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.

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.

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.

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.

Targeting 15d-prostaglandin J2 to hepatic stellate cells: two options evaluated.

PURPOSE: Delivery of apoptosis-inducing compounds to hepatic stellate cells (HSC) may be an effective strategy to reverse liver fibrosis. The aim of this study was therefore to examine the selective targeting of the apoptosis-inducing drug 15-deoxy-delta12,14-prostaglandin J2 (15dPGJ2) with two different HSC-carriers: human serum albumin modified with the sugar mannose-6-phosphate (M6PHSA) or albumin modified with PDGF-receptor recognizing peptides (pPBHSA). METHODS AND RESULTS: After chemical conjugation of 15dPGJ2 to the carriers, the constructs displayed pharmacological activity and specific receptor-mediated binding to HSC in vitro. Unlike 15dPGJ2-pPBHSA, the cellular binding of 15dPGJ2-M6PHSA was reduced by a scavenger receptor antagonist. In vivo, both conjugates rapidly accumulated in fibrotic livers. Intrahepatic analysis revealed that 15dPGJ2-M6PHSA mainly accumulated in HSC, and to a lesser extent in Kupffer cells. 15dPGJ2-pPBHSA also predominantly accumulated in HSC with additional uptake in hepatocytes. Assessment of target receptors in human cirrhotic livers revealed that M6P/IGFII-receptor expression was present in fibrotic areas. PDGF-P receptor expression was abundantly expressed on human fibroblasts. CONCLUSIONS: These studies show that 15dPGJ2 coupled to either M6PHSA or pPBHSA is specifically taken up by HSC and is highly effective within these cells. Both carriers differ with respect to receptor specificity, leading to differences in intrahepatic distribution. Nevertheless, both carriers can be used to deliver the apoptosis-inducing drug 15dPGJ2 to HSC in vivo.