Eduard Post

Novel engineered targeted interferon-gamma blocks hepatic fibrogenesis in mice.

Liver fibrogenesis is a process tightly controlled by endogenous anti‐ and pro‐fibrogenic factors. Interferon gamma (IFNγ) is a potent antifibrogenic cytokine in vitro and might therefore represent a powerful therapeutic entity. However, its poor pharmacokinetics and adverse effects, due to the presence of IFNγ receptors on nearly all cells, prevented its clinical application so far. We hypothesized that delivery of IFNγ specifically to the disease‐inducing cells and concurrently avoiding its binding to nontarget cells might increase therapeutic efficacy and avoid side effects. We conjugated IFNγ to a cyclic peptide recognizing the platelet‐derived growth factor beta receptor (PDGFβR) which is strongly up‐regulated on activated hepatic stellate cells (HSC), the key effector cells responsible for hepatic fibrogenesis. The IFNγ conjugates were analyzed in vitro for PDGFβR‐specific binding and biological effects and in vivo in acute (early) and chronic (progressive and established) carbon‐tetrachloride‐induced liver fibrosis in mice. The targeted‐IFNγ construct showed PDGFβR‐specific binding to fibroblasts and HSC and inhibited their activation in vitro. In vivo, the targeted‐IFNγ construct attenuated local HSC activation in an acute liver injury model. In the established liver fibrosis model, it not only strongly inhibited fibrogenesis but also induced fibrolysis. In contrast, nontargeted IFNγ was ineffective in both models. Moreover, in contrast to unmodified IFNγ, our engineered targeted‐IFNγ did not induce IFNγ‐related side effects such as systemic inflammation, hyperthermia, elevated plasma triglyceride levels, and neurotropic effects. Conclusion: This study presents a novel HSC‐targeted engineered‐IFNγ, which in contrast to systemic IFNγ, blocked liver fibrogenesis and is devoid of side effects, by specifically acting on the key pathogenic cells within the liver. (HEPATOLOGY 2011;)

A novel approach to deliver anticancer drugs to key cell types in tumors using a PDGF receptor-binding cyclic peptide containing carrier.

Tumor stromal cells have been recently recognized to contribute to tumor growth. Therefore, we hypothesized that delivery of anticancer drugs to these cells in addition to the tumor cells might treat cancer more effectively. Stromal cells abundantly expressed Platelet-Derived Growth Factor Receptor-beta (PDGFR-beta) in different human tumors as shown with immunohistochemistry. To achieve targeting through PDGFR-beta, we developed a carrier by modifying albumin with a PDGFR-beta recognizing cyclic peptide (pPB-HSA). pPB-HSA specifically bound to PDGFR-beta-expressing 3T3 fibroblasts, C26 and A2780 cancer cells in vitro. Subsequently, doxorubicin was conjugated to pPB-HSA through an acid-sensitive hydrazone linkage. In vitro, Dox-HSA-pPB was taken up by fibroblasts and tumor cells and a short exposure of the conjugate induced cell death in these cells. In vivo, the conjugate rapidly accumulated into PDGFR-beta expressing cells in C26 tumors. Treatment with Dox-HSA-pPB significantly reduced the C26 tumor growth in mice while free doxorubicin treated mice had lower response to the therapy. Furthermore, in contrast to free doxorubicin the conjugate did not induce loss in body weight. In conclusion, the present study reveals a novel approach to target key cell types in tumors through PDGFR-beta, which can be applied to enhance the therapeutic efficacy of anticancer drugs.

PEGylation improves pharmacokinetic profile, liver uptake and efficacy of Interferon gamma in liver fibrosis

Interferon gamma (IFNγ) is a potent cytokine that displays a variety of anti-viral, anti-proliferative, immunomodulatory, apoptotic and anti-fibrotic functions. However, its clinical use is limited to the treatment of few diseases due to the rapid clearance from the body. PEGylated IFN-alpha formulations are shown to be beneficial in viral hepatitis, but PEGylation of IFNγ to enhance its therapeutic effects in liver fibrosis is not yet explored. Liver fibrosis is characterized by the extensive accumulation of an abnormal extracellular matrix and is the major cause of liver-related morbidity and mortality worldwide. To date, there is no pharmacotherapy available for this disease. We modified IFNγ with different-sized linear PEG molecules (5, 10 and 20kDa) and assessed the biological activity in vitro and in vivo. All PEGylated IFNγ constructs were biologically active and activated IFNγ signaling in vitro as determined with a nitric oxide release assay and a pGAS-Luc reporter plasmid assay, respectively. Similar to IFNγ, all PEGylated IFNγ induced a significant reduction of fibrotic parameters in mouse NIH3T3 fibroblasts as shown with immunohistochemical staining and quantitative PCR analyses. In vivo, the pharmacokinetic profile of radiolabeled (125)I-IFNγ-PEG conjugates revealed a decreased renal clearance and an increased plasma half-life with an increase of PEG size. Moreover, the liver accumulation of PEGylated IFNγ constructs was significantly higher than the unmodified IFNγ, which was also confirmed by increased MHC-II expression in the livers. Furthermore, in a CCl(4)-induced acute liver injury model in mice, PEGylated constructs reduced the early fibrotic parameters more drastically than unmodified IFNγ. Of note, these effects were stronger with higher PEG-sized IFNγ constructs. These data nicely correlated with the pharmacokinetic data. In conclusion, PEGylation significantly improved the pharmacokinetics, liver uptake and anti-fibrotic effects of IFNγ. This study opens new opportunities to exploit the therapeutic applications of PEGylated IFNγ for the treatment of liver fibrosis and other diseases.

Reduction of Fibrogenesis by Selective Delivery of a Rho Kinase Inhibitor to Hepatic Stellate Cells in Mice

One of the pathways activated during liver fibrosis is the Rho kinase pathway, which regulates activation, migration, and contraction of hepatic stellate cells (HSC). Inhibition of this kinase by the Rho kinase inhibitor Y27632 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] has been shown to reduce fibrosis in animal models. However, kinase expression is ubiquitous, so any inhibitor may affect many cell types. We hypothesize that cell-specific delivery of a kinase inhibitor will be beneficial. Therefore, we conjugated Y27632 to the carrier mannose-6-phosphate (M6P) human serum albumin (HSA), which is taken up specifically in activated HSC through the M6P/insulin-like growth factor II receptor. This conjugate decreased protein expression of phosphorylated myosin light chain 2 (pMLC2) and vinculin, downstream of Rho kinase, in activated primary HSC and decreased the migration and contraction of HSC. In an ex vivo model, free Y27632 decreased contractility of rat aortas, whereas the Y27-conjugate did not, showing that the Y27-conjugate does not affect nontarget tissue. In chronic CCl4-induced liver fibrosis, both free drug and conjugate reduced HSC activation; however, only the Y27-conjugate significantly reduced collagen deposition. Treatment with the Y27-conjugate, but not with free drug, reduced pMLC2 expression in livers 24 h after injection, demonstrating prolonged inhibition of the Rho kinase pathway. The Rho kinase inhibitor Y27632 can be specifically targeted to HSC using M6PHSA, decreasing its effects in nontarget tissues. The targeted drug effectively reduced fibrotic parameters in vivo via the inhibition of the Rho kinase pathway.

Selective delivery of IFN-γ to renal interstitial myofibroblasts: a novel strategy for the treatment of renal fibrosis

Renal fibrosis leads to end‐stage renal disease demanding renal replacement therapy because no adequate treatment exists. IFN‐γ is an antifibrotic cytokine that may attenuate renal fibrosis. Systemically administered IFN‐γ causes side effects that may be prevented by specific drug targeting. Interstitial myofibroblasts are the effector cells in renal fibrogenesis. Here, we tested the hypothesis that cell‐specific delivery of IFN‐γ to platelet‐derived growth factor receptor β (PDGFRβ)‐expressing myofibroblasts attenuates fibrosis in an obstructive nephropathy [unilateral ureteral obstruction (UUO)] mouse model. PEGylated IFN‐γ conjugated to PDGFRβ‐recognizing peptide [(PPB)‐polyethylene glycol (PEG)‐IFN‐γ] was tested in vitro and in vivo for antifibrotic properties and compared with free IFN‐γ. PDGFRβ expression was >3‐fold increased (P < 0.05) in mouse fibrotic UUO kidneys and colocalized with α‐smooth muscle actin‐positive (SMA+) myofibroblasts. In vitro, PPB‐PEG‐IFN‐γ significantly inhibited col1a1, col1a2, and α‐SMA mRNA expression in TGF‐β‐activated NIH3T3 fibroblasts (P < 0.05). In vivo, PPB‐PEG‐IFN‐γ specifically accumulated in PDGFRβ‐positive myofibroblasts. PPB‐PEG‐IFN‐γ treatment significantly reduced renal collagen I, fibronectin, and α‐SMA mRNA and protein expression. Compared with vehicle treatment, PPB‐PEG‐IFN‐γ preserved tubular morphology, reduced interstitial T‐cell infiltration, and attenuated lymphangiogenesis (all P < 0.05) without affecting peritubular capillary density. PPB‐PEGIFN‐γ reduced IFN‐γ‐related side effects as manifested by reduced major histocompatibility complex class II expression in brain tissue (P < 0.05 vs. free IFN‐γ). Our findings demonstrate that specific targeting of IFN‐γ to PDGFRβ‐expressing myofibroblasts attenuates renal fibrosis and reduces systemic adverse effects.—Poosti, F., Bansal, R., Yazdani, S., Prakash, J., Post, E., Klok, P., vanden Born J. deBorst M.H. vanGoor H. Poelstra K. Hillebrands, J.‐L. Selective delivery of IFN‐γ to renal interstitial myofibroblasts: a novel strategy for the treatment of renal fibrosis. FASEB J. 29, 1029–1042 (2015). www.fasebj.org

Increased Liver Uptake and Reduced Hepatic Stellate Cell Activation with a Cell-Specific Conjugate of the Rho-kinase Inhibitor Y27632

ABSTRACTPurposeRho-kinase regulates activation of hepatic stellate cells (HSC) during liver fibrosis, but the ubiquitous presence of this kinase may hinder examination of its exact role and the therapeutic use of inhibitors. We therefore coupled the Rho-kinase inhibitor Y27632 to a drug carrier that binds the mannose-6-phosphate insulin-like growth factor II (M6P/IGFII)-receptor which is upregulated on activated HSC.MethodsY27632 was coupled to mannose-6-phosphate human serum albumin (M6PHSA), and in vitro experiments were performed on primary rat HSC. Biodistribution and effect studies were performed in an acute CCl4 model in mice.ResultsY27-conjugate remained stable in serum, while drug was efficiently released in liver homogenates. Receptor-blocking studies revealed that it was specifically taken up through the M6P/IGFII-receptor on fibroblasts, and it inhibited expression of fibrotic markers in activated HSC. In vivo, liver drug levels were significantly higher after injection of Y27-conjugate as compared to Y27632, and the conjugate accumulated specifically in HSC. After acute CCl4-induced liver injury, Y27-conjugate reduced the local activation of HSC, whereas an equimolar dose of free drug did not.ConclusionsWe conclude that specific targeting of a Rho-kinase inhibitor to HSC leads to enhanced accumulation of the drug in HSC, reducing early fibrogenesis in the liver.

PGE2-treated macrophages inhibit development of allergic lung inflammation in mice

In healthy lungs, many macrophages are characterized by IL‐10 production, and few are characterized by expression of IFN regulatory factor 5 (formerly M1) or YM1 and/or CD206 (formerly M2), whereas in asthma, this balance shifts toward few producing IL‐10 and many expressing IFN regulatory factor 5 or YM1/CD206. In this study, we tested whether redressing the balance by reinstating IL‐10 production could prevent house dust mite‐induced allergic lung inflammation. PGE2 was found to be the best inducer of IL‐10 in macrophages in vitro. Mice were then sensitized and challenged to house dust mites during a 2 wk protocol while treated with PGE2 in different ways. Lung inflammation was assessed 3 d after the last house dust mite challenge. House dust mite‐exposed mice treated with free PGE2 had fewer infiltrating eosinophils in lungs and lower YM1 serum levels than vehicle‐treated mice. Macrophage‐specific delivery of PGE2 did not affect lung inflammation. Adoptive transfer of PGE2‐treated macrophages led to fewer infiltrating eosinophils, macrophages, (activated) CD4+, and regulatory T lymphocytes in lungs. Our study shows that the redirection of macrophage polarization by using PGE2 inhibits development of allergic lung inflammation. This beneficial effect of macrophage repolarization is a novel avenue to explore for therapeutic purposes.

Enhanced Effectivity of an ALK5-Inhibitor after Cell-Specific Delivery to Hepatic Stellate Cells in Mice with Liver Injury

Transforming growth factor-β (TGF-β) is a major pro-fibrotic cytokine, causing the overproduction of extracellular matrix molecules in many fibrotic diseases. Inhibition of its type-I receptor (ALK5) has been shown to effectively inhibit fibrosis in animal models. However, apart from its pro-fibrotic effects, TGF-β also has a regulatory role in the immune system and influences tumorigenesis, which limits the use of inhibitors. We therefore explored the cell-specific delivery of an ALK5-inhibitor to hepatic stellate cells, a key cell in the development of liver fibrosis. We synthesized a conjugate of the ALK5-inhibitor LY-364947 coupled to mannose-6-phosphate human serum albumin (M6PHSA), which binds to the insulin-like growth factor II receptor on activated HSC. The effectivity of the conjugate was evaluated in primary HSC and in an acute liver injury model in mice. In vitro, the free drug and the conjugate significantly inhibited fibrotic markers in HSC. In hepatocytes, TGF-β-dependent signaling was inhibited by free drug, but not by the conjugate, thus showing its cell-specificity. In vivo, the conjugate localized in desmin-positive cells in the liver and not in hepatocytes or immune cells. In the acute liver injury model in mice, the conjugate reduced fibrogenic markers and collagen deposition more effectively than free drug. We conclude that we can specifically deliver an ALK5-inhibitor to HSC using the M6PHSA carrier and that this targeted drug reduces fibrogenic parameters in vivo, without affecting other cell-types.

Upregulation of Epac-1 in Hepatic Stellate Cells by Prostaglandin E-2 in Liver Fibrosis Is Associated with Reduced Fibrogenesiss

Exchange protein activated by cAMP (Epac-1) is an important signaling mechanism for cAMP-mediated effects, yet factors that change Epac-1 levels are unknown. Such factors are relevant because it has been postulated that Epac-1 directly affects fibrogenesis. Prostaglandin E2 (PGE2) is a well-known cAMP activator, and we therefore studied the effects of this cyclo-oxygenase product on Epac-1 expression and on fibrogenesis within the liver. Liver fibrosis was induced by 8 weeks carbon tetrachloride (CCL4) administration to mice. In the last 2 weeks, mice received vehicle, PGE2, the cyclo-oxygenase-2 inhibitor niflumic acid (NFA), or PGE2 coupled to cell-specific carriers to hepatocytes, Kupffer cells, or hepatic stellate cells (HSC). Results showed antifibrotic effects of PGE2 and profibrotic effects of NFA in CCL4 mice. Western blot analysis revealed reduced Epac-1 protein expression in fibrotic livers of mice and humans compared with healthy livers. PGE2 administration to fibrotic mice completely restored intrahepatic Epac-1 levels and also led to reduced Rho kinase activity, a downstream target of Epac-1. Cell-specific delivery of PGE2 to either hepatocytes, Kupffer cells, or HSC identified the latter cell as the key player in the observed effects on Epac-1 and Rho kinase. No significant alterations in protein kinase A expressions were found. In primary isolated HSC, PGE2 elicited Rap1 translocation reflecting Epac-1 activation, and Epac-1 agonists attenuated platelet-derived growth factor–induced proliferation and migration of these cells. These studies demonstrate that PGE2 enhances Epac-1 activity in HSC, which is associated with significant changes in (myo)fibroblast activities in vitro and in vivo. Therefore, Epac-1 is a potential target for antifibrotic drugs.

Targeting tumor stromal cells through a PDGF-beta receptor binding carrier

Tumor stromal cells significantly contribute to tumor growth and abundantly express platelet-derived growth factor-beta receptor (PDGF-βR). In this study, we targeted stromal as well as tumor cells using our PDGF-βR binding carrier (pPB-HSA). pPB-HSA showed PDGF-βR-specific binding in-vitro and, in-vivo it rapidly accumulated in C26 tumors in mice after i.v. injection. We conjugated doxorubicin to pPB-HSA and, the conjugate showed antitumor effects in-vitro in tumor and stromal cells and in-vivo in C26-tumor bearing mice.