PKC inhibitors for the treatment of septic cholestasis by targeting polymethine dyes

Abstract

The present invention relates to inhibitors 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 polymethine dye and at least one polymer and / or at least one lipid and / or at least one virus-like particle, wherein the at least one polymethine dye is a symmetric or asymmetric polymethine. [Selection diagram] None

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. 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, a scoring scale called "Sequential Organ Failure Assessment" (SOFA) 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 for 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, notes 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, 118, 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 or higher (≥3) adverse reactions reported in 5% or more (≥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] The present invention in a first aspect thereof 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 polymethine dye and at least one polymer and / or at least one lipid and / or at least one virus-like particle, wherein the at least one polymethine dye is a symmetric or asymmetric polymethine of general structure I or II: TIFF0007672973000001.tif90170 formula, an represents the numerical value 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; b. R1-R17 may be the same or different and may be hydrogen or deuterium, one or more alkyl, tert-alkyl, cycloalkyl- ("alkyl" and "cycloalkyl" radicals also include olefinic structures) or aryl, carboxyaryl, dicarboxyaryl, heteroaryl or heteroalicyclic radicals, alkyloxy, alkylmercapto, arlyoxy, arylmercapto, heteroaryloxy, heteroarylmercapto groups, hydroxyl, halogen, nitro or cyano groups, alkyl-substituted or cyclic amine functional groups, and / or two ortho-positioned radicals, e.g., R3 and R4, R13 and R14, and / or R1 and R2, and R11 and R12, and / or R7 and R9, may together form an additional aromatic, heteroaromatic, aliphatic or heteroaliphatic ring; c. At least one of the R1-R17 substituents determines the hydrophilic properties of these polymethine dyes, SO3 - , (-SO3H), PO3 2- , COOH, OH or NR3 + , a cyclodextrin or a sugar, which may also be attached to the polymethine dye by a spacer group; d. at least one of the R1-R17 substituents has a reactive group (linker) such as isocyanate, isothiocyanate, hydrazine, amine, mono- and dichloro- or mono- and dibromo triazine, aziridine, epoxide, sulfonyl halides, acid halides, carboxylic acid anhydrides, N-hydroxy-succinimide esters, imido esters, carboxylic acids, glyoxal, aldehydes, maleimides or iodoacetamides and phosphoramidite derivatives or azides, alkynes or olefins, where this substituent may also be linked to the polymethine dye by a spacer group; e. The aromatic, heteroaromatic, aliphatic or heteroaliphatic spacer group is [(CH2) a -Y-(CH2) b ] c or [(C6H4) a -Y-(C6H4) b ], where Y may be the same or different and contain a CR2-, O-, S-, -SO2, SO2NH-, NR-, COO- or CONR functional group, which is attached to one of the R1-R17 substituents, a.) and b.) may be the same or different and have a numerical value of 0-18, and c has a numerical value of 0-18, f. The corresponding R8 and R9 substituents with n=2, 3, 4 or 5 may also be present 2, 3, 4 or 5 times, which may be the same or different.

[0015] In a preferred embodiment of the invention, at least one polymethine dye is an ICG derivative.

[0016] Thus, a preferred embodiment of 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 ICG derivative, and at least one polymer and / or at least one lipid and / or at least one virus-like particle, wherein the at least one ICG derivative or ICG-like polymethine dye is a symmetric or asymmetric polymethine of general structure I or II as disclosed herein (as shown and further defined above and in the claims).

[0017] The term "ICG derivative" refers to any polymethine dye having two indole moieties according to structures I and II, where at least one residue is available for coupling reaction and the net charge after coupling is -1 or + / -0.

[0018] Thus, the ICG derivatives, ICG-like polymethine dyes, ICG-based polymethine dyes according to the present invention relate to symmetric or asymmetric polymethine dyes corresponding to the general structures I or II as disclosed herein.

[0019] The terms "ICG derivatives", "ICG-like polymethine dyes" and "ICG-based polymethine dyes" are used synonymously with the same meaning.

[0020] 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.

[0021] 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 the basal site of the hepatocyte.

[0022] 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., lc; Li et al., lc). These kinase inhibitors significantly affect cell proliferation and immune cell signaling, acting as immunosuppressants.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Direct inhibition or reduction of the activity of PKC according to the present invention means influencing the activity by 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 morpholino, etc. According to known biochemical approaches, the respective nucleic acid constructs are designed and directly linked to polymethine-based targeting moieties, either by covalent bonding or by encapsulation in nanocarriers carrying the polymethine-based targeting moieties.

[0030] 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.

[0031] 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.

[0032] Currently, PKC inhibitors are primarily used in the treatment of cancer and autoimmune diseases. Currently, two PKC inhibitors are available as tested drugs: Rydapt® (midostaurin) and Arxxant® (ruboxistaurin).

[0033] 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.

[0034] 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.

[0035] 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 the therapeutic agent 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 targeting moieties derived from polymethine dyes 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.

[0036] 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.

[0037] 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 polymethine dye-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, in which the systemic adverse effects that commonly occur when kinase inhibitors are administered in the treatment of frontline infections are greatly reduced.

[0038] 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 the terms "anticholestatic agent" and "cholestatic agent" and "drug" or "drugs". Further, the terms "agent" and "drug" are used synonymously according to the present invention.

[0039] 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.

[0040] 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).

[0041] 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."

[0042] Polymethine dyes or polymethine-like dyes such as indocyanine green (ICG) are known to interact with several organic anion transporting polypeptides (OATPs) that are mainly present in the liver. Therefore, ICG is widely used in liver function tests. The so-called indocyanine green plasma disappearance rate reflects the capacity of biliary excretion rate and is used as a standard method in clinics.

[0043] Furthermore, it is known that some polymethine dyes or polymethine-based dyes, such as indocyanine green (ICG), can be used as photosensitizers in cancer therapy, since these compounds generate singlet oxygen and free radicals when excited by light. Therefore, such dyes are often described not only for diagnosis, but also for model compounds in the photodynamic therapy of cancer.

[0044] Almost all dyes, regardless of their structure, favorably accumulate in cancer tissues due to the so-called “enhanced permeability and retention” or EPR effect of spontaneously formed dye-liposome conjugates (Ngoune et al., Accumulating nanoparticles by EPR: A route of no return, J. Contr. Rel. 2016).

[0045] The EPR effect means that tumor tissue is usually characterized by enhanced angiogenesis and leaky vasculature that facilitates the entry of nanoparticles or albumin particles. In addition, lymphatic flow is usually greatly reduced in tumor tissue. These two factors ultimately contribute to the accumulation of particles such as liposomes and albumin or lipoprotein conjugated small molecules. When such particles accumulate in tumor tissue, no active carrier or transport system is involved, so this process is called "passive targeting". The EPR effect has been widely utilized in cancer therapy and diagnosis: For diagnostic reasons, dyes (e.g. Evans Blue, ICG, Toluidine Blue, Fluorescein) For photodynamic therapy of tumors, several NIR dyes are used that are complexed with lipoproteins or albumin. For enhanced tumor treatment, drugs are encapsulated in liposomes or particles to enhance accumulation in tumor tissue. For example, the approved drug Doxel®.

[0046] Keeping in mind the EPR effect, it is clear that this type of targeting is not an active process and that the dyes described above cannot be used for active tumor targeting, even if particles containing these dyes accumulate within tumors.

[0047] However, some polymethine-based dyes, such as indocyanine green (ICG), are actively and rapidly taken up into the liver by OATP-mediated endocytosis, and this property could be considered for active hepatocyte / liver targeting. However, ICG cannot be conjugated to polymers or lipids by chemical means to engineer an active liver / hepatocyte-targeted nanostructured delivery system, because the ends of the alkyl chains contain two sulfonate residues that represent the "ends" of chemical modification or coupling.

[0048] Therefore, the design and synthesis of polymethine dyes with modified ICG-like scaffolds that can be coupled to other chemical moieties (i.e., polymers, proteins, small molecules) using known coupling reaction methods is a key step of the present invention. Furthermore, the dyes are designed to have a net charge of -1 or ±0 after coupling.

[0049] The inventors have found that hepatocyte targeting of ICG or ICG-like polymethine dyes is highly dependent on the ratio of the net charges of the molecules under physiological conditions and the surface or shape of the molecules; and not just the scaffold. When ICG-like polymethine dyes are coupled to polymers or lipids or carriers with functional groups, the net charges of the unbound functional groups are lost during the coupling reaction. Thus, the ratio of the net charges on the dye molecules is changed during coupling, and hepatocyte targeting is reduced or lost.

[0050] For the design of ICG or ICG-like polymethine dyes covalently bound to polymers or lipids or carriers, therefore, when charged groups are used in the coupling reaction, it is necessary to use coupling positions that do not contribute charge or to introduce additional charged groups. Functional groups that generate a negative net charge under physiological conditions are preferably sulfate, sulfonate, sulfonamide, phosphate, phosphonate, borate, boronate, BR4, perchlorate, or carboxylic acid, strong CH-, OH-, or NH-acidic groups. Functional groups that generate a positive net charge under physiological conditions are preferably amines, quaternary salts, guanidines, amidines and their respective quaternary salts, oxonium ions, pyridinium salts, pyridines. All these groups can also be part of a cyclic structure (e.g., indolinium ions, tetrazolium or cyclic oxonium ions, pyrylium salts). The use of metal salt complexes to generate a permanent charge may also be applicable (eg, complexed zinc).

[0051] For OATP-mediated targeting of hepatocytes, we found that the overall charge of the conjugated construct should be zero or -1. Since the chemical nature of the compound inherently results in one permanent positive charge on the indole nitrogen, this can be compensated by a permanently charged residue such as a sulfonate or phosphonate residue. Hydrogen phosphates or hydrogen sulfates are also possible, but are less stable. General structures I and II shown in Figure 2 and included in the claims show the structures of polymethine dyes with two indole moieties.

[0052] The cell selectivity of the polymethine dyes of the present invention is obtained through the specific interaction of the polymethine dyes bound to nanoparticles or liposomes or virus-like particles with the liver-specific influx transporters, which are defined for hepatocytes.

[0053] The polymethine dyes according to the invention, which are specifically taken up by influx transporters in the basolateral membrane of hepatocytes, make the nanoparticles specific to hepatocytes. According to current information and the FDA, the following exemplary transporters fall into the category of influx transporters in hepatocytes: TIFF0007672973000002.tif99170

[0054] Consequently, a preferred embodiment of the present invention relates to an inhibitor of the PKC signaling pathway for use in the treatment of septic cholestasis, wherein at least one polymethine dye has at least one tissue-specific transporter that induces uptake of the nanostructured delivery system into cells of a target tissue. According to a further preferred embodiment of the present invention, said at least one tissue-specific transporter is selected from the group consisting of OATP1B1, OATP-C, OATP2, LST-1, OATP1B3, OATP8, OATP2B1, OATP1A2, NaDC3, SDCT2, NTCP, OCT1, OCT3, OAT2, OAT1, OAT3, PGT, OCT2, OAT1, OATP4A1, OATP4C1.

[0055] The polymethine dyes or polymethine targeting moieties (PTMs) according to the present invention relate to chemical polymethines (trimethine, pentamethine or heptamethine structures (see Figures 1 and 2)) that are recognized by specialized surface molecules (OATPs), preferably OATP1B2 or OATP1B3, OATO2B1 and NTCP. Upon recognition, the nanostructured delivery system together with the PTM becomes internalized or endocytosed, preferably by a clathrin-mediated mechanism.

[0056] In accordance with the present invention, the term "polymethine dye" is used synonymously with the term "polymethine targeting moiety (PTM)". Further synonymous terms are "polymethine dye targeting moiety", "polymethine-based targeting moiety", and "polymethine-like targeting moiety". All of these terms have the same meaning.

[0057] The terms "tissue-specific transporter", "transporter" and "influx transporter" are used synonymously according to the present invention.

[0058] To connect the polymethine targeting moiety (PTM) to the nanostructured delivery system or to the PKC reducer, construct or polymer, known standard methods can be used, as shown in FIG. 3. To modify the attachment point of the PKC reducer, construct or polymer, standard methods can be applied. The most common standard method is shown in FIG. 4, which describes the interconversion of carboxylic acid to amine, alcohol to carboxylic acid or maleimide. Detailed illustrated procedures for interconversion of carboxylic acid derivatives to amine or maleimide are described in Example 2 and shown in FIG. 6. For individual applications, 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 7).

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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, Go 6983, K252a, ANA-12, lestaurtinib, stauprimide, CEP-701, alciliaflavin A, chelerythrine chloride, and bisindolylmaleimides I-XII, also known as BIM I-XII.

[0063] 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.

[0064] 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 polymethine 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., FIG. 1).

[0065] The polymethine targeting moieties of the invention as selective liver targeting moieties can be attached to the agents of the 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 reactions, Huisgen reactions (e.g., 1,3-cycloaddition of alkynes to azides), Diels-Alder reactions (e.g., trans-cyclooctene coupling to tetrazine derivatives), mono- or di-halogenated triazines, aziridines, epoxides, sulfonyl halides, imidoesters, phosphoramidites, maleimide-thiol reactions, isocyanate-, isothiocyanate coupling, carbodiimide coupling, halo-acetamide coupling. Alternatively, reactive carbonyl compounds, preferably ketones, aldehyde acetals or hemiacetals with amines that form Schiff bases that can be reduced to the corresponding amines, can be used in accordance with the present invention (see, for example, FIG. 3).

[0066] The term "nanostructured delivery system" according to the present invention is characterized by at least one polymethine targeting moiety (PTM) 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, comprising contacting the target tissue with said nanostructured delivery system.

[0067] At least one polymethine targeting moiety (PTM) as a targeting unit induces active and selective transport of the nanostructured delivery system into the target tissue.

[0068] At least one polymethine targeting moiety (PTM) 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.

[0069] 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.

[0070] The nanostructured delivery system of the present invention comprises a combination of a nanostructured carrier and a polymethine targeting moiety. The nanostructured carrier 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 by specialized microscopy methods, such as SEM, STEM, or cryo-TEM, AFM. The shape can be preferably, but not limited to, either spherical, ellipsoidal, rod-shaped, barrel-shaped, discoidal, or polyhedral. The size preferably varies from 1 nm to 800 nm.

[0071] In a preferred embodiment, at least one of the polymers, lipids, virus-like particles and / or active agents contains functional groups that allow for chemical modification and attachment of polymethine targeting moieties (PTMs) (see, e.g., FIG. 4). 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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).

[0078] 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]

[0079] [Figure 1A]Figure 1 shows exemplary building blocks for generating / preparing a variety of different nanostructured delivery systems useful for the treatment of septic cholestasis. Figure 1A shows a variety of potential compounds that reduce PKC activity. [Figure 1B] FIG. 1B shows several representative polymethine dye targeting moieties (PTMs). [Figure 1C] FIG. 1C shows an example of a targeted nanostructured delivery system. [Diagram 2] 2A and 2B show Markush formulas of the claimed polymethine dyes, which are preferably useful as polymethine-based targeting moieties (PTMs). [Figure 3-1] Figure 3 shows examples of polymethine targeting moieties (PTMs) useful for hepatocyte targeting, where "R" represents a possible attachment point of a carrier system (polymer, virus-like particle, lipid, or gene construct). Figure 3 further shows a general synthetic approach for the synthesis / attachment of polymethine targeting moieties (PTMs) to a drug, drug construct, carrier polymer, virus-like particle, or linker. [Figure 3-2] Figure 3 shows examples of polymethine targeting moieties (PTMs) useful for hepatocyte targeting, where "R" represents a possible attachment point of a carrier system (polymer, virus-like particle, lipid, or gene construct). Figure 3 further shows a general synthetic approach for the synthesis / attachment of polymethine targeting moieties (PTMs) 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 the scheme for synthesizing two representative PTMs (PTM1 and PTM2) suitable for conjugation to nanostructured delivery systems for liver targeting. Detailed protocols are shown in Examples 1a and 1b. Similar procedures can be adopted for the synthesis of various PTMs. [Figure 6]6 shows examples of how COOH-functionalized PTMs can be converted to their respective amine- (7a) or maleimide-functionalized (13a) PTMs. Detailed procedures are given in Example 2. [Figure 7] 7 shows the coupling of an amino-terminated PTM (7a) to the terminal carboxylic acid from PLGA. A detailed procedure is given in Example 3. [Figure 8] Figure 8 shows a strategy for direct coupling of nucleic acid entities to maleimide-functionalized PTM (13b). Figure 8A: 3'-end labeling strategy for primarily DNA-like constructs; Figure 8B: 5'-end labeling strategy for DNA, RNA, or modified nucleotides. Detailed procedures are given in Example 4. [Figure 9] Figure 9 shows the preparation of nanoparticles by emulsion, double emulsion and nanoprecipitation: Figure 9A: double emulsion, Figure 9B: emulsion; Figure 9C: nanoprecipitation (dropping). [Figure 10] FIG. 10 shows the toxicity of targeted nanoparticles and free drug (BIM-1) in HepG2 cells. [Figure 11] FIG. 11 shows a Kaplan-Meier-Schatzer plot showing mouse survival in a peritoneal contamination and infection (PCI) model using two different stool batches. [Figure 12] Figure 12 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 13] Figure 13 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 14] Figure 14 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] Figure 1A-C show exemplary building blocks for preparing various different nanostructured delivery systems according to the present invention that are useful for the inventive treatment of septic cholestasis. Figure 1A 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 1B shows different preferred polymethine targeting moieties (PTMs) that can be used according to the present invention. The blue dots represent the connection points to the drug / drug construct or polymer. Possible chemical linkages are shown in Figure 3. Figure 1C shows different carrier systems carrying polymethine 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. Essentially, PTMs 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 polymethine targeting moieties (PTMs) of the present invention and therefore can themselves form targeted nanostructured delivery systems after ligation to a nucleic acid construct. Such targeted nanostructured delivery systems are preferably formed when the PTMs are directly bound to the inhibitors of the PKC signaling pathway of the present invention (preferably nucleic acid constructs) and these constructs form the nanostructured delivery systems (with or without helper polymers).

[0081] Polymethine dye-based targeting moieties contain chemical moieties that are recognized by specific recognition units, preferably OATPs or solute carrier transporter proteins. Balanced charge of the molecules is important for recognition. In a preferred embodiment, after binding to the final construct / carrier, the net charge of the PTM is + / -0 or -1. Some representative polymethine targeting moieties (PTMs) are shown in Figure 1B.

[0082] Figures 2 and 3 show the structures of polymethine dyes of the present invention having two indole moieties. Figure 2 shows the general structure I or II of symmetric or asymmetric polymethine dyes according to the present invention, where: a.) n represents the number 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; b.) R1-R17 may be the same or different and may be hydrogen or deuterium, one or more alkyl, tert-alkyl, cycloalkyl- ("alkyl" and "cycloalkyl" radicals also include olefinic structures) or aryl, carboxyaryl, dicarboxyaryl, heteroaryl or heteroalicyclic radicals, alkyloxy, alkylmercapto, arlyoxy, arylmercapto, heteroaryloxy, heteroarylmercapto groups, hydroxyl, halogen, nitro or cyano groups, alkyl-substituted or cyclic amine functional groups, and / or two ortho-positioned radicals, e.g., R3 and R4, R13 and R14, and / or R1 and R2, and R11 and R12, and / or R7 and R9, may together form an additional aromatic, heteroaromatic, aliphatic or heteroaliphatic ring; c.) At least one of the R1-R17 substituents determines the hydrophilic character of these polymethine dyes, SO3 - , (-SO3H), PO3 2- , COOH, OH or NR3 + , a cyclodextrin or a sugar, which may also be attached to the polymethine dye by a spacer group; d.) at least one of the R1-R17 substituents bears a reactive group (linker) such as an isocyanate, isothiocyanate, hydrazine, amine, mono- and dichloro- or mono- and dibromo triazine, aziridine, epoxide, sulfonyl halides, acid halides, carboxylic acid anhydrides, N-hydroxy-succinimide esters, imido esters, carboxylic acids, glyoxals, aldehydes, maleimides or iodoacetamides and phosphoramidite derivatives or azides, alkynes or olefins, where this substituent may also be attached to the polymethine dye by a spacer group; e.) The aromatic, heteroaromatic, aliphatic or heteroaliphatic spacer group is [(CH2) a -Y-(CH2) b ] c or [(C6H4) a -Y-(C6H4) b ], where Y may be the same or different and contain a CR2-, O-, S-, -SO2, SO2NH-, NR-, COO- or CONR functional group, which is attached to one of the R1-R17 substituents, a.) and b.) may be the same or different and have a numerical value of 0-18, and c has a numerical value of 0-18, f.) The corresponding R8 and R9 substituents with n=2, 3, 4 or 5 may also be present 2x, 3x, 4x or 5x, which may be the same or different.

[0083] Figure 3 shows an example of a conjugation strategy of a drug / drug construct with a polymer or targeting moiety according to the present invention. The polymethine-driven targeting moiety selective liver targeting moiety of the present invention 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 person, i.e. chemists, can be applied. In a preferred embodiment, as shown in Figure 3, a reactive carbonyl compound can be used, preferably a ketone, an aldehyde acetal or a hemiacetal, together with an amine that forms a Schiff base that can be reduced to the corresponding amine.

[0084] The polymethine moiety-induced endocytosis according to the present invention can be employed for tissue-specific transport of drugs themselves. For this purpose, the drug of interest is coupled with a moiety suitable for coupling reaction, including polymethine dyes for active hepatocyte / liver targeting. According to the present invention, inhibitors of the PKC signaling pathway are coupled to polymers or nanostructured delivery systems (i.e., polymer particles) directly or using spacers that include polymethine dyes or ligand constructs as shown in Figure 3.

[0085] The polymethine dyes of the present invention are important inventive tools for targeted delivery and cell / tissue / organ specificity of drugs, i.e., inhibitors, drug constructs or carriers. As outlined in Figures 1B, 2, and 3, such recognition ligands can be seen as derivatives of ICG. They are useful for hepatocyte targeting.

[0086] Different approaches can be applied to attach the polymethine dye targeting moieties to drugs, drug constructs or carriers. Depending on the functional groups present in the respective drug, drug construct or nanostructured delivery system of the invention and in the respective recognition ligand, the most appropriate method must be evaluated. 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 polymethine 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.

[0087] To increase the distance between the targeting moiety and the drug / drug construct, polymer and / or carrier system, the polymethine targeting moiety can be attached directly or via an additional spacer. An exemplary synthesis of Dye-PLGA useful as a nanocarrier is shown in FIG. 7. Further disclosure is given in Example 3. The polymethine dye-labeled PLGA (FIG. 7 and Example 3) is preferably useful for encapsulation of PKC inhibitors by nanoprecipitation, emulsion or double emulsion as shown in FIG. 9 and described in Example 5.

[0088] Alternatively, polymethine targeting moieties can be attached to the final particles (nanostructured delivery systems) after encapsulation of the inhibitors of the present invention. In this case, inhibitors of the PKC signaling pathway are encapsulated accordingly. After preparation of the nanoparticles, the functional groups in the polymer are activated and coupled to the PTMs similar to coupling as shown in Figure 3. 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 useful coupling strategy that is preferred according to the present invention is shown in Figure 4.

[0089] This approach can be employed with small molecules, nucleic acid constructs such as si-RNA, or carriers of the invention such as organic or inorganic liposomes or nanoparticles.

[0090] The polymethine targeting moieties (PTMs) according to the present invention are 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 polymethine targeting moiety (PTM) containing a maleimide functional group can be attached to the 3' or 5' EndTAG TMThe nucleic acid construct is attached by known labeling methods such as. In either method, the preferred functional group on the PTM is maleimide, which can be generated as shown in FIG. 4. Two EndTAG coupling strategies for selectively attaching a nucleic acid construct to a polymethine targeting moiety (PTM) are shown in FIG. 4 and illustrated in FIG. 6. Strategies for coupling of nucleic acid entities to PTMs are shown in FIG. 8. FIG. 8A shows a 3'-end labeling strategy for primarily DNA-like constructs; FIG. 8B 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.

[0091] To mimic septic cholestasis, systemic inflammation was induced using an established peritoneal contamination and infection (PCI) model, in which a human fecal suspension is applied intraperitoneally (i.p.) to rapidly induce sepsis accompanied by liver dysfunction.

[0092] For each batch of human stool, the dose is carefully titrated to achieve a survival rate of 0% to 20% within two weeks.

[0093] 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 11 shows the survival of mice with the two different batches used in the Kaplan-Meier-Schatzer plot.

[0094] Figures 12, 13, and 14 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 12 shows the effect of the PKC inhibitor BIM-1 as free drug and as cargo in a targeted nanoparticle formulation.

[0095] FIG. 13 shows the effect of the PI3-kinase inhibitor AS605240 as free drug and as cargo in a targeted nanoparticle formulation.

[0096] FIG. 14 shows the effect of the PKC inhibitor midostaurin as a free drug and as the cargo of a targeted nanoparticle formulation.

[0097] The present invention is further illustrated below on the basis of examples, but the present invention is not limited thereto. EXAMPLES

[0098] Working Example Example 1A and B: Synthesis of polymethine targeting moieties (PTMs) useful for hepatocyte targeting. The synthetic scheme is shown in Figures 5A and B. The synthetic scheme for PTM1(7) is outlined in Figure 5A. Step 1: 1.6 g (10 mmol) of trimethylindolane (1) was dissolved in 40 mL of acetonitrile and a solution of 2.53 g (11 mmol) of tosylethylene glycol methyl ether (2) in 10 mL of acetonitrile was added. The reaction mixture was stirred at room temperature under argon for 4 days. 1-(2-Methoxyethyl)-2,3,3-trimethyl-3H-indol-1-ium (3) precipitated from the solution, filtered off, washed with a mixture of acetonitrile and diethyl ether and dried to give 2.18 g (73%). Steps 2 and 3: A mixture of 111 mg (5 mmol) of malonaldehydedianile hydrochloride (4) and 110 mg (5 mmol) of 1-(2-methoxyethyl)-2,3,3-trimethyl-3H-indol-1-ium (3) in a mixture of 5 mL of glacial acetic acid and 0.5 mL of acetic anhydride was heated to 120° C. for 2.5 h to give intermediate 5 as a purple solution. The solution was cooled to about 70° C. and 177 mg of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium-5-sulfonate (6) and 350 mg of KOAc were added. After the addition, the reaction mixture was again heated to 120° C. for 3 h. The reaction was monitored by TLC. The cooled mixture was poured into 50 mL of EtOAc to precipitate the crude polymethine dye 7 as a deep blue solid. The solid was washed with EtOAc (3×30 mL) and dried in vacuum. (Yield: 83%). Further purification was carried out by preparative C18-RP-HPLC with acetonitrile and 0.1% formic acid. C 35 H 43 Mass (ES+) m / z of O6N2S, [M+1] + ,Calculated value: 607.2836, Actual value: 608.2833

[0099] The synthetic scheme for PTM2(13) is shown in Figure 5B. Step 1: A solution of 3 mmol of 2,3,3-trimethyl-3H-indole (1) in 9 mmol of 1,4-butanesulfone (2) is heated at 120° C. for 2 h. After cooling, the residue is washed with acetone (3×50 ml), filtered and dried to give 9 in 75% yield. Step 2: A mixture of (9) (0.9 mmol) and N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline monohydrochloride (10) (1 mmol) (from Sigma) in 4 mL of acetic anhydride and 1 mL of glacial acetic acid is heated at 100 °C for 2 h. After cooling, the solution is carefully precipitated in diethyl ether and the crude product is filtered off. For further purification, the crude product is dissolved in 3 mL of DCM and precipitated again in diethyl ether. The purple solid is filtered, washed with water and dried to give 11 with sufficient purity for the next step. Yield: 72%. Step 3: A solution of 3 mmol of compound 11 and 3.3 mmol of commercially available 6-(2,3,3-trimethylindol-1-yl)-hexanoic acid bromide (12) (AKos Chemicals) in a mixture of 20 mL of pyridine and 2 mL of acetic acid is heated for 4 h at 50° C. After cooling, (13) is precipitated from diethyl ether, redissolved in pyridine, and precipitated with diethyl ether. The crude product was dissolved in DCM, washed with 0.1M HCl and brine and evaporated to dryness. Further purification was carried out by preparative C18-RP-HPLC with acetonitrile and 0.1% formic acid. C 40 H 49 Mass (ES+) m / z of O5N2ClS, [M+1]+, calculated value: 705.3454, observed value: 705.3449

[0100] Example 2: Interconversion of Functional Groups in PTM Step 4: (also shown in Figure 6) For amine functionalization of carboxylic acid dye 7, 1 mmol is dissolved in 10 mL of DCM. To this solution, 1.2 mmol of HBTU, 1 mmol of Hunig's base and 1.5 mmol of ethylenediamine (free base) are successively added and the mixture is stirred at room temperature for 12 h. The final product is purified by precipitation from diethyl ether, a second precipitation from DMF in diethyl ether and finally by C18RP column chromatography (acetonitrile / formic acid) to give 7a in 68% yield as a deep blue solid. Step 4a (also shown in Figure 6) For maleimide functionalization of carboxylic acid dye 4, 1 mmol is dissolved in 10 mL of DCM. To this solution, 1.2 mmol of HBTU, 1.5 mmol of Hunig's base and 1.5 mmol of aminoethylmaleimide x TFA are successively added and the mixture is stirred at room temperature for 12 h. The final product is purified by precipitation from diethyl ether, a second precipitation from DMF in diethyl ether and finally by C18RP column chromatography (acetonitrile / formic acid) to give 13a in 77% yield as a dark green solid.

[0101] Example 3: Coupling of PTM1 to PLGA Dissolve 1 g of PLGA (Resomer RG 502 H) MW: 12.000 in 5 mL of CHCl3 and 8 mg of EDCxHCl predissolved in 1 mL of chloroform, add 4.8 mg of NHS in 1 mL of DMF. Stir the activated mixture for 4 h, then add 1 mL of a solution of amine-functionalized dye (7a) (1 mg / mL in DMF) and 80 μL of trimethylamine under inert atmosphere. After stirring the mixture for 18 h at room temperature, add 58 μL of glacial acetic acid and remove the solvent under strong vacuum. Wash the resulting sticky oil 5 times with 50 mL of water and twice with 50 mL of methanol. To remove the free dye, dissolve the residue in 350 mL of acetonitrile and add 10 g of freshly washed Amberlite IR120H (5x water wash, then 3x acetonitrile wash). Rotate the flask on a rotary evaporator for 40 min without vacuum or heat. Filter off the solution and evaporate under reduced pressure. The residue is dissolved again in 6 mL of acetonitrile and precipitated in 100 mL of cold water (0° C.). The precipitated polymer is filtered off and freeze-dried. This dye-labeled PLGA was used for the preparation of nanoparticles, which were described in the in vivo experiments according to standard nanoprecipitation or emulsion procedures for the encapsulation of appropriate drugs. For the skilled person, the usual process according to standard protocols can be carried out without any problems.

[0102] Example 4: Coupling of PTMs to genetic material Direct Coupling of Genetic-Based Inhibitors to Maleimide-Functionalized Polymethine Targeting Moieties (EndTaq®) According to vectorlabs®, 1 μg of PKC-siRNA (custom made by JenaBioscience) is incubated with T4 polynucleotide kinase and ATPγS in reaction buffer at 37° C. for 30 min. 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 maleimide functionalized PTM (13b) is added and shaken at 65° C. for 30 min. The final construct is purified again under sterile conditions using a ThermoFischer RNA purification kit.

[0103] Example 5: Preparation of nanoparticles After functionalization of the polymer with polymethine targeting moieties (see Example 3), nanoparticles were produced by nanoprecipitation using polyvinyl alcohol (PVA) as a surfactant. The PTM1-labeled polymer and the PKC inhibitor BIM-1 and 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 nanoparticles formed contain 3 wt% BIM-1, 6 wt% midostaurin, or 12 wt% AS605240 encapsulated in PTM-labeled PGLA. The solution is purified and concentrated by cross-flow filtration (Sartorius). The preparation methods of the nanoparticles of the present invention by emulsion, double emulsion and nanoprecipitation are further exemplarily shown in FIG. 9.

[0104] Assessment and visualization of hepatocyte targeting was performed according to the intravital microscopy method described in WO 2015 / 035974, the disclosure of which is hereby incorporated by reference in its entirety.

[0105] Example 6: Characterization of the nanoparticles of the invention Nanoparticles of PTM1-PLGA (Example 3) 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: Shape determined 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.

[0106] Table 1: Results: A: Non-targeted nanoparticles (PLGA / PVA) containing 2.5% encapsulated neutral lipid orange. B: PTM1-targeted nanoparticles from Example 5 containing 4% BIM-1 (PKC inhibitor) C: PTM1-targeted nanoparticles from Example 5 containing 12% AS605230 (a PI3 kinase inhibitor) D: PTM1-targeted nanoparticles from Example 5 containing 6% midostaurin (PKC inhibitor) TIFF0007672973000003.tif29170

[0107] Example 7: 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.

[0108] 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 7PBMCs 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.

[0109] 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. 2 The 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).

[0110] To obtain more meaningful data compared to static single-cell cultures, we used several scalable co-culture models, which more closely resemble the in vivo situation than static single-cell cultures.

[0111] 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 -1 Monocytes 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 2The 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 (Raasch et al., Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions, 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.

[0112] 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 :119-131.

[0113] Preparation of HepaRG and endothelial cells for the Dynamic42 Sinusoid model 2.7x10 HepaRG cells 4 Cells / cm 2Cells were seeded at a density of 1000 x g / 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% 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.

[0114] 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.

[0115] 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 2Cells 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.

[0116] 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 5 HUVECs / 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 0.9x104 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.

[0117] Table 2: Dimensions of the sinusoidal tip TIFF0007672973000004.tif56170

[0118] Table 3: Flow velocity within the sinusoidal chip TIFF0007672973000005.tif42170

[0119] 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.

[0120] Example 8: Determination of Cytotoxicity Cytotoxicity tests were performed with 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 (Life technologies, Darmstadt, Germany) prepared according to the manufacturer's instructions. After a further incubation of 4 h, fluorescence was measured at Ex 570 / Em 610 nm, using untreated cells on the same well plate as a negative control. The negative control was normalized as 0% metabolic inhibition and taken as 100% viability. Cell viability below 70% was considered to indicate cytotoxicity. Data are expressed as the mean ± SD of triplicate determinations. Figure 10 shows that the encapsulated drug is less toxic at high concentrations compared to the free drug (BIM-1).

[0121] Example 9: Survival in a Cholestatic Model Under Septic Conditions "Peritoneal Contamination and Infection (PCI)" Experimental setup: By using the PCI model, sepsis with systemic infection / organ failure was induced in male C57 / BL6 mice. 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 11, 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. 11, 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. 11.

[0122] 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.

[0123] 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).

[0124] 9a) PTM1-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: 66% PVA: 31% BIM-1: 4% Size / Zeta potential: 93nm / -18

[0125] The particle suspension was diluted with 45% glucose solution to give a final glucose concentration of 5%.

[0126] 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 12 and show that survival increased from 10% to 80% within 7 days.

[0127] 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: 30% AS605240:12% Size / Zeta potential: 166nm / -15

[0128] The particle suspension was diluted with 45% glucose solution to give a final glucose concentration of 5%.

[0129] 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 13 and show that survival increased from 0% to 40% within 7 days.

[0130] 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: 69% PVA: 25% Midostaurin: 6% Size / Zeta potential: 101nm / -25

[0131] The particle suspension was diluted with 45% glucose solution to give a final glucose concentration of 5%.

[0132] Five mice were treated with the targeted nanoparticles and evaluated with two sham mice to test the tolerability of the nanoparticles in healthy mice.

[0133] The results are shown in FIG. 14 and demonstrate that survival increased from 10% to 60% within 7 days.

Claims

1. 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 polymethine dye and at least one polymer and / or at least one lipid and / or at least one virus-like particle, wherein said at least one polymethine dye is chemically bound to said polymer or lipid nanoparticle or liposome, or to said virus-like particle, in which said inhibitor of the PKC signaling pathway is encapsulated or entrapped, and wherein said at least one polymethine dye is a symmetric or asymmetric polymethine of general structure I or II: During the ceremony, a. n represents the numbers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; b. R1-R17 may be the same or different and may be hydrogen or deuterium, one or more alkyl, tert-alkyl, cycloalkyl- ("alkyl" and "cycloalkyl" radicals also include olefinic structures) or aryl, carboxyaryl, dicarboxyaryl, heteroaryl or heteroalicyclic radicals, alkyloxy, alkylmercapto, aryloxy, arylmercapto, heteroaryloxy, heteroarylmercapto groups, hydroxyl, halogen, nitro or cyano groups, alkyl-substituted or cyclic amine functional groups, and / or two ortho radicals may join together to form an additional aromatic, heteroaromatic, aliphatic or heteroaliphatic ring; c. At least one of the R1-R17 substituents is a SO ３ － , (-SO ３ H), P.O. ３ ２－ , COOH, OH or NR ３ ＋ , a cyclodextrin or a sugar, which may also be attached to the polymethine dye by a spacer group; d. at least one of the R1-R17 substituents has a reactive group (linker) such as an isocyanate, isothiocyanate, hydrazine, amine, mono- and dichloro- or mono- and dibromo triazine, aziridine, epoxide, sulfonyl halides, acid halides, carboxylic acid anhydrides, N-hydroxy-succinimide esters, imido esters, carboxylic acids, glyoxals, aldehydes, maleimides or iodoacetamides and phosphoramidites or azides, alkynes or olefins, where this substituent may also be attached to the polymethine dye by a spacer group; e. The aromatic, heteroaromatic, aliphatic or heteroaliphatic spacer group is [(CH ２ ) ａ -Y-(CH ２ ) ｂ ] ｃ Or [(C ６ H ４ ) ａ -Y-(C ６ H ４ ) ｂ ], where Y can be the same or different, and CR ２ -, O-, S-, -SO ２ , S.O. ２ contains an NH-, NR-, COO-, or CONR functional group, which is attached to one of the R1-R17 substituents, a.) and b.) may be the same or different and have a value of 0-18, and c is a value of 0-18; f. The corresponding R8 and R9 substituents with n=2, 3, 4 or 5 may also occur 2, 3, 4 or 5 times, which may be the same or different; Selective nanostructured delivery systems.

2. 2. The selective nanostructured delivery system of claim 1, wherein at least one polymethine dye is ICG.

3. 3. The selective nanostructured delivery system of claim 1 or 2, wherein at least one polymethine dye has at least one tissue-specific transporter that induces uptake of the nanostructured delivery system into cells of the target tissue.

4. 4. The selective nanostructured delivery system of claim 3, wherein the at least one tissue-specific transporter is selected from the group consisting of OATP1B1, OATP-C, OATP2, LST-1, OATP1B3, OATP8, OATP2B1, OATP1A2, NaDC3, SDCT2, NTCP, OCT1, OCT3, OAT2, OAT1, OAT3, PGT, OCT2, OAT1, OATP4A1, and OATP4C1.

5. The selective nanostructure delivery system of 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, and an inhibitor comprising siRNA, shRNA, miRNA, morpholino, an antisense construct, or RNase H.

6. 6. The selective nanostructured delivery system of 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, alcyaflavin A, or inhibitors including bisindolylmaleimides I-XII, also known as BIM I-XII.

7. 6. The selective nanostructured delivery system of claim 5, 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.

8. 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.

9. 9. The selective nanostructured delivery system of any one of claims 1 to 8, 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.

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

11. 11. The selective nanostructured delivery system of 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. The selective nanostructured delivery system of 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, phospholipids, sphingolipids, lipoproteins and glycolipids.

13. 13. The selective nanostructured delivery system of any one of claims 1 to 12, 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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