted 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 or selective liver targeting moiety of the present invention can be attached to the drug or drug construct of the present invention by conventional chemical coupling reactions, such as those mentioned above. For the coupling reaction, all reactions familiar to skilled chemists can be applied. In a preferred embodiment, as shown in Figure 6, a reactive carbonyl compound, preferably a ketone, aldehyde acetal, or hemiacetal can be used together with an amine to form 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 a target tissue, preferably the liver. Preferred lectins for recognition include ASGPR and GalNAc constructs as carbohydrate targeting moieties. Additionally, galactose-terminal glycoproteins, arabinoglycans, pullulans, and sitosterol glycosides (also known as sitoG) are useful as lectin recognition constructs. Figure 7b shows some representative carbohydrate targeting moieties.

[0074] Figures 7a-c show exemplary building blocks for preparing various 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. They are 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 trivalent construct configuration is merely exemplary; the chains can also include PEG, amide, triazole, or other moieties, and the chain length can vary between 2 and 30 atoms. Figure 7c shows different delivery systems carrying carbohydrate targeting moieties (shown as asterisks). Shown above are polymers (organic or inorganic), lipids, and virus-like particles that can act as vehicles for targeted drug delivery. Essentially, the 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 themselves form targeted nanostructured delivery systems after ligation to nucleic acid constructs. Such targeted nanostructured delivery systems are preferably formed when the CTMs directly bind to the inhibitors of the PKC signaling pathway of the present invention (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 of 0% to 20% within 2 weeks.

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

[0077] Figures 15, 16, and 17 show Kaplan-Meier-Schatzer plots of survival in mice treated with PKC activity-reducing compounds. These figures show the effects of drug 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 targeted nanoparticle formulations.

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

[0080] The present invention is further illustrated below with reference to examples, but the present invention is not limited thereto. [Example]

[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. After aqueous workup and recrystallization from EtOAc, the chain-functionalized carbohydrate (3) was obtained in 85% yield. This product 3 (0.85 mmol) was treated with 25% NaOMe solution (0.08 mmol) in 5 mL of MeOH. After stirring with Amberlite resin (500 mg) for 1 h, filtration, and solvent removal, all acyl-protected hydroxyl groups were completely deprotected in quantitative yield. CBZ-amine 4 (0.85 mmol) was deprotected by catalytic hydrogenation under atmospheric pressure using 20% ​​Pd / C (20 mg) in 5 mL of MeOH. After filtration and solvent removal, 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 aminotriester (1 g) was dissolved in 10 mL of DMF and 5 equivalents of HBTU and DIEA were added at room temperature. Under nitrogen, 1 equivalent of 5-maleimidovaleric acid was added and stirred at room temperature for 24 hours. The reaction mixture was poured into 250 mL of 10% NaHCO3 and extracted three times with ethyl acetate. The combined organic phases were evaporated to dryness, dissolved in 25 mL of 1 M TFA in DCM, and stirred for 24 hours. After evaporation of the solvent, the residue was dissolved in 300 mL of acetone and 3.5 equivalents of NaOMe were added. The precipitated compound was filtered off, dried, and used without further purification. 1 g of the trisodium salt was dissolved in water, acidified to pH 2, and extracted three times with chloroform. The combined organic phases were evaporated to a final volume of 50 mL. 5 equivalents of Pfp-TFA and 20 equivalents of DIEA were added to the resulting tri-acid, and the reaction mixture was stirred for 2 hours. After the reaction, the mixture is poured into 500 mL of water, extracted three 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 the mixture is 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 Jena Bioscience) is incubated in reaction buffer with T4 polynucleotide kinase and ATPγS at 37°C for 30 minutes. The reaction is purified using a ThermoFischer RNA purification kit and carefully stored, as required for RNA. (Cold, 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 at 65°C for 30 minutes. The final construct is again purified 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) and DIEA (5.00 mmol) were added at room temperature. CBZ-5-aminovaleric acid 15 (5.00 mmol) was added under nitrogen and stirred at room temperature for 24 h. The reaction mixture was poured into 250 mL of 10% aqueous NaHCO3 solution 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 the 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 of reaction time, 3.5 equivalents of fully acylated carbohydrate 3 bearing an amine chain were added to the reaction mixture, which was then 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 the eluent to afford the fully protected trivalent GalNAc construct 18.

[0087] This product 18 (0.85 mmol) was treated with a solution of 25% NaOMe (0.08 mmol) in 5 mL of MeOH. After stirring with Amberlite resin (500 mg) for 1 h, filtration, and solvent removal, 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 using 20% ​​Pd / C (20 mg) in 5 mL of MeOH. After filtration and solvent removal, the desired product 19 was purified by semi-prep C18 RP-HPLC using acetonitrile containing 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 sequentially to this solution. After stirring at room temperature for 3 h, 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 equivalents, 223.81 μmol). After 5 minutes, 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 appropriate drugs. In the case of nucleic acid derivatives, polyplexes consisting of PEI or any other basic polymer are formed. Encapsulation is then carried out via double emulsion in Gal-Nac PLGA. The coupling of CTM1 to PLGA is outlined in Figure 11.

[0089] Example 4: Preparation of nanoparticles After functionalization of the polymer with carbohydrate-targeting moieties (see Example 5), nanoparticles were generated 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 PVA solution. The formed nanoparticles contained 4 wt% BIM-1, 6 wt% midostaurin, or 10 wt% AS605240 encapsulated in GalNAc-targeted (CTM1) PLGA. The solution was purified and concentrated by cross-flow filtration. The methods for preparing nanoparticles of the present invention by emulsion, double emulsion, and nanoprecipitation are further illustrated in Figure 12.

[0090] To demonstrate cell / tissue targeting, neutral lipid orange (DYOMICS) was encapsulated in place of the PKC reducer using the same procedure. Evaluation and visualization of hepatocyte targeting was 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 nanoparticles of the present invention Nanoparticles of Gal-Nac-PLGA were produced 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 various nanostructured delivery systems dissolved in deionized water using a Zetasizer (Malvern Instruments GmbH) by measuring the electrophoretic signal (zeta potential, surface charge). Endotoxin: The 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 hemoglobin concentration in red blood cells incubated with particles in physiological buffer for 1 hour. If the red blood cell membrane is damaged, the measurable hemoglobin concentration in the supernatant increases. - 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 uniformly 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: TIFF2026000924000002.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 occur. Interactions between NPs and macrophages can significantly reduce the efficacy of NPs. Furthermore, interactions can lead to macrophage activation, which can subsequently harm the host and surrounding tissues. Therefore, the interaction between NPs and macrophages must first be demonstrated. Particle size, shape, coating, and surface charge are important determining factors. Two assays were performed under static conditions.

[0095] A. Human peripheral blood mononuclear cell (PBMC) culture and macrophage differentiation. PBMCs were freshly isolated immediately after donor blood collection from healthy volunteers. Donors were informed of the purpose of the study and provided written informed consent. Blood sample volume was diluted 1:1 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 isolation solution (Biochrom AG, Germany). PBMCs were obtained by 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) were used. 2 ) per 10 7 PBMCs were plated in 6-well plates (or smaller wells at equivalent cell densities) in 2 mL of 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). After a 3-hour incubation, cells were washed with simple X-VIVO 15 medium, and fresh medium containing the supplements (as described above) was added. Macrophage (Mf) differentiation was performed for 5 days, including 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% CO / 95% air atmosphere at 37°C. 2Macrophages 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 cell culture flasks. Medium changes were performed after 2–4 days (depending on cell confluency). For experiments, macrophages were detached by Accutase treatment, seeded, and cultured for 24 hours. Then, particles, i.e., Neutral Lipid Orange-loaded NPs, were incubated in phenol red-free medium for individual periods. 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 the BCA assay (Thermo Fisher Scientific, USA).

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

[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 under physiological shear stress conditions in a co-culture model of endothelial cells and macrophages. Briefly, monocytes were cultured at 4 mg mL -1Monocytes were harvested 24 hours after isolation by treatment with 5 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) in serum-free X-VIVO 15 for 45 minutes. Subsequently, monocytes and HUVECs were cultured in 10% autologous serum, 10 ng mL -1 of GM-CSF and 100UmL -1 Penicillin and 100 μg mL -1 Pool 1.3x10 cells at 1:3 in endothelial growth medium MV supplemented with streptomycin 5 HUVECs cm-2 and 0.43x10 5 monocytes cm 2 Cells were seeded into diamond-chamber chips at a density of 0.7, 3.0, 6.0, and 10.0 dyn cm. The medium was changed daily. Mf differentiation was performed under static culture conditions in the presence of GM-CSF for 72 hours. HUVECs were perfused using a peristaltic pump (Ismatec REGLO digital MS-CA-4 / 12-100, Germany). Shear stress within 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 hours after uptake of the nanoparticles for 60 minutes at a concentration of 1000 mg / mL. Negatively charged nanoparticles containing Nile Red were dissolved in additive-free endothelial cell growth medium MV.

[0099] B.Dvnamic42 Sinusoid-Chip based microfluidic model Cell specificity and targeting are determined in a chip-based microfluidically supported multicellular culture system consisting of macrophages, hepatocytes, stellate cells, and endothelial cells. Cell culture and assembly of the Dynamic42 Sinusoid model were 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 μg / ml 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% CO 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 provided 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 Liver Diseases, Mount Sinai School of Medicine, New York, NY, USA) were added to 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 hours of incubation in a humidified cell incubator at 5% CO2 and 37°C, the cells were washed twice with X-VIVO15 medium. Adherent monocytes were cultured in X-VIVO15 medium for 24 hours and then seeded into the liver sinusoids.

[0103] Assembly of Dvnamic42 Sinusoid The liver sinusoidal model was assembled by seeding alternating layers of blood vessels and hepatocytes. In each sterile biochip, 2.7 x 10 cells were 5 HUVECs / cm 2 (Total 3.0 10 5 cells) and 0.9x10 5 pieces / cm 2 of monocytes (total 1x10 5HUVECs and monocytes (2.7 x 10 cells) were mixed and seeded on the membrane of the upper chamber. HUVECs and monocytes were co-cultured for at least 3 days with daily medium changes 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 x 10 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 (number of cells) were seeded on the membrane opposite the HUVEC cells and cultured for 24 hours 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, containing 50 μM hydrocortisone and 5 μg / ml insulin, before use in experiments.

[0104] Table 2: Dimensions of the sinusoidal tip TIFF2026000924000003.tif57170

[0105] Table 3: Flow velocity within the sinusoidal chip TIFF2026000924000004.tif42170

[0106] The hepatic sinusoid model was perfused at a flow rate of 50 μl / min for up to 72 hours and then allowed to equilibrate after 7 days of static incubation. Subsequently, drug constructs and controls (at least in triplicate) were incubated in the hepatic sinusoid model under variable dynamic conditions for individual periods. The hepatic sinusoids were then fixed with paraformaldehyde, methanol, or both and analyzed by immunofluorescence staining. Different cell layers were examined under a fluorescent microscope to analyze the enrichment of the constructs in or on different cell types. Furthermore, the vascular and hepatic cell layers could be separately lysed and cell-specific nanoparticle uptake measured using a microplate reader equipped with a fluorescence detection system.

[0107] Example 7: Determination of cytotoxicity Cytotoxicity tests were performed using L929 mouse fibroblasts and HepG2 cells (human hepatoma cell line) as recommended by ISO 10993-5. 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, and 100 mg / mL streptomycin and incubated at 37°C in a humidified 5% (v / v) CO2 atmosphere for 24 hours. The test substances (polymers) were added to the cells at the indicated concentrations (0.5 μg / mL to 50 μg / mL), and the plates were incubated for an additional 24 hours. Control cells were incubated in fresh medium. The medium was then replaced with 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 4 h of incubation at 37 °C (30 min for PrestoBlue), fluorescence was measured at Ex 570 / Em 610 nm (560 / 590 for PrestoBlue) using untreated cells in the same well plate as a negative control. The negative control was normalized to 0% metabolic inhibition and assigned a viability of 100%. Cell viability below 70% was considered to indicate cytotoxicity. Data are presented as the mean ± SD of triplicate determinations. After 24 h, Figure 13 shows more or less similar toxicity for all drugs, regardless of 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 and organ failure was induced in male C57 / BL6 mice using the PCI model. To this end, human fecal suspension (2.5 μl / g BW for fecal batch 1 and 6 μl / g BW for fecal batch 2, respectively) was injected intraperitoneally (without anesthesia) at weight-adapted doses, resulting in 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 daily starting 6 hours after infection. Animals were closely monitored and scored every 6 hours for signs of infection to ensure timeliness. For fecal batch 1, 70% of mice died within the first 2 days at the 2.5 μg / g dose, 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 provided 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 the nanoparticles, we used 0.5 mg / kg, which in all cases resulted in even more pronounced effects. 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 was performed with different drugs capable of reducing PKC activity (BIM-1 and midostaurin as PKC inhibitors and AS605240 as a PI3 kinase inhibitor) or a control formulation (once daily, intraperitoneally or intravenously) and a combination of dose and antibiotic therapy (twice daily, subcutaneously). Drug treatment was scheduled for 5 days. The dose / antibiotic therapy was performed for 7 days (2 days longer than drug treatment). Observation for the first 5 days was performed 24 hours a day, with 3-hour intervals. Animals were then observed until the 14th day (twice daily).

[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 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 demonstrate an increase in survival 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 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 demonstrate an increase in survival rate 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 concentrations / loading efficiencies: PTM-PLGA: 41% PVA: 52% Midostaurin: 7% Size / Zeta potential: 185nm / -1

[0116] The particle suspension was diluted with 45% glucose solution to 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. 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, 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.

2. 2. The inhibitor of PKC signaling pathway for use according to claim 1, wherein 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.

3. 3. An inhibitor of the PKC signaling pathway for use according to claim 1 or 2, wherein the carbohydrate targeting moiety binds to a recognition unit located on the liver.

4. The inhibitor of the PKC signaling pathway for use according to claim 4, wherein the recognition unit is a receptor, preferably a lectin, more preferably the asialoglycoprotein receptor (ASGPR) or also known as the Ashwell-Morell receptor.

5. 5. The inhibitor of the PKC signaling pathway for use according to any one of claims 1 to 4, 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, an siRNA, an shRNA, an miRNA, a modified oligo analogue, an antisense construct, and an RNAse H.

6. 6. The inhibitor of the PKC signaling pathway for use according to claim 5, 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, and bisindolylmaleimides I-XII, also known as BIM I-XII.

7. 6. The inhibitor of the PKC signaling pathway for use according to claim 5, wherein the inhibitor is a PI3 kinase inhibitor selected from the group consisting of copanlisib, idelalisib, wortmannin derivatives, bryostain derivatives, taselisib, omipalisib, AS605240, GSK1059615, bupallisib, alpelisib, pictilisib, cerabilisib, dactolisib, dihydrosphingosine, calphostin C and melittin.

8. 8. An inhibitor of the PKC signalling pathway for use according to any one of claims 1 to 7, wherein the inhibitor directly or indirectly inhibits or reduces the activity of a PKC or a PKC subtype.

9. 9. The inhibitor of the PKC signaling pathway for use according to any one of claims 1 to 8, wherein the at least one polymer is selected from the group consisting of polyesters, polyacrylates, polystyrene derivatives, polyamides, polyurethanes, polyacrylonitrile, polytetrafluoroethylene, silicones, silica particles, cerium oxide, aluminum oxide or apatite particles, polyethylene glycol, polyethylene oxide and polyoxazolines, and copolymers thereof, preferably copolymers of various compositions such as random, gradient, alternating, block, graft or star copolymers.

10. 10. The inhibitor of the PKC signaling pathway for use according to claim 9, wherein at least one polymer is an organic, inorganic, hydrophobic, hydrophilic, amphiphilic, anionic and / or cationic polymer.

11. The inhibitor of the PKC signaling pathway for use according to claim 9 or 10, 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.

12. 12. An inhibitor of the PKC signaling pathway for use according to any one of claims 1 to 11, wherein at least one lipid is selected from the group consisting of saturated and unsaturated fatty acids, cholesterol derivatives, phospholipids, sphingolipids, lipoproteins and glycolipids.

13. 13. The inhibitor of PKC signaling pathway for use according to any one of claims 1 to 12, wherein 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).

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