Hepatic Stellate Cell Targeting Using Peptide-Modified Biologicals.

Liver diseases are a leading cause of death worldwide and are rising exponentially due to increasing prevalence of metabolic disorders. Hepatic stellate cells (HSCs) are recognized as a key therapeutic target in liver diseases as these cells, upon activation during liver damage and ongoing liver inflammation, secrete excessive amounts of extracellular matrix that leads to liver tissue scarring (fibrosis) responsible for liver dysfunction (end-stage liver disease) and desmoplasia in hepatocellular carcinoma. Targeting of HSCs to reverse fibrosis progression has been realized by several experts in the field, including us. We have developed strategies to target activated HSCs by utilizing the receptors overexpressed on the surface of activated HSCs. One well-known receptor is platelet derived growth factor receptor-beta (PDGFR-ß). Using PDGFR-ß recognizing peptides (cyclic PPB or bicyclic PPB), we can deliver biologicals, e.g., interferon gamma (IFNγ) or IFNγ activity domain (mimetic IFNγ), to the activated HSCs that can inhibit their activation and reverse liver fibrosis. In this chapter, we provide the detailed methods and the principles involved in the synthesis of these targeted (mimetic) IFNγ constructs. These methods can be adapted for synthesizing constructs for targeted/cell-specific delivery of peptides/proteins, drugs, and imaging agents useful for various applications including diagnosis and treatment of inflammatory and fibrotic diseases and cancer.

Drug Targeting and Nanomedicine: Lessons Learned from Liver Targeting and Opportunities for Drug Innovation.

Drug targeting and nanomedicine are different strategies for improving the delivery of drugs to their target. Several antibodies, immuno-drug conjugates and nanomedicines are already approved and used in clinics, demonstrating the potential of such approaches, including the recent examples of the DNA- and RNA-based vaccines against COVID-19 infections. Nevertheless, targeting remains a major challenge in drug delivery and different aspects of how these objects are processed at organism and cell level still remain unclear, hampering the further development of efficient targeted drugs. In this review, we compare properties and advantages of smaller targeted drug constructs on the one hand, and larger nanomedicines carrying higher drug payload on the other hand. With examples from ongoing research in our Department and experiences from drug delivery to liver fibrosis, we illustrate opportunities in drug targeting and nanomedicine and current challenges that the field needs to address in order to further improve their success.

Blood group AB is associated with poor outcomes in infants with necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is a neonatal disease associated with necrosis and perforation of the bowel. We investigated the association between blood group and NEC outcomes and the potential contribution of fetal-maternal blood group incompatibility. METHODS: Retrospective study including all preterm-born infants with NEC (≥ Bell's stage IIa) admitted to our NICU between January 2008 and October 2019. We analyzed the association between infants' blood groups and fetal-maternal blood group incompatibility with Bell stage severity, need for surgery, and mortality due to NEC. RESULTS: We included 237 NEC patients. In univariable analyses both AB blood group and fetal-maternal blood group incompatibility increased infants' risk of severe outcomes, with odds ratios (OR) ranging from 6.57 to 12.06 and 1.97 to 2.38, respectively. When adjusted for gestational age only AB blood group remained significant with OR 7.47 (95% confidence interval, 1.95-28.53, P = 0.003), 12.37 (2.63-58.20, P = 0.001), and 8.16 (2.28-29.14, P = 0.001) for NEC Bell's stage III, need for surgery, and NEC related mortality, respectively. Blood group incompatibility adjusted for gestational age was not related to worse outcomes with OR 1.84 (0.87-3.89, P = 0.11, 2.08 (0.98-4.41, P = 0.06) 1.52 (0.68-3.42, P = 0.31), for NEC Bell's stage III, need for surgery, and NEC related mortality, respectively. CONCLUSION: Our data confirm an association between blood group AB and worse outcomes in NEC infants, but this is not based on fetal-maternal blood group incompatibility.

Phosphate Groups in the Lipid A Moiety Determine the Effects of LPS on Hepatic Stellate Cells: A Role for LPS-Dephosphorylating Activity in Liver Fibrosis.

Alkaline phosphatase (AP) activity is highly upregulated in plasma during liver diseases. Previously, we demonstrated that AP is able to detoxify lipopolysaccharide (LPS) by dephosphorylating its lipid A moiety. Because a role of gut-derived LPS in liver fibrogenesis has become evident, we now examined the relevance of phosphate groups in the lipid A moiety in this process. The effects of mono-phosphoryl and di-phosphoryl lipid A (MPLA and DPLA, respectively) were studied in vitro and LPS-dephosphorylating activity was studied in normal and fibrotic mouse and human livers. The effects of intestinal AP were studied in mice with CCL4-induced liver fibrosis. DPLA strongly stimulated fibrogenic and inflammatory activities in primary rat hepatic stellate cells (rHSCs) and RAW264.7 macrophages with similar potency as full length LPS. However, MPLA did not affect any of the parameters. LPS-dephosphorylating activity was found in mouse and human livers and was strongly increased during fibrogenesis. Treatment of fibrotic mice with intravenous intestinal-AP significantly attenuated intrahepatic desmin+- and αSMA+ -HSC and CD68+- macrophage accumulation. In conclusion, the lack of biological activity of MPLA, contrasting with the profound activities of DPLA, shows the relevance of LPS-dephosphorylating activity. The upregulation of LPS-dephosphorylating activity in fibrotic livers and the protective effects of exogenous AP during fibrogenesis indicate an important physiological role of intestinal-derived AP during liver fibrosis.

Cell type-specific pharmacological kinase inhibition for cancer chemoprevention.

Safety is prerequisite for preventive medicine, but non-toxic agents are generally ineffective as clinical chemoprevention. Here we propose a strategy overcoming this challenge by delivering molecular-targeted agent specifically to the effector cell type to achieve sufficient potency, while circumventing toxicity in the context of cancer chemoprevention. Hepatic myofibroblasts drive progressive fibrosis that results in cirrhosis and liver cancer. In a rat model of cirrhosis-driven liver cancer, a small molecule epidermal growth factor receptor inhibitor, erlotinib, was delivered specifically to myofibroblasts by a versatile nanoparticle-based system, targeting platelet-derived growth factor receptor-beta uniquely expressed on their surface in the liver. With systemic administration of erlotinib, tumor burden was reduced to 31%, which was further improved to 21% by myofibroblast-targeted delivery even with reduced erlotinib dose (7.3-fold reduction with equivalent erlotinib dose) and less hepatocyte damage. These findings demonstrate a strategy, cell type-specific kinase inhibition, for more effective and safer precision cancer chemoprevention.

Upregulation of Epac-1 in Hepatic Stellate Cells by Prostaglandin E2 in Liver Fibrosis Is Associated with Reduced Fibrogenesis.

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.

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.

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 ß (PDGFRß)-expressing myofibroblasts attenuates fibrosis in an obstructive nephropathy [unilateral ureteral obstruction (UUO)] mouse model. PEGylated IFN-γ conjugated to PDGFRß-recognizing peptide [(PPB)-polyethylene glycol (PEG)-IFN-γ] was tested in vitro and in vivo for antifibrotic properties and compared with free IFN-γ. PDGFRß 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-ß-activated NIH3T3 fibroblasts (P < 0.05). In vivo, PPB-PEG-IFN-γ specifically accumulated in PDGFRß-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-PEG-IFN-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 PDGFRß-expressing myofibroblasts attenuates renal fibrosis and reduces systemic adverse effects.

Targeted elimination of activated hepatic stellate cells by an anti-epidermal growth factor-receptor single chain fragment variable antibody-tumor necrosis factor-related apoptosis-inducing ligand (scFv425-sTRAIL).

BACKGROUND: Progressive liver fibrosis is the result of chronic liver injury and is characterized by the excessive accumulation of extracellular matrix that may result in liver failure. Activated hepatic stellate cells are known to play a central role in this process and their elimination is a crucial step towards the resolution and reversion of liver fibrosis. In the present study, we investigated the potential application of an anti-epidermal growth factor receptor single chain fragment variable antibody-tumor necrosis factor-related apoptosis-inducing ligand (scFv425-sTRAIL) fusion protein in the targeted elimination of activated hepatic stellate cells. METHODS: Activated hepatic stellate cells (LX2 cells) were treated by adenovirus-derived scFv425-sTRAIL to evaluate its effect on the viability and extracellular matrix production of this type of cells. RESULTS: In vitro treatment of activated hepatic stellate cells with scFv425-sTRAIL induced a significant reduction in viability (up to 100% reduction) and extracellular matrix production (60% reduction), yet no significant effect was observed on hepatic parenchymal cells. Blockage of the epidermal growth factor receptor (EGFR) by a monoclonal antibody significantly reduced the effectiveness of scFv425-sTRAIL in activated hepatic stellate cells, whereas a reduced effectivity was also observed after inhibition of the caspase pathway. CONCLUSIONS: Evidence is presented for the successful application of the scFv425-sTRAIL fusion protein in the targeted elimination of activated hepatic stellate cells via EGFR and simultaneous activation of the caspase pathway. scFv425-sTRAIL may thus represent a new therapeutic compound against liver fibrosis.

Enhanced effectivity of an ALK5-inhibitor after cell-specific delivery to hepatic stellate cells in mice with liver injury.

Transforming growth factor-ß (TGF-ß) 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-ß 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-ß-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.

Selective targeting of interferon γ to stromal fibroblasts and pericytes as a novel therapeutic approach to inhibit angiogenesis and tumor growth.

New approaches to block the function of tumor stromal cells such as cancer-associated fibroblasts and pericytes is an emerging field in cancer therapeutics as these cells play a crucial role in promoting angiogenesis and tumor growth via paracrine signals. Because of immunomodulatory and other antitumor activities, IFNγ, a pleiotropic cytokine, has been used as an anticancer agent in clinical trials. Unfortunately only modest beneficial effects, but severe side effects, were seen. In this study, we delivered IFNγ to stromal fibroblasts and pericytes, considering its direct antifibrotic activity, using our platelet-derived growth factor-beta receptor (PDGFßR)-binding carrier (pPB-HSA), as these cells abundantly express PDGFßR. We chemically conjugated IFNγ to pPB-HSA using a heterobifunctional PEG linker. In vitro in NIH3T3 fibroblasts, pPB-HSA-IFNγ conjugate activated IFNγ-signaling (pSTAT1α) and inhibited their activation and migration. Furthermore, pPB-HSA-IFNγ inhibited fibroblasts-induced tube formation of H5V endothelial cells. In vivo in B16 tumor-bearing mice, pPB-HSA-IFNγ rapidly accumulated in tumor stroma and pericytes and significantly inhibited the tumor growth while untargeted IFNγ and pPB-HSA carrier were ineffective. These antitumor effects of pPB-HSA-IFNγ were attributed to the inhibition of tumor vascularization, as shown with α-SMA and CD-31 staining. Moreover, pPB-HSA-IFNγ induced MHC-II expression specifically in tumors compared with untargeted IFNγ, indicating the specificity of this approach. This study thus shows the impact of drug targeting to tumor stromal cells in cancer therapy as well as provides new opportunities to use cytokines for therapeutic application.

PEGylation of interleukin-10 improves the pharmacokinetic profile and enhances the antifibrotic effectivity in CCl4-induced fibrogenesis in mice.

Liver fibrosis represents a scar formation process as a response to chronic injury and a major cause of death worldwide. To date, no drug is available for this condition. Interleukin-10 (IL-10) has potent anti-inflammatory and antifibrotic properties but its short half-life in the circulation hampers its clinical use. Our aim was therefore to modify IL-10 with polyethylene glycol (PEG) to prolong its circulation time and enhance its effectivity. IL-10 was modified with 5 or 20 kDa PEG. The biological activity was preserved after PEGylation as assessed by inhibition of TNF-α production by macrophages. In vivo, during CCl(4)-induced fibrogenesis in mice, both 5PEG-IL-10 and 20PEG-IL-10 showed a longer circulation time compared to IL-10, which was associated with a significant increased liver accumulation. Immunohistochemical analysis of fibrotic livers of mice receiving treatment with IL-10 or its PEGylated forms, revealed a decrease in markers reflecting HSC and KC activation induced by 5PEG-IL10. Transcription levels of IL-6 were decreased upon treatment with IL-10 and both PEGylated forms, whereas IL-1ß levels were only down-regulated by 5PEGIL-10 and 20PEGIL-10. We conclude that PEGylation of IL-10 is a good strategy to attenuate liver fibrosis and that 5PEGIL-10 is the most effective conjugate.

Specific delivery of kinase inhibitors in nonmalignant and malignant diseases.

INTRODUCTION: Kinase inhibitors have been hailed as a breakthrough in the treatment of cancer. Extensive research is now being devoted to the development of kinase inhibitors as a treatment for many nonmalignant diseases. However, the use of kinase inhibitors in both malignant and nonmalignant diseases is also associated with side effects and the development of resistance. It may be worthwhile to explore whether cell-specific delivery of kinase inhibitors improves therapeutic efficacy and reduces side effects. AREAS COVERED: This review aims to provide an overview of the preclinical studies performed to examine the specific targeting of kinase inhibitors in vitro and in vivo. It gives an introduction to kinase signaling pathways induced during disease, along with the possible problems associated with their inhibition. It also discusses the studies on specific delivery and shows that altering the specificity of kinase inhibitors by targeting methods improves their effectivity and safety. EXPERT OPINION: Compared with the delivery of cytotoxic compounds, the specific delivery of kinase inhibitors has not yet been studied extensively. The studies discussed in this review provide an insight into methods used to target kinase inhibitors to different organs. The targeting of different kinase inhibitors has improved their therapeutic possibilities, but many questions still remain to be studied.

Anticancer effects of 15d-prostaglandin-J2 in wild-type and doxorubicin-resistant ovarian cancer cells: novel actions on SIRT1 and HDAC.

15-Deoxy-delta-12,14-prostaglandin-J(2) (15d-PGJ(2)), an arachidonic metabolite and a natural PPARγ agonist, is known to induce apoptosis in tumor cells. In this study, we investigated new therapeutic potentials of 15d-PGJ(2) by determining its anticancer effects in wild-type and doxorubicin-resistant ovarian carcinoma cells. Despite high expression of resistance-inducing genes like MDR1, Bcl2 and Bcl-xl, 15d-PGJ(2) strongly induced apoptosis in doxorubicin-resistant (A2780/AD) cells similar to the wild-type (A2780). This was found to be related to caspase-3/7- and NF-κB pathways but not to its PPARγ agonistic activity. 15d-PGJ(2) also was able to reduce the doxorubicin resistance of A2780/AD cells at low doses as confirmed by the inhibition of gene expression of MDR1 (p-glycoprotein) and SIRT1 (a drug senescence gene). We also investigated effects of 15d-PGJ(2) on cell migration and transformation using a wound-healing assay and morphological analyses, respectively. We found that 15d-PGJ(2) inhibited migration most likely due to NF-κB inhibition and induced transformation of the round-shape A2780/AD cells into elongated epithelial cells due to HDAC1 inhibition. Using a 15d-PGJ(2) analog, we found the mechanism of action of these new activities of 15d-PGJ(2) on SIRT1 and HDAC1 gene expressions and enzyme activities. In conclusion, the present study demonstrates that 15d-PGJ(2) has a high therapeutic potential to kill drug-resistant tumor cells and, the newly described inhibitory effects of this cyclo-oxygenase product on SIRT1 and HDAC will provide new opportunities for cancer therapeutics.

A role for intestinal alkaline phosphatase in the maintenance of local gut immunity.

BACKGROUND AND AIMS: Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor known to dephosphorylate lipopolysaccharide (LPS); however, the role of IAP in the gut response to luminal bacteria remains poorly defined. We investigated immune responses of wild-type (WT) and IAP-knockout (IAP-KO) mice to LPS and Salmonella typhimurium challenges. METHODS: Cryostat sectioning and standard indirect immunohistochemical staining for major histocompatibility complex (MHC) class II molecules were performed on liver tissue from WT and IAP-KO mice. WT and IAP-KO mice were orally gavaged with S. typhimurium; bacterial translocation to mesenteric nodes, liver, and spleen was determined by tissue homogenization and plating. In other experiments, WT and IAP-KO mice received intraperitoneal injections of LPS, with subsequent quantification of complete blood counts and serum interleukin (IL)-6 by enzyme-linked immunosorbent assay (ELISA). WT and IAP-KO whole blood were plated and stimulated with LPS and Pam-3-Cys, followed by cytokine assays. RESULTS: Immunohistologic liver examinations showed increased expression of MHC class II molecules in IAP-KO mice. Following S. typhimurium challenge, WT mice appeared moribund compared with IAP-KO mice, with increased bacterial translocation. WT mice had >50% decrease (P<.005) in platelets and 1.8-fold (P<.05) increased serum IL-6 compared with IAP-KO mice in response to LPS injections. IAP-KO whole-blood stimulation with LPS and Pam-3-Cys resulted in increased IL-6 and tumor necrosis factor (TNF)-alpha secretion compared with WT. CONCLUSIONS: IAP-KO mice exhibit characteristics consistent with local LPS tolerance. Whole-blood response of IAP-KO mice did not reflect systemic tolerance. These data suggest that IAP is a local immunomodulating factor, perhaps regulating LPS-toll-like receptor 4 (TLR4) interaction between commensal microflora and intestinal epithelium.

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.

Tumor-targeted intracellular delivery of anticancer drugs through the mannose-6-phosphate/insulin-like growth factor II receptor.

Tumor-targeting of anticancer drugs is an interesting approach for the treatment of cancer since chemotherapies possess several adverse effects. In the present study, we propose a novel strategy to deliver anticancer drugs to the tumor cells through the mannose-6-phosphate/insulin-like growth factor receptor (M6P/IGF-IIR) which are abundantly expressed in several human tumors. We developed a drug carrier against M6P/IGF-II receptor by modifying human serum albumin (HSA) with M6P moieties. M6P-HSA specifically bound and internalized into M6P/IGF-IIR-expressing B16 melanoma cells as demonstrated with radioactive studies and anti-HSA immunostaining. In vivo, M6P-HSA rapidly accumulated in subcutaneous tumors in tumor and stromal components after an intravenous injection. To demonstrate the application of M6P-HSA as a drug carrier, we coupled doxorubicin to it. Dox-HSA-M6P conjugate could release doxorubicin at lysosomal pH and showed M6P-specific binding and uptake in tumor cells. In vitro, a short exposure with Dox-HSA-M6P induced killing of tumor cells, which could be blocked by excess M6P-HSA. In vivo, Dox-HSA-M6P distributed to tumors and some other organs while free doxorubicin distributed to all organs but slightly to tumors. In B16 tumor-bearing mice, Dox-HSA-M6P significantly inhibited the tumor growth whereas an equimolar dose of free doxorubicin did not show any anti-tumor effect. In addition, targeted doxorubicin did not show any side-effects on liver and kidney function tests, body weight and blood cell counts. In conclusion, M6P-HSA is a suitable carrier for delivery of anticancer drugs to tumors through M6P/IGF-IIR. Improved antitumor effects of the targeted doxorubicin by M6P-HSA suggest that this novel approach may be applied to improve the therapeutic efficacy of anticancer drugs.

Albumin-binding and tumor vasculature determine the antitumor effect of 15-deoxy-Delta-(12,14)-prostaglandin-J(2) in vivo.

15-Deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)), a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, induces cell death in tumor cells in vitro; however, no study showed its in vivo effect on tumors. Here, we report that 15d-PGJ(2) shows antitumor effects in vivo in mice. However, its effects correlate with tumor uptake of albumin, to which it reversibly binds. 15d-PGJ(2) induces cell death in B16F10 melanoma and C26 colon carcinoma cells in vitro. These effects were not elicited through PPARgamma-dependent pathways because an irreversible PPARgamma antagonist GW9662 did not inhibit these effects. Caspase- and nuclear factor kappaB- (NF-kappaB) dependent pathways were found to be involved as determined with caspase-3/7 fluorescent assay and NF-kappaB containing plasmid transfection assay, respectively. Noticeably, 15d-PGJ(2) had significantly stronger effects in C26 cells compared with B16 cells in all assays. However, in vivo, there was no effect on C26 tumors, yet it significantly inhibited the B16 tumor growth in mice by 75%. We found that 15d-PGJ(2) rapidly bound to albumin and in vivo albumin greatly distributed to B16 tumors compared with C26 tumors, shown with gamma-camera imaging and immunohistochemical staining. Albumin accumulation can be attributed to the large blood vessel diameter in B16 tumors and an enhanced permeability and retention effect. These findings suggest that 15d-PGJ(2) can be an effective therapeutic agent for cancer, although its effects seem to be limited to the tumors allowing albumin penetration.