Polyethyleneimine-coating enhances adenoviral transduction of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential, which makes them attractive targets for regenerative medicine applications. Efficient gene transfer into MSCs is essential for basic research in developmental biology and for therapeutic applications involving gene-modification in regenerative medicine. Adenovirus vectors (Advs) can efficiently and transiently introduce an exogenous gene into many cell types via their primary receptors, the coxsackievirus and adenovirus receptors (CARs), but not into MSCs, which lack CAR expression. To overcome this problem, an Adv coated with cationic polymer polyethyleneimine (PEI) was developed. In this study, we demonstrated that PEI coating with an optimal ratio can enhance adenoviral transduction of MSCs without cytotoxicity. We also investigated the physicochemical properties and internalization mechanisms of the PEI-coated Adv. These results could help to evaluate the potentiality of the PEI-coated Adv as a prototype vector for efficient and safe transduction into MSCs.

An inactivated whole-virion vaccine for Enterovirus D68 adjuvanted with CpG ODN or AddaVax elicits potent protective immunity in mice.

Enterovirus D68 (EV-D68), a pathogen that causes respiratory symptoms, mainly in children, has been implicated in acute flaccid myelitis, which is a poliomyelitis-like paralysis. Currently, there are no licensed vaccines or treatments for EV-D68 infections. Here, we investigated the optimal viral inactivation reagents, vaccine adjuvants, and route of vaccination in mice to optimize an inactivated whole-virion (WV) vaccine against EV-D68. We used formalin, ß-propiolactone (BPL), and hydrogen peroxide as viral inactivation reagents and compared their effects on antibody responses. Use of any of these three viral inactivation reagents effectively induced neutralizing antibodies. Moreover, the antibody response induced by the BPL-inactivated WV vaccine was enhanced when adjuvanted with cytosine phosphoguanine oligodeoxynucleotide (CpG ODN) or AddaVax (MF59-like adjuvant), but not with aluminum hydroxide (alum). Consistent with the antibody response results, the protective effect of the inactivated WV vaccine against the EV-D68 challenge was enhanced when adjuvanted with CpG ODN or AddaVax, but not with alum. Further, while the intranasal inactivated WV vaccine induced EV-D68-specific IgA antibodies in the respiratory tract, it was less protective against EV-D68 challenge than the injectable vaccine. Thus, an injectable inactivated EV-D68 WV vaccine prepared with appropriate viral inactivation reagents and an optimal adjuvant is a promising EV-D68 vaccine.

Lipid Nanoparticle with 1,2-Di-O-octadecenyl-3-trimethylammonium-propane as a Component Lipid Confers Potent Responses of Th1 Cells and Antibody against Vaccine Antigen.

Adjuvants are effective tools to enhance vaccine efficacy and control the type of immune responses such as antibody and T helper 1 (Th1)- or Th2-type responses. Several studies suggest that interferon (IFN)-γ-producing Th1 cells play a significant role against infections caused by intracellular bacteria and viruses; however, only a few adjuvants can induce a strong Th1-type immune response. Recently, several studies have shown that lipid nanoparticles (LNPs) can be used as vaccine adjuvants and that each LNP has a different adjuvant activity. In this study, we screened LNPs to develop an adjuvant that can induce Th1 cells and antibodies using a conventional influenza split vaccine (SV) as an antigen in mice. We observed that LNP with 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA) as a component lipid (DOTMA-LNP) elicited robust SV-specific IgG1 and IgG2 responses compared with SV alone in mice and was as efficient as SV adjuvanted with other adjuvants in mice. Furthermore, DOTMA-LNPs induced robust IFN-γ-producing Th1 cells without inflammatory responses compared to those of other adjuvants, which conferred strong cross-protection in mice. We also demonstrated the high versatility of DOTMA-LNP as a Th1 cell-inducing vaccine adjuvant using vaccine antigens derived from severe acute respiratory syndrome coronavirus 2 and Streptococcus pneumoniae. Our findings suggest the potential of DOTMA-LNP as a safe and effective Th1 cell-inducing adjuvant and show that LNP formulations are potentially potent adjuvants to enhance the effectiveness of other subunit vaccines.

Optimization and internalization mechanisms of PEGylated adenovirus vector with targeting peptide for cancer gene therapy.

We have previously developed a novel adenovirus vector (Adv) that targeted tumor tissues/vasculatures after systemic administration. The surface of this Adv is conjugated with CGKRK tumor homing peptide by the cross-linking reaction of polyethyleneglycol (PEG). In this study, we showed that the condition of PEG modification was important to minimize the gene expression in normal tissues after systemic treatment. When Adv was modified only with PEG-linked CGKRK, its luciferase expression was enhanced even in the liver tissue, as well as the tumor tissue. However, in the reaction with the mixture of non-cross-linking PEG and PEG-linked CGKRK, we found out that the best modification could suppress its gene expression in the liver, without losing that in the tumor. We also studied the internalization mechanisms of CGKRK-conjugated Adv. Results suggested that there is a specific interaction of the CGKRK peptide with a receptor at the cell surface enabling efficient internalization of CGKRK-conjugated Adv. The presence of cell-surface heparan sulfate is important receptor for the cellular binding and uptake of CGKRK-conjugated Adv. Moreover, macropinocytosis-mediated endocytosis is also important in endocytosis of CGKRK-conjugated Adv, aside from clathrin-mediated and caveolae-mediated endocytosis. These results could help evaluate the potentiality of CGKRK-conjugated Adv as a prototype vector with suitable efficacy and safety for systemic cancer gene therapy.

Tumor vascular targeted delivery of polymer-conjugated adenovirus vector for cancer gene therapy.

Previously, we generated a cancer-specific gene therapy system using adenovirus vectors (Adv) conjugated to polyethylene glycol (Adv-PEG). Here, we developed a novel Adv that targets both tumor tissues and tumor vasculatures after systemic administration by conjugating CGKRK tumor vasculature homing peptide to the end of a 20-kDa PEG chain (Adv-PEG(CGKRK)). In a primary tumor model, systemic administration of Adv-PEG(CGKRK) resulted in ~500- and 100-fold higher transgene expression in tumor than that of unmodified Adv and Adv-PEG, respectively. In contrast, the transgene expression of Adv-PEG(CGKRK) in liver was about 400-fold lower than that of unmodified Adv, and was almost the same as that of Adv-PEG. We also demonstrated that transgene expression with Adv-PEG(CGKRK) was enhanced in tumor vessels. Systemic administration of Adv-PEG(CGKRK) expressing the herpes simplex virus thymidine kinase (HSVtk) gene (Adv-PEG(CGKRK)-HSVtk) showed superior antitumor effects against primary tumors and metastases with negligible side effects by both direct cytotoxic effects and inhibition of tumor angiogenesis. These results indicate that Adv-PEG(CGKRK) has potential as a prototype Adv with suitable efficacy and safety for systemic cancer gene therapy against both primary tumors and metastases.

Intravenous administration of polyethylene glycol-coated (PEGylated) proteins and PEGylated adenovirus elicits an anti-PEG immunoglobulin M response.

A single intravenous administration of polyethylene glycol-coated (PEGylated) bovine serum albumin (BSA) and ovalbumin (OVA) elicited an anti-PEG immunoglobulin M (IgM) response, similar to that from PEGylated liposomes, although the administration did not elicit specific neutralizing antibodies to BSA and OVA. A cross-reactivity was observed between anti-PEG IgMs elicited by PEG-BSA and PEGylated liposomes. The anti-PEG IgM level induced by PEGylated proteins (BSA and OVA) reached the maximum at day 5 following intravenous injection. This production pattern was consistent with that induced by PEGylated liposomes. Splenectomy suppressed the anti-PEG IgM response against PEG-BSA and PEGylated liposomes. These observations relating PEG-BSA and PEGylated liposomes indicate that PEGylated proteins might promote the immune responses against PEG with a mechanism similar to that of PEGylated liposomes. In addition, a single intravenous administration of PEGylated adenovirus (PEG-Ad) also elicited an anti-PEG IgM response in a PEG-modification ratio dependent manner. To the best of our knowledge, this is the first report showing that an intravenous administration can elicit an anti-PEG IgM response against PEGylated substances. It appears that anti-PEG IgMs can be produced by the systemic administration of a PEGylated substance and may limit the efficacy of PEGylated substances such as proteins, Ad vector and nanoparticles, due to a cross-reactivity seen in some patients. The immunogenicity of PEGylated substances is usually tested against those very substances, rather than against covalently attached PEG. Our study suggests that the PEG immunogenicity of PEGylated therapeutic agents and particles merits further investigation.

Lysine-deficient lymphotoxin-α mutant for site-specific PEGylation.

The cytokine lymphotoxin-α (LTα) is a promising anticancer agent; however, its instability currently limits its therapeutic potential. Modification of proteins with polyethylene glycol (PEGylation) can improve their in vivo stability, but PEGylation occurs randomly at lysine residues and the N-terminus. Therefore, PEGylated proteins are generally heterogeneous and have lower bioactivity than their non-PEGylated counterparts. Previously, we created phage libraries expressing mutant LTαs in which the lysine residues of wild-type LTα (wtLTα) were substituted for other amino acids. Here, we attempted to create a lysine-deficient mutant LTα with about the same bioactivity as wtLTα by using these libraries and site-specific PEGylation of the N-terminus. We isolated a lysine-deficient mutant LTα, LT-K0, with almost identical bioactivity to that of wtLTα against mouse LM cells. The bioactivity of wtLTα decreased to 10% following random PEGylation, whereas that of LT-K0 decreased to 50% following site-specific PEGylation; PEGylated LT-K0 retained five times the bioactivity of randomly PEGylated wtLTα. These results suggest that site-specific PEGylated LT-K0 may be useful in cancer therapy.

Creation of a lysine-deficient LIGHT mutant with the capacity for site-specific PEGylation and low affinity for a decoy receptor.

The cytokine LIGHT is a promising candidate for cancer therapy. However, the therapeutic effect of LIGHT as a systemic anticancer agent is currently insufficient because of its instability and its binding to nonfunctional soluble decoy receptor 3 (DcR3), which is overexpressed in various tumors. Modification of proteins with polyethylene glycol (PEGylation) can improve their in vivo stability, but PEGylation may occur randomly at all lysine residues and the NH(2)-terminus; therefore, PEGylated proteins are generally heterogeneous and have decreased bioactivity. In this study, we attempted to create a lysine-deficient LIGHT mutant that could be PEGylated site-specifically and would have lower affinity for DcR3. We prepared phage libraries expressing LIGHT mutants in which all the lysine residues were replaced with other amino acids. A lysine-deficient LIGHT mutant [mLIGHT-Lys(-)] was isolated by panning against lymphotoxin beta receptor (LTbetaR). mLIGHT-Lys(-) could be site-specifically PEGylated at its NH(2)-terminus, yielding molecular uniformity and in vitro bioactivity equal to that of non-PEGylated, wild-type LIGHT. Furthermore, mLIGHT-Lys(-) was not trapped by the nonfunctional DcR3, despite binding to its functional receptors. These results suggest that mLIGHT-Lys(-) might be a useful candidate for cancer therapy.

Simple PEG conjugation of SPIO via an Au-S bond improves its tumor targeting potency as a novel MR tumor imaging agent.

Gold/iron oxide magnetic nanoparticles are hybrid nanoparticles containing a core of magnetic iron oxide and surface colloidal gold, which allows for various biomaterials to be immobilized on the surface of the iron oxide nanoparticles via colloidal gold. Here, we developed a novel magnetic resonance (MR) imaging agent to broaden the MR tumor-imaging spectrum of superparamagnetic iron oxide nanoparticles (SPIO), e.g., Feridex(), a clinical MR imaging agent for diagnosing liver cancer. Au/Feridex was synthesized by electron beam irradiation, and thiol-modified poly(ethylene glycol) (PEG-SH) was easily conjugated to its surface via an Au-S bond without the need for any chemical reactions. PEG conjugation of Au/Feridex enhanced its accumulation in Meth-A tumor tissue and decreased its accumulation in normal liver tissue. In addition, MRI using PEG-Au/Feridex, in contrast to MRI using unmodified Au/Feridex and Feridex, detected B16BL6 and Meth-A tumor tissues in vivo. This finding indicates that PEG-Au/Feridex is useful for diagnosing various types of tumors. In addition, because the synthesis of PEG-Au/Feridex is simple and high yields are easily produced, PEG-modified SPIO for tumor diagnosis can be prepared on an industrial scale with low cost.

Adenovirus vector covalently conjugated to polyethylene glycol with a cancer-specific promoter suppresses the tumor growth through systemic administration.

Cancer gene therapy with adenovirus vectors (Adv) is limited to local administration because systemic administration of Adv produces a weak therapeutic effect and severe side effects. Previously, we generated a dual cancer-specific Adv system by using Adv covalently conjugated to polyethylene glycol (PEG) for transductional targeting and the telomere reverse transcriptase (TERT) promoter as a cancer-specific promoter for transcriptional targeting (PEG-Ad-TERT). We demonstrated that systemic administration of PEG-Ad-TERT showed superior antitumor effects against lung metastatic cancer with negligible side effects. Here, we investigated the therapeutic efficacy of systemic administration of PEG-Ad-TERT for the treatment of primary tumors. We first evaluated the transgene expression of PEG-Ad-TERT containing the luciferase gene (PEG-Ad-TERT/Luc) in primary tumors. Systemic administration of PEG-Ad-TERT/Luc resulted high transgene expression, similar to that observed in tumors for the conventional cytomegalovirus (CMV) promoter-driven Adv containing the luciferase gene (Ad-CMV/Luc). By comparison, transgene expression was 2500-fold lower than that of Ad-CMV/Luc in liver. We then examined the therapeutic effect of systemic administration of PEG-Ad-TERT containing the herpes simplex virus thymidine kinase (HSVtk) gene (PEG-Ad-TERT/HSVtk) for the treatment of primary tumors. We showed that PEG-Ad-TERT/HSVtk produced a notable antitumor effect against primary tumors with negligible side effects. These results demonstrated that PEG-Ad-TERT can be regarded as a prototype Adv with suitable efficacy and safety for systemic cancer gene therapy against both metastatic and primary tumors.

Optimized PEGylated adenovirus vector reduces the anti-vector humoral immune response against adenovirus and induces a therapeutic effect against metastatic lung cancer.

Application of adenovirus vectors (Adv) in metastatic cancer treatment is limited. We previously demonstrated that covalent conjugation of polyethleneglycol (PEG) to Adv enhances therapeutic effects and decreases toxic side-effects after systemic administration, but the level of immune response to PEGylated Adv (PEG-Ad) was not examined. Here, we examined the effect of PEGylation of Adv on the production of anti-Adv antibodies and antitumor response. We constructed a set of PEG-Ad using 5-kDa PEG, with modification rates of 30%, 45% and 90%. After systemic administration of Advs to rats, we examined the level of anti-Adv immunoglobulin (Ig)G and IgM in serum. The levels of anti-Adv IgG and anti-Adv IgM in rats treated with unmodified Adv were higher than those in control group. Rats treated with PEG-Ad that had a 90% modification rate showed lower level of anti-Adv IgG and anti-Adv IgM than those treated with unmodified Adv, whereas rats treated with PEG-Ad that had a 30% or 45% modification rate showed a similar level of anti-Adv IgG and IgM to those treated with unmodified Adv. Systemic administration of PEG-Ad that had a 90% modification rate, and expressed tumor necrosis factor-alpha, significantly reduced the number of metastatic colonies in the lung compared to unmodified Adv, with negligible side effects. These results suggest that systemic administration of PEG-Ad with an appropriate PEG modification rate has the potential to reduce the production of antibodies against Adv and increase the therapeutic response against metastatic cancer.

Assessment of influence of smoking, drinking, leukoplakia and dental irritation on local control of early oral tongue carcinoma treated with brachytherapy: age and dental factors are potential prognostic factors.

BACKGROUND: To examine the background characteristics of elderly patients (65 years or older) with node-negative mobile tongue cancer (T1-2N0M0) who showed worse local control than a younger group. MATERIALS AND METHODS: We retrospectively analyzed background data for 265 patients treated with brachytherapy with or without external radiotherapy between 1967 and 1999. We examined dental factors (such as irritation by prosthesis), leukoplakia, tobacco smoking and alcohol consumption for comparisons between the elderly (age > or = 65 years; n = 83) and a control group (64 years or younger; n = 182). RESULTS: The elderly patients showed a worse outcome than the control group (respectively 86% and 70% at 5 years; P < 0.05). Incidence of dental factors tended to be higher for elderly patients (53%) than the control group (40%, P = 0.07). Dental factors proved to have prognostic importance for local control. Five-year local control rate was 85% for patients with and 76% for patients without dental factors (P = 0.04). The elderly group positive for dental factors showed a lower 5-year local control rate (61%) than the other three groups [(elderly without the dental factor (-) group (80%), control with the dental factor (+) group (84%), and control without the dental factor (-) group (87%)] (P < 0.05). Leukoplakia was found more frequently in the control (23%) than in the elderly group (5%) (P = 0.006) but had no effect on treatment outcome. CONCLUSIONS: Age and dental factors (including prosthesis irritation) are potentially important prognostic factors for local control of oral tongue cancer treated with brachytherapy, especially for elderly patients.

Evaluation of organ-at-risk dose reduction with jaw tracking technique in flattening filter-free beams in lung stereotactic body radiation therapy.

PURPOSE: (i) to investigate the capability of organ-at-risk (OAR) dose reduction with the jaw tracking (JT) technique in flattening filter-free (FFF) beams in lung stereotactic body radiation therapy (SBRT), (ii) to propose a novel metric to quantify the jaw movements during JT, and (iii) to examine the relationships between the quantified jaw movements and reduction rate of OAR doses. METHODS: The individual SBRT plans with volumetric modulated arc therapy using the JT technique (JT-VMAT) and VMAT plans with a fixed jaw (FJ-VMAT) were created for 15 patients, and dosimetric parameters were compared. A jaw tracking complexity score (JTCS) was defined and compared with the multi-leaf collimator (MLC) modulation complexity score (MCS). The correlations between the JTCS and reduction rate of OAR doses were examined. RESULTS: The decrease of OARs doses was statistically significant in the JT-VMAT plans (1.2% in V20 of the lung and <1% in all other OARs). The correlations between the JTCS and MCS were not significant. There were significant correlations between the JTCS and the reduction rates in V20, V2.5, and Dmean of the lung, D1% of the spinal cord, and D90% of the body. CONCLUSIONS: A significant decrease of dosimetric parameters of OARs was found with JT-VMAT in FFF beams. This reduction is very small and probably not clinically relevant. JTCS, a novel metric to quantify the jaw movements during JT, was proposed, and the complexity of jaw movements did not correlate with that of the movements of MLC leaves. There were significant correlations between the JTCS and some dosimetric parameters of OARs.

Development of Normal Tissue Complication Probability Model for Trismus in Head and Neck Cancer Patients Treated With Radiotherapy: The Role of Dosimetric and Clinical Factors.

BACKGROUND/AIM: The aim of this study was to develop a normal tissue complication probability (NTCP) model for trismus in head and neck cancer (HNC) patients treated with radiotherapy (RT). PATIENTS AND METHODS: Prospective measurements of maximum inter-incisal opening (MIO) were performed at baseline and 6 months after definitive RT in 132 HNC patients. The primary endpoint of this study was defined when a patient fulfilled both of the following criteria: 1) MIO at 6 months after RT ≤35 mm and 2) MIO at 6 months after RT ≤80% of baseline MIO. Eleven clinical factors and a wide range of dosimetric factors (mean dose, maximum dose, V5, V10, V20, and V40) in twelve organs at risk (OARs) were chosen as candidate prognostic variables. RESULTS: Thirty out of 132 patients (23%) developed the primary endpoint. Multivariate logistic regression analysis revealed that the mean dose to the contralateral mandible joint (p=0.001) and baseline MIO (p=0.027) were independent prognostic factors. CONCLUSION: A multivariable NTCP model for trismus in HNC patients treated with RT was established including the mean dose to contralateral mandible joint and baseline MIO.

Development of PEGylated adenovirus vector with targeting ligand.

For effective gene therapy, a vector system that transduces the therapeutic gene into target cells efficiently and safely is essential. Adenovirus (Ad) vectors frequently are used for gene therapy research, especially cancer gene therapy, because of their high transduction efficiency. However, broad clinical utility of Ad vectors have not yet been achieved owing to problems related to several properties inherent to Ads. Systemic administration of Ad vectors leads to acute virus accumulation and undesirable transgene expression in the liver, with subsequent inefficient systemic cancer-targeted therapy and pronounced hepatotoxicity. Furthermore, most people have Ad-neutralizing antibodies, which hamper gene expression efficiency. Chemical conjugation of Ad surface with polyethylene glycol (PEG) (PEGylation) is one of the promising strategies to overcome these problems. Furthermore, PEGylation of Ad vectors with targeting ligands on the tip of PEG, which alter the transfection range of Ad vectors will improve the safety and efficiency of Ad gene-delivery vectors. In this review, we describe the molecular biology of Ads and outline this PEGylation approach including our data.

[Development of pegylated adenovirus vector for cancer gene therapy].

Adenovirus vectors (Ad) have been frequently used for cancer gene therapy research because of their high gene transduction efficiency. However, systemic administration of conventional Ad can lead to the acute accumulation of virus particles and transgene expression in the liver, which may cause severe hepatotoxicity. For these reasons, clinical application of Ad for systemic administration has been limited, although intratumor administration of Ad has shown marked antitumor effects. Therefore, to promote the application of Ad in systemic cancer gene therapy, especially against the distant metastatic cancer, a novel Ad with marked accumulation in tumors and minimal hepatic distribution is needed. From this perspective, bioconjugation with polyethylene glycol (PEGylation) to Ad surface is a promising strategy, and we are trying to develop cancer targeted Ad by PEGylation approach. Through our study, we particularly clarified that PEGylated Ad (PEG-Ad) with optimized PEG modification ratio exhibited the enhanced distribution and gene expression in tumor tissue via systemic injection, which was based on the enhanced permeability and retention (EPR) effect. Moreover, PEG-Ad encoding therapeutic gene demonstrated not only stronger tumor-suppressive activity but also fewer hepatotoxic side effects compared with conventional Ad. In addition, we further attempted the active targeting using targeting ligand on the tip of PEG. We revealed that PEG-Ad with transferrin as a tumor targeting ligand could transduce more efficiently into tumor cells, which express transferrin receptor, compared with conventional PEG-Ad. In this symposium, I will present our approach for development of cancer targeted Ad by PEGylation.

A strategy for efficient cross-presentation of CTL-epitope peptides leading to enhanced induction of in vivo tumor immunity.

The activation of antitumor cytotoxic T-lymphocytes (CTLs) depends on how efficiently the relevant tumor antigen peptides are delivered into the major histocompatibility complex (MHC) class I presentation pathway in antigen presenting cells (APCs). An elegant approach to promote the peptide-MHC class I association has been described for enhanced peptide transportation into the endoplasmic reticulum (ER) by adding an ER insertion signal sequence (Eriss). Nevertheless, this approach does not appear potent enough to induce in vivo tumor protective immunity. Herein, we present a novel peptide-vaccine strategy based on the combined utilization of Eriss and fusogenic liposomes (FLs) capable of directly introducing encapsulated CTL-epitope peptides into the MHC class I pathway of APCs. APCs pulsed with free peptides, FL-encapsulated peptides, or FL-encapsulated Eriss-conjugated peptides exhibited comparable levels of antigen-presenting activity at early phases after pulsing. Interestingly, whereas in the first two methods the APC ability began to decline 40 to 60 h after pulsing, FL-encapsulated Eriss(+) peptides allowed APCs to retain peptide-presentation activity for at least 140 h. This advantage of FL-encapsulated Eriss(+) peptides correlated with the induction of more potent antitumor immunity compared with soluble Eriss(+) or Eriss(-) peptides or FL-encapsulated Eriss(-) peptides when they were administered in vivo. Thus, Eriss-conjugated CTL-epitope peptides encapsulated in FLs provide a highly efficient tumor-vaccine to enhance the induction of in vivo tumor immunity.

Synthesis of a poly(vinylpyrrolidone-co-dimethyl maleic anhydride) co-polymer and its application for renal drug targeting.

We have synthesized a polymeric drug carrier, polyvinylpyrrolidone-co-dimethyl maleic anhydride [poly(VP-co-DMMAn)], for use in renal drug delivery. About 80% of the 10-kDa poly(VP-co-DMMAn) selectively accumulated in the kidneys 24 h after intravenous administration to mice. Although this accumulated poly(VP-co-DMMAn) was gradually excreted in the urine, about 40% remained in the kidneys 96 h after treatment. Poly(VP-co-DMMAn) was taken up by the renal proximal tubular epithelial cells and no cytotoxicity was noted. Higher doses did not produce toxicity in the kidneys or other tissues. In contrast, polyvinylpyrrolidone of the same molecular weight did not show any tissue-specific distribution. Poly(VP-co-DMMAn)-modified superoxide dismutase accumulated in the kidneys after intravenous administration and accelerated recovery from acute renal failure in a mouse model. In contrast, polyvinylpyrrolidone-modified superoxide dismutase and native superoxide dismutase were not as effective. Thus, poly(VP-co-DMMAn) is a useful candidate as a targeting carrier for renal drug delivery systems.

Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity.

Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostly nonspecific and may occur at all lysine residues, some of which may be within or near an active site. Resultant PEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop a strategy for site-specific mono-PEGylation using tumor necrosis factor-alpha (TNF-alpha). We prepared phage libraries expressing TNF-alpha mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-alpha (mTNF-alpha-Lys(-)) was isolated by panning against TNF-alpha-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-alpha-Lys(-) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-alpha-Lys(-), with superior molecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-alpha. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach.

Edema worsens target coverage in high-dose-rate interstitial brachytherapy of mobile tongue cancer: a report of two cases.

PURPOSE: We report our study on two patients to highlight the risk of underdosage of the clinical target volume (CTV) due to edema during high-dose-rate interstitial brachytherapy (HDR-ISBT) of mobile tongue cancer. MATERIAL AND METHODS: To treat the lateral side of the CTV, flexible applicator tubes were implanted on the mouth floor. Two-dimensional planning was performed using X-ray images for Case 1, and three-dimensional (3D) planning was performed using computed tomography (CT) for Case 2. Prescribed doses for both cases were 54 Gy in nine fractions. CASE REPORTS: Case 1 was treated for cancer of the right lateral border of the tongue in 2005. Tongue edema occurred after implantation, and part of the lateral border of the tongue protruded between the applicator tubes. Acute mucosal reaction abated in the protruded area earlier than in the other parts of the CTV. In this case, the tumor recurred in this area 5 months after the treatment. Case 2 was treated for cancer of the left lateral border of the tongue. Because tongue edema occurred in this case also, plastic splints were inserted between the applicator tubes to push the edematous region into the irradiated area. The mucosal surface of the CTV was covered by the 70% isodose, and 100% isodose line for before and after splint insertion. Local control of the tumor was achieved 4 years after treatment. DISCUSSION AND CONCLUSIONS: To ensure sufficient target coverage, 3D image-based planning using CT should be performed, followed by re-planning using repeated CT as needed. Also, the development of devices to prevent protrusion of the edematous tissue outside the target area will help to ensure the full dosing of CTV.