Radiometal-Labeled Chitosan Microspheres as Transarterial Radioembolization Agents against Hepatocellular Carcinoma.

Transarterial radioembolization (TARE) is an emerging treatment for patients with unresectable hepatocellular carcinoma (HCC). This study successfully developed radiometal-labeled chitosan microspheres (111In/177Lu-DTPA-CMS) with a diameter of 36.5 ± 5.3 µm for TARE. The radiochemical yields of 111In/177Lu-DTPA-CMS were greater than 90% with high radiochemical purities (>98%). Most of the 111In/177Lu-DTPA-CMS were retained in the hepatoma and liver at 1 h after intraarterial (i.a.) administration. Except for liver accumulation, radioactivity in each normal organ was less than 1% of the injected radioactivity (%IA) at 72 h after injection. At 10 days after injection of 177Lu-DTPA-CMS (18.6 ± 1.3 MBq), the size of the hepatoma was significantly reduced by around 81%, while that of the rats in the control group continued to grow. This study demonstrated the effectiveness of 177Lu-DTPA-CMS in the treatment of N1-S1 hepatoma. 111In/177Lu-DTPA-CMS have the potential to be a superior theranostic pair for the treatment of clinical hepatoma.

Effect of Cerenkov Radiation-Induced Photodynamic Therapy with 18F-FDG in an Intraperitoneal Xenograft Mouse Model of Ovarian Cancer.

Ovarian cancer (OC) metastases frequently occur through peritoneal dissemination, and they contribute to difficulties in treatment. While photodynamic therapy (PDT) has the potential to treat OC, its use is often limited by tissue penetration depth and tumor selectivity. Herein, we combined Cerenkov radiation (CR) emitted by 18F-FDG accumulated in tumors as an internal light source and several photosensitizer (PS) candidates with matched absorption bands, including Verteporfin (VP), Chlorin e6 (Ce6) and 5'-Aminolevulinic acid (5'-ALA), to evaluate the anti-tumor efficacy. The in vitro effect of CR-induced PDT (CR-PDT) was evaluated using a cell viability assay, and the efficiency of PS was assessed by measuring the singlet oxygen production. An intraperitoneal ES2 OC mouse model was used for in vivo evaluation of CR-PDT. Positron emission tomography (PET) imaging and bioluminescence-based imaging were performed to monitor the biologic uptake of 18F-FDG and the therapeutic effect. The in vitro studies demonstrated Ce6 and VP to be more effective PSs for CR-PDT. Moreover, VP was more efficient in the generation of singlet oxygen and continued for a long time when exposed to fluoro-18 (18F). Combining CR emitted by 18F-FDG and VP treatment not only significantly suppressed tumor growth, but also prolonged median survival times compared to either monotherapy.

Investigation of biodistribution and tissue penetration of PEGylated gold nanostars and their application for photothermal cancer treatment in tumor-bearing mice.

Gold nanostars (AuNSs), with unique physicochemical properties, are thought to be a promising agent for photothermal therapy (PTT). In this study, we prepared PEGylated gold nanostars (pAuNSs) using the HEPES-reduction method. The high photothermal conversion efficiency (∼80%) and photothermal stability of pAuNSs were demonstrated in vitro and in vivo. 111In-DTPA-pAuNSs were prepared as a radioactive surrogate for the biodistribution studies of pAuNSs. In both microSPECT/CT images and the biodistribution study, the tumor-to-muscle (T/M) ratio reached a maximum at 24 h post intravenous injection of 111In-DTPA-pAuNSs. The high linear correlation between the 111In radioactivity and the gold content in the tumors (R2 0.86-0.99) indicated that 111In-DTPA-pAuNSs were appropriate for noninvasively tracking pAuNSs in vivo after systemic administration. Histological examination after silver enhancement staining clearly illustrated that the accumulated pAuNSs in the tumors were mainly located on the luminal surface of vessels. The mice bearing a SKOV-3 xenograft exhibited remarkable therapeutic efficacy with negligible organ damage after receiving pAuNS-mediated photothermal therapy. Our findings suggested that pAuNSs, together with their radioactive surrogate 111In-DTPA-pAuNSs, are promising for applications in image-guided photothermal therapy.

Specific activation of pro-Infliximab enhances selectivity and safety of rheumatoid arthritis therapy.

During rheumatoid arthritis (RA) treatment, long-term injection of antitumor necrosis factor α antibodies (anti-TNFα Abs) may induce on-target toxicities, including severe infections (tuberculosis [TB] or septic arthritis) and malignancy. Here, we used an immunoglobulin G1 (IgG1) hinge as an Ab lock to cover the TNFα-binding site of Infliximab by linking it with matrix metalloproteinase (MMP) -2/9 substrate to generate pro-Infliximab that can be specifically activated in the RA region to enhance the selectivity and safety of treatment. The Ab lock significantly inhibits the TNFα binding and reduces the anti-idiotypic (anti-Id) Ab binding to pro-Infliximab by 395-fold, 108-fold compared with Infliximab, respectively, and MMP-2/9 can completely restore the TNFα neutralizing ability of pro-Infliximab to block TNFα downstream signaling. Pro-Infliximab was only selectively activated in the disease site (mouse paws) and presented similar pharmacokinetics (PKs) and bio-distribution to Infliximab. Furthermore, pro-Infliximab not only provided equivalent therapeutic efficacy to Infliximab but also maintained mouse immunity against Listeria infection in the RA mouse model, leading to a significantly higher survival rate (71%) than that of the Infliximab treatment group (0%). The high-selectivity pro-Infliximab maintains host immunity and keeps the original therapeutic efficiency, providing a novel strategy for RA therapy.

Evaluation of the Biological Behavior of a Gold Nanocore-Encapsulated Human Serum Albumin Nanoparticle (Au@HSANP) in a CT-26 Tumor/Ascites Mouse Model after Intravenous/Intraperitoneal Administration.

Colorectal cancer is one of the major causes of cancer-related death in Taiwan and worldwide. Patients with peritoneal metastasis from colorectal cancer have reduced overall survival and poor prognosis. Hybrid protein-inorganic nanoparticle systems have displayed multifunctional applications in solid cancer theranostics. In this study, a gold nanocore-encapsulated human serum albumin nanoparticle (Au@HSANP), which is a hybrid protein-inorganic nanoparticle, and its radioactive surrogate 111In-labeled Au@HSANP (111In-Au@HSANP), were developed and their biological behaviors were investigated in a tumor/ascites mouse model. 111In-Au@HSANP was injected either intravenously (iv) or intraperitoneally (ip) in CT-26 tumor/ascites-bearing mice. After ip injection, a remarkable and sustained radioactivity retention in the abdomen was noticed, based on microSPECT images. After iv injection, however, most of the radioactivity was accumulated in the mononuclear phagocyte system. The results of biodistribution indicated that ip administration was significantly more effective in increasing intraperitoneal concentration and tumor accumulation than iv administration. The ratios of area under the curve (AUC) of the ascites and tumors in the ip-injected group to those in the iv-injected group was 93 and 20, respectively. This study demonstrated that the ip injection route would be a better approach than iv injections for applying gold-albumin nanoparticle in peritoneal metastasis treatment.

Presence of Gold Nanoparticles in Cells Associated with the Cell-Killing Effect of Modulated Electro-Hyperthermia.

The efficacy of gold nanoparticle (AuNP)-assisted radiofrequency (RF)-induced hyperthermia employing the Kanzius device remains controversial. Different from the Kanzius device, modulated electro-hyperthermia (mEHT) utilizes the capacitive-impedance coupled 13.56 MHz radiofrequency (RF) current and has been approved for clinical cancer treatment. In this study, we investigated the heating characteristics of spherical-, urchin-, and rod-like AuNPs of a similar 50 nm size upon exposure to a 13.56 MHz radiofrequency using the LabEHY-105CL, an in vivo mEHT device. We found that, regardless of the AuNPs' sphere-, urchin- or rod-like shape, purified gold nanoparticle solution would not promote heat generation. The temperature elevation during radiofrequency irradiation was solely attributed to the ionic background of the solution. The AuNPs present in the medium (≤25 ppm) showed no effect on selective cell killing of malignant cells, whereas the AuNPs incorporated in the cells diminished the cell selectivity as well as cell death and acted as protectors in mEHT cancer treatment. Our study suggested that (1) the temperature elevation induced by 50 nm AuNPs in the 13.56 MHz radiofrequency field was negligible and was shape-independent, and (2) the presence of AuNPs would alter the cell-killing effect of modulated electro-hyperthermia.

Pre-existing anti-polyethylene glycol antibody reduces the therapeutic efficacy and pharmacokinetics of PEGylated liposomes.

Rationale: Increasing frequency of human exposure to PEG-related products means that healthy people are likely to have pre-existing anti-PEG antibodies (pre-αPEG Ab). However, the influence of pre-αPEG Abs on the pharmacokinetics (PK) and therapeutic efficacy of LipoDox is unknown. Methods: We generated two pre-αPEG Ab mouse models. First, naïve mice were immunized with PEGylated protein to generate an endogenous αPEG Ab titer (endo αPEG). Second, monoclonal αPEG Abs were passively transferred (αPEG-PT) into naïve mice to establish a αPEG titer. The naïve, endo αPEG and αPEG-PT mice were intravenously injected with 111in-labeled LipoDox to evaluate its PK. Tumor-bearing naïve, endo αPEG and αPEG-PT mice were intravenously injected with 111in-labeled LipoDox to evaluate its biodistribution. The therapeutic efficacy of LipoDox was estimated in the tumor-bearing mice. Results: The areas under the curve (AUC)last of LipoDox in endo αPEG and αPEG-PT mice were 11.5- and 15.6- fold less, respectively, than that of the naïve group. The biodistribution results suggested that pre-αPEG Ab can significantly reduce tumor accumulation and accelerate blood clearance of 111In-labeled LipoDox from the spleen. The tumor volumes of the tumor-bearing endo αPEG and αPEG-PT mice after treatment with LipoDox were significantly increased as compared with that of the tumor-bearing naïve mice. Conclusions: Pre-αPEG Abs were found to dramatically alter the PK and reduce the tumor accumulation and therapeutic efficacy of LipoDox. Pre-αPEG may have potential as a marker to aid development of personalized therapy using LipoDox and achieve optimal therapeutic efficacy.

Synthesis and biological evaluation of 2-(3,4-dimethoxyphenyl)-6-(2-[18F]fluoroethoxy)benzothiazole ([18F]FEDBT) for PET imaging of breast cancer.

Given the ever-present demand for improved PET radiotracer in oncology imaging, we have synthesized 2-(3,4-dimethoxyphenyl)-6-(2-[18F]fluoroethoxy)benzothiazole ([18F]FEDBT), a fluorine-18-containing fluoroethylated benzothiazole to explore its utility as a PET imaging tracer. [18F]FEDBT was prepared via kryptofix-mediated nucleophilic substitution of the tosyl group precursor. Fractionated ethanol-based solid-phase (SPE cartridge-based) purification afforded [18F]FEDBT in 60% radiochemical yield (EOB), with radiochemical purity in excess of 98% and the specific activity was 35GBq/µmol. The radiotracer displayed clearly higher cellular uptake ratio in various breast cancer cell lines MCF7, MDA-MB-468 and MDA-MB-231. However, both biodistribution and microPET studies have showed an higher abdominal accumulation of [18F]FEDMBT and the tumor/muscle ratio of 1.8 was observed in the MDA-MB-231 xenograft tumors mice model. Further the lipophilic improvement is needed for the reducement of hepatobilliary accumulation and to promote the tumor uptake for PET imaging of breast cancer.

Monitoring Tumor Response after Liposomal Doxorubicin in Combination with Liposomal Vinorelbine Treatment Using 3'-Deoxy-3'-[18F]Fluorothymidine PET.

PURPOSE: Surgical resection is the standard treatment for localized colorectal cancer, which is the most common type of gastrointestinal cancer. However, over 40 % cases are diagnosed metastasized and apparently inoperable. Systemic chemotherapy provides an alternative to these patients. This study aims to evaluate the therapeutic potential of liposomal doxorubicin (lipoDox) in combination with liposomal vinorelbine (lipoVNB) in a CT-26 colon carcinoma-bearing mouse model. PROCEDURES: The in vitro cytotoxicity of Dox and VNB on CT-26 cancer cells was determined by MTT and colony formation assays. Mice were subcutaneously inoculated with 2 × 105 of CT-26 cells in the right hind flank. When tumor size reached 200 ± 50 mm3, mice were assigned to receive different treatment protocols. The pharmacokinetics, micro single-photon emission computed tomography/x-ray computed tomography imaging, biodistribution, and immunohistochemical staining studies were performed to survey the therapeutic efficacy of each regimen. RESULTS: Based on the results of pharmacokinetic study, co-administration of lipoDox and lipoVNB did not affect their individual systemic distribution, while lipoDox retained longer in blood than lipoVNB did. Superior tumor growth retardation was observed in the group received lipoDox plus lipoVNB administration (1 mg/kg each, namely D1V1) than those injected with lipoDox plus VNB (1 mg/kg each, namely D1fV1). No severe side effects were detected in each group. The tumor-to-muscle ratio (T/M) derived from 3'-dexoy-3'-[18F]fluorothymidine ([18F]FLT) micro positron emission tomography (PET) images of D1V1- and D1fV1-treated mice and the controls on day 7 was 6.88 ± 0.54, 7.50 ± 0.84, and 9.87 ± 0.73, respectively, suggesting that D1V1 is a more efficacious regimen against CT-26 xenografts. The results of proliferating cell nuclear antigen (PCNA) immunohistochemical staining were consistent with those findings obtained from [18F]FLT microPET imaging. CONCLUSION: This study demonstrated that lipoDox in combination with lipoVNB was more efficacious than clinically used regimen, lipoDox plus VNB, in the treatment of colon carcinoma and [18F]FLT-PET is a promising approach in monitoring the treatment outcome at early stage.

Using anti-poly(ethylene glycol) bioparticles for the quantitation of PEGylated nanoparticles.

Attachment of polyethylene glycol (PEG) molecules to nanoparticles (PEGylation) is a widely-used method to improve the stability, biocompatibility and half-life of nanomedicines. However, the evaluation of the PEGylated nanomedicine pharmacokinetics (PK) requires the decomposition of particles and purification of lead compounds before analysis by high performance liquid chromatography (HPLC), mass spectrometry, etc. Therefore, a method to directly quantify un-decomposed PEGylated nanoparticles is needed. In this study, we developed anti-PEG bioparticles and combined them with anti-PEG antibodies to generate a quantitative enzyme-linked immunosorbent assay (ELISA) for direct measurement of PEGylated nanoparticles without compound purification. The anti-PEG bioparticles quantitative ELISA directly quantify PEG-quantum dots (PEG-QD), PEG-stabilizing super-paramagnetic iron oxide (PEG-SPIO), Lipo-Dox and PEGASYS and the detection limits were 0.01 nM, 0.1 nM, 15.63 ng/mL and 0.48 ng/mL, respectively. Furthermore, this anti-PEG bioparticle-based ELISA tolerated samples containing up to 10% mouse or human serum. There was no significant difference in pharmacokinetic studies of radiolabeled PEG-nanoparticles (Nano-X-111In) through anti-PEG bioparticle-based ELISA and a traditional gamma counter. These results suggest that the anti-PEG bioparticle-based ELISA may provide a direct and effective method for the quantitation of any whole PEGylated nanoparticles without sample preparation.

Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine.

BACKGROUND: The enzyme-prodrug system is considered a promising tool for tumor treatment when conjugated with a targeting molecule. The asparagine-glycine-arginine (NGR) motif is a developing and interesting targeting peptide that could specifically bind to aminopeptidase N (APN), which is an NGR receptor expressed on the cell membrane of angiogenic endothelial cells and a number of tumor cells within the tumor tissues. The objective of this study was to develop a novel targeted enzyme-prodrug system using 5-fluorocytosine (5-FC) and an NGR-containing peptide fused with yeast cytosine deaminase (yCD), i.e. CNGRC-yCD fusion protein, to target APN-expressing cells within the tumor tissues and to convert 5-FC into 5-fluorouracil (5-FU) to kill tumors. RESULTS: Both yCD and CNGRC-yCD proteins were cloned into the pET28a vector and expressed by an Escherichia coli host. Both yCD and CNGRC-yCD proteins were purified and the yields were approximately 20 mg/L with over 95 % purity. The binding assay demonstrated that the CNGRC-yCD fusion protein had specific binding affinity toward purified APN recombinant protein and high-APN-expressing cells, including human endothelial cells (HUVECs) and various types of human tumor cell lines, but not low-APN-expressing tumor cell lines. Moreover, the enzyme activity and cell viability assay showed that the CNGRC-yCD fusion protein could effectively convert 5-FC into 5-FU and resulted in significant cell death in both high-APN-expressing tumor cells and HUVECs. CONCLUSIONS: This study successfully constructs a new targeting enzyme-prodrug system, CNGRC-yCD fusion protein/5-FC. Systematic experiments demonstrated that the CNGRC-yCD protein retained both the APN-binding affinity of NGR and the enzyme activity of yCD to convert 5-FC into 5-FU. The combined treatment of the CNGRC-yCD protein with 5-FC resulted in the significantly increased cell death of high-APN-expressing cells as compared to that of low-APN-expressing cells.

Development and Characterization of the Recombinant Human VEGF-EGF Dual-Targeting Fusion Protein as a Drug Delivery System.

The design, preparation, as well as structural and functional characterizations of the recombinant fusion protein hVEGF-EGF as a dual-functional agent that may target both EGFR (R: receptor) and angiogenesis are reported. hVEGF-EGF was found to bind to EGFR more strongly than did EGF, and to bind to VEGFR similarly to VEGF. Mass spectrometry measurements showed that the sites of DTPA (diethylenetriaminepentaacetic acid) conjugated hVEGF-EGF (for radiolabeling) were the same as those of its parent hEGF and hVEGF proteins. All DTPA-conjugated proteins retained similar binding capacities to their respective receptors as compared to their respective parent proteins. In vitro cell binding studies using BAEC (a bovine aortic endothelial cell) and MDA-MB-231 (a human breast cancer) cells expressing both EGFR and VEGFR confirmed similar results. Treating BAEC cells with hVEGF-EGF induced remarkable phosphorylation of EGFR, VEGFR, and their downstream targets ERK1/2. Nevertheless, the radiolabeled (111)In-DTPA-hVEGF-EGF showed cytotoxicity against MDA-MB-231 cells. Pharmacokinetic studies using (111)In-DTPA-hVEGF-EGF in BALB/c nude mice showed that appreciable tracer activities were accumulated in liver and spleen. In all, this study demonstrated that the fusion protein hVEGF-EGF maintained the biological specificity toward both EGFR and VEGFR and may be a potential candidate as a dual-targeting moiety in developing anticancer drugs.

Pharmacokinetic analysis and uptake of 18F-FBPA-Fr after ultrasound-induced blood-brain barrier disruption for potential enhancement of boron delivery for neutron capture therapy.

UNLABELLED: Boronophenylalanine has been applied in clinical boron neutron capture therapy for the treatment of high-grade gliomas. The purpose of this study was to evaluate the pharmacokinetics of 4-borono-2-(18)F-fluoro-L-phenylalanine-fructose ((18)F-FBPA-Fr) in F98 glioma-bearing Fischer 344 rats by means of intravenous injection of (18)F-FBPA-Fr both with and without blood-brain barrier disruption (BBB-D) induced by focused ultrasound (FUS). METHODS: Dynamic PET imaging of (18)F-FBPA-Fr was performed on the ninth day after tumor implantation. Blood samples were collected to obtain an arterial input function for tracer kinetic modeling. Ten animals were scanned for approximately 3 h to estimate the uptake of (18)F radioactivity with respect to time for the pharmacokinetic analysis. Rate constants were calculated by use of a 3-compartment model. RESULTS: The accumulation of (18)F-FBPA-Fr in brain tumors and the tumor-to-contralateral brain ratio were significantly elevated after intravenous injection of (18)F-FBPA-Fr with BBB-D. (18)F-FBPA-Fr administration after sonication showed that the tumor-to-contralateral brain ratio for the sonicated tumors (3.5) was approximately 1.75-fold higher than that for the control tumors (2.0). Furthermore, the K1/k2 pharmacokinetic ratio after intravenous injection of (18)F-FBPA-Fr with BBB-D was significantly higher than that after intravenous injection without BBB-D. CONCLUSION: This study demonstrated that radioactivity in tumors and the tumor-to-normal brain ratio after intravenous injection of (18)F-FBPA-Fr with sonication were significantly higher than those in tumors without sonication. The K1/k2 ratio may be useful for indicating the degree of BBB-D induced by FUS. Further studies are needed to determine whether FUS may be useful for enhancing the delivery of boronophenylalanine in patients with high-grade gliomas.

Tumor burden talks in cancer treatment with PEGylated liposomal drugs.

PURPOSE: PEGylated liposomes are important drug carriers that can passively target tumor by enhanced permeability and retention (EPR) effect in neoplasm lesions. This study demonstrated that tumor burden determines the tumor uptake, and also the tumor response, in cancer treatment with PEGylated liposomal drugs in a C26/tk-luc colon carcinoma-bearing mouse model. METHODS: Empty PEGylated liposomes (NanoX) and those encapsulated with VNB (NanoVNB) were labeled with In-111 to obtain InNanoX and InVNBL in high labeling yield and radiochemical purity (all >90%). BALB/c mice bearing either small (58.4±8.0 mm(3)) or large (102.4±22.0 mm(3)) C26/tk-luc tumors in the right dorsal flank were intravenously administered with NanoVNB, InNanoX, InVNBL, or NanoX as a control, every 7 days for 3 times. The therapeutic efficacy was evaluated by body weight loss, tumor growth inhibition (using calipers and bioluminescence imaging) and survival fraction. The scintigraphic imaging of tumor mouse was performed during and after treatment. RESULTS: The biodistribution study of InVNBL revealed a clear inverse correlation (r (2) = 0.9336) between the tumor uptake and the tumor mass ranged from 27.6 to 623.9 mg. All three liposomal drugs showed better therapeutic efficacy in small-tumor mice than in large-tumor mice. Tumor-bearing mice treated with InVNBL (a combination drug) showed the highest tumor growth inhibition rate and survival fraction compared to those treated with NanoVNB (chemodrug only) and InNanoX (radionuclide only). Specific tumor targeting and significantly increased tumor uptake after periodical treatment with InVNBL were evidenced by scintigraphic imaging, especially in mice bearing small tumors. CONCLUSION: The significant differences in the outcomes of cancer treatment and molecular imaging between animals bearing small and large tumors revealed that tumor burden is a critical and discriminative factor in cancer therapy using PEGylated liposomal drugs.

Pharmacokinetic analysis of 111 in-labeled liposomal Doxorubicin in murine glioblastoma after blood-brain barrier disruption by focused ultrasound.

The goal of this study was to evaluate the pharmacokinetics of targeted and untargeted (111)In-doxorubicin liposomes after these have been intravenously administrated to tumor-bearing mice in the presence of blood-brain barrier disruption (BBB-D) induced by focused ultrasound (FUS). An intracranial brain tumor model in NOD-scid mice using human brain glioblastoma multiforme (GBM) 8401 cells was developed in this study. (111)In-labeled human atherosclerotic plaque-specific peptide-1 (AP-1)-conjugated liposomes containing doxorubicin (Lipo-Dox; AP-1 Lipo-Dox) were used as a microSPECT probe for radioactivity measurements in the GBM-bearing mice. Compared to the control tumors treated with an injection of (111)In-AP-1 Lipo-Dox or (111)In-Lipo-Dox, the animals receiving the drugs followed by FUS exhibited enhanced accumulation of the drug in the brain tumors (p<0.05). Combining sonication with drugs significantly increased the tumor-to-normal brain doxorubicin ratio of the target tumors compared to the control tumors. The tumor-to-normal brain ratio was highest after the injection of (111)In-AP-1 Lipo-Dox with sonication. The (111)In-liposomes micro-SPECT/CT should be able to provide important information about the optimum therapeutic window for the chemotherapy of brain tumors using sonication.

Pharmacokinetics and dosimetry of (111)In/(188)Re-labeled PEGylated liposomal drugs in two colon carcinoma-bearing mouse models.

PEGylated liposomes are important drug carriers for nanomedicine cancer therapy. PEGylated liposomes can encapsulate radio- and chemo-drugs and passively target tumor sites via enhanced permeability and retention effect. This study estimated the pharmacokinetics and dosimetry after administration of radio-chemotherapeutics ((111)In-labeled vinorelbine [VNB]-encapsulated liposomes, InVNBL, and (188)Re-labeled doxorubicin [DXR]-encapsulated liposomes, ReDXRL) for radionuclide therapy in two colon carcinoma-bearing mouse models. A C26 colon carcinoma tumor/ascites mouse model and a subcutaneous solid tumor-bearing mouse model were employed. Biodistribution studies of InVNBL and ReDXRL after intraperitoneal administration in tumor/ascites-bearing mice (protocol A) and intravenous administration in subcutaneous solid tumor-bearing mice (protocol B) were performed. The radiation dose to normal tissues and tumors were calculated based on the results of distribution studies in mice, using the OLINDA/EXM program. The cumulated activities in most organs after administration of InVNBL in either the tumor/ascites-bearing mice (protocol A) or the subcutaneous solid tumor-bearing mice (protocol B) were higher than those of ReDXRL. Higher tumor-to-normal-tissues absorption dose ratios (T/NTs) were observed after administration of InVNBL than those of ReDXRL for protocol A. The T/NTs for the liver, spleen, and red marrow after injection of InVNBL for protocol B were similar to those of ReDXRL. The critical organ was found to be red marrow, and thus the red marrow absorption dose defined the recommended maximum administration activity of these liposomal drugs. Characterization of pharmacokinetics and dosimetry is needed to select the appropriate radiotherapeutics for specific tumor treatment applications. The results suggest that InVNBL is a promising therapeutic agent, which is as good as ReDXRL, in two mouse tumor models.

Evaluation of pharmacokinetics of 111In-labeled VNB-PEGylated liposomes after intraperitoneal and intravenous administration in a tumor/ascites mouse model.

Nanoliposomes are important drug carriers that can passively target tumor sites by the enhanced permeability and retention (EPR) effect in neoplasm lesions. This study evaluated the biodistribution and pharmacokinetics of 111In-labeled vinorelbine (VNB)-encapsulated PEGylated liposomes (IVNBPL) after intraperitoneal (i.p.) and intravenous (i.v.) administration in a C26/tk-luc colon carcinoma ascites mouse model. IVNBPL was prepared by labeling VNB-encapsulated PEGylated liposomes with 111In-oxine. BALB/c mice were i.p. inoculated with 2 x 10(5) C26/tk-luc cells in 500 muL of phosphate-buffered saline. Peritoneal tumor lesions were confirmed by 124I-FIAU/micro-PET (positron emission tomography) and bioluminescence imaging. Ascites production was examined by ultrasound imaging on day 10 after tumor cell inoculation. The pharmacokinetics and biodistribution studies of IVNBPL in a tumor/ascites mouse model were conducted. The labeling efficiency was more than 90%. The in vitro stability in human plasma at 37 degrees C for 72 hours was 83% +/- 3.5%. For i.p. administration, the areas under curves (AUCs) of ascites and tumor were 6.78- and 1.70-fold higher, whereas the AUCs of normal tissues were lower than those via the i.v. route. This study demonstrates that i.p. administration is a better approach than i.v. injection for IVNBPL, when applied to the treatment of i.p. malignant disease in a tumor/ascites mouse model.

Prevention of TGF-beta-induced apoptosis by interlukin-4 through Akt activation and p70S6K survival signaling pathways.

In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-beta (TGF-beta). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-beta-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-beta-induced apoptosis.

Proteomics analysis of a novel compound: cyclic RGD in breast carcinoma cell line MCF-7.

In studies of cell adhesion, migration, growth, differentiation, and apoptosis, synthetic peptides containing the RGD (Arg-Gly-Asp) motif have been extensively used as the inhibitors of integrin-ligand interactions. The RGD motif is an integrin-recognition motif found in many ligands, so that the RGD-containing peptides can be used to probe integrin functions in various biological systems. A linear RGD is a tripeptide consisting of a flexible structure that makes the motif bind to its receptor with inefficient chelating affinity. Therefore, we designed a cyclic-RGD peptide (Tpa-RGDWPC, cRGD) with rigid skeleton to closely bind with its receptor. The cRGD was obtained by solid-phase peptide synthesis method using Rink amide resin. We showed that the cRGD exerts more potency than linear RGD on inhibiting cell growth of MCF-7 breast carcinoma cells. This stimulated us to question how cRGD inhibits cell growth of MCF-7 cells. Moreover, understanding what molecular mechanism underlies the effect that RGD motif exerts on MCF-7 cells is also of considerable importance. We used proteomics and bioinformatics to survey the global changes in proteins after cRGD treatment in MCF-7 cells. The classification of these proteins is shown according to the different biological processes in which they are involved. Most of the proteins that appear to be strongly influenced by cRGD treatment are involved in metabolism, cell growth, responsive to external stimulus, cell communication, reproduction and cell death. This is the first report which monitors the protein expression profile of MCF-7 cells in response to treatment with RGD-containing peptides in a time-course analysis. The clustering data indicated temporal patterns of altered protein expression that can be categorized into early, intermediate and late response proteins. These patterns of protein expression may be important for predicting its response to cRGD. In summary, these results provide a molecular explanation for the properties of cRGD in breast cancer cells and present a valuable in-depth description of their possible impact on breast cancer therapy.