Chemical modification of bradykinin-polymer conjugates for optimum delivery of nanomedicines to tumors.

We recently prepared pH-responsive HPMA copolymer conjugates of bradykinin (P-BK), which release BK in response to the acidic tumor microenvironment, and found that administration of P-BK increased the tumor accumulation and therapeutic efficacy of nanomedicine. Because the release of BK from P-BK determines its onset of action, P-BKs with different release rates were prepared, and their properties were evaluated. The release kinetics were significantly altered by substitution proximal to hydrazone bond, release constant of methyl-substituted P-BK (P-MeBK) was approximately 4- and 80-fold higher than that of cyclopropyl-substituted P-BK (P-CPBK) and phenyl-substituted P-BK (P-PhBK). None of the P-BKs were active, but the release of BK restored their BK-like activity. Pre-administration of the P-BKs increased the tumor accumulation of nanomedicine in C26 tumor-bearing mice by 2- and 1.4-fold for P-MeBK and P-PhBK at 3 and 6 h. Altogether, this study provides insights into the design of pH-responsive nanodrugs with the desired release properties to target acidic lesions such as cancer and inflammation.

Peptidyl-prolyl cis-trans isomerase A participates in the selenium transport into the rat brain.

Selenium, an essential micronutrient, plays vital roles in the brain. Selenoprotein P (SELENOP), a major plasma selenoprotein, is thought to transport selenium to the brain. However, Selenop-knockout mice fed a diet containing an adequate amount of selenium shows no objective neurological dysfunction which is observed in the selenium-deficient diet-fed Selenop-knockout mice. This fact indicated that selenium from low-mass selenium-source compounds can be transported by SELENOP-independent alternative pathways to the brain. In this study, to obtain the basic information about the SELENOP-independent transport pathways, we performed ex vivo experiments in which the rat brain cell membrane fraction was analyzed to find selenium-binding and/or -interactive proteins using its reactive metabolic intermediate, selenotrisulfide (STS), and MALDI TOF-mass spectrometry. Several membrane proteins with the cysteine (C) thiol were found to be reactive with STS through the thiol-exchange reaction. One of the C-containing proteins in the brain cell membrane fraction was identified as peptidyl-prolyl cis-trans isomerase (PPIase) A from tryptic fragmentation experiments and database search. Among the 4 C residues in rat PPIase A, 21st C was proved to react with STS by assessment using C mutated recombinant proteins. PPIase A is ubiquitously expressed and also associates with a variety of biologically important events such as immunomodulation, intracellular signaling, transcriptional regulation and protein trafficking. Consequently, PPIase A was thought to participate in the selenium transport into the rat brain.

Discovery of inner centromere protein-derived small peptides for cancer imaging and treatment targeting survivin.

Survivin belongs to the inhibitor of apoptosis protein family, which is consistently overexpressed in most cancer cells but rarely expressed in normal adult tissues. Therefore, the detection and inhibition of survivin are regarded as attractive strategies for cancer-specific treatment. In this study, we designed and synthesized 7-19 residues of inner centromere protein (INCENP)-derived small peptides (INC peptides) as novel survivin-targeting agents. The INC peptides showed binding affinity for the human survivin protein (Kd  = 91.4-255 nmol L-1 ); INC16-22 , which contains residues 16-22 of INCENP, showed the highest affinity (91.4 nmol L-1 ). Confocal fluorescence imaging showed consistent colocalization of FITC-INC16-22 and survivin in cell lines. Nona-arginine-linked INC16-22 (r9-INC16-22 ) rendered INC16-22 cells penetrable and strongly inhibited cell growth of MIA PaCa-2 cells (52% inhibition at 1.0 µmol L-1 ) and MDA-MB-231 cells (60% inhibition at 10 µmol L-1 ) as determined by MTT assays. The exposure of MIA PaCa-2 cells to 40 µmol L-1 r9-INC16-22 apparently reduced the intracellular protein expression levels of survivin. However, cleaved caspase-3 was significantly increased in cells treated with r9-INC16-22 , even at 10 µmol L-1 , compared to untreated cells. Flow cytometry revealed that r9-INC16-22 strongly induced apoptosis in MIA PaCa-2 cells. These results indicate that the cytotoxic effects of r9-INC16-22 could be mediated mainly through the disruption of survivin-dependent antiapoptotic functions and partly because of the direct degradation of the survivin protein. Our findings suggest that INC peptides can act as useful scaffolds for novel cancer imaging and anticancer agents.

Superior Penetration and Cytotoxicity of HPMA Copolymer Conjugates of Pirarubicin in Tumor Cell Spheroid.

N-(2-Hydroxypropyl)methacrylamide copolymer conjugates of pirarubicin (THP), P-THP, accumulates selectively in solid tumor tissue by the enhanced permeability and retention (EPR) effect. Despite of high accumulation in solid tumors, some macromolecular antitumor agents show poor therapeutic outcome because of poor tissue diffusion into the tumor as well as obstructed tumor blood flow. Here, we confirmed that cellular uptake of P-THP was 25 times less than that of free THP at 1-4 h incubation time in vitro. The passage of P-THP through the confluent tight-monolayer cells junction was 12 times higher than free THP, and P-THP penetrated deeper into the tumor cell spheroid (1.3-1.7-fold) than free THP in 4 h. In addition, P-THP showed cytotoxicity comparable to that of free THP to tumor-cells in spheroid form, despite of 7 times lower cytotoxicity of P-THP to the monolayer cells to that of free THP in vitro. These results indicate that P-THP administration can exhibit deeper diffusion into the tumor cell spheroid than free THP. As a consequence, P-THP exhibits more efficient antitumor activity than free THP in vivo, which is also supported by better pharmacokinetics and tumor accumulation of P-THP than free THP.

Synthesis and characterization of radioiodinated 3-phenethyl-2-indolinone derivatives for SPECT imaging of survivin in tumors.

Survivin, overexpressed in most cancers, is associated with poor prognosis and resistance to radiation therapy and chemotherapy. Herein, we report the synthesis of three 3-phenethyl-2-indolinone derivatives and their application as in vivo imaging agents for survivin. Of these, 3-(2-(benzo[d][1,3]dioxol-5-yl)-2-oxoethyl)-3-hydroxy-5- iodoindolin-2-one (IPI-1) showed the highest binding affinity (Kd = 68.3 nM) to recombinant human survivin, as determined by quartz crystal microbalance (QCM). In vitro studies demonstrated that the [125I]IPI-1 binding in survivin-positive MDA-MB-231 cells was significantly higher than that in survivin-negative MCF-10A cells. In addition, uptake of [125I]IPI-1 by MDA-MB-231 cells decreased in a dose-dependent manner in the presence of the high-affinity survivin ligand S12; this is indicative of specific binding of [125I]IPI-1 to cellular survivin protein in vitro. Biodistribution studies in MDA-MB-231 tumor-bearing mice demonstrated the moderate uptake of [125I]IPI-1 in the tumor tissue (1.37% ID/g) at 30 min that decreased to 0.32% ID/g at 180 min. Co-injection of S12 (2.5 mg/kg) slightly reduced tumor uptake and the tumor/muscle ratio of [125I]IPI-1. Although further structural modifications are necessary to improve pharmacokinetic properties, our results indicate that PI derivatives may be useful as tumor-imaging probes targeting survivin.

Preparation and in Vitro Analysis of Human Serum Albumin Nanoparticles Loaded with Anthracycline Derivatives.

Nanoparticles prepared using human serum albumin (HSA) have emerged as versatile carriers for improving the pharmacokinetic profile of drugs. The desolvation of HSA using ethanol followed by stabilization through crosslinking with glutaraldehyde is a common technique for preparing HSA nanoparticles, but our knowledge concerning the characteristics (or functions) of HSA nanoparticles and their efficiency when loaded with drugs is limited. To address this issue in more detail, we prepared anthracycline-loaded HSA nanoparticles. Doxorubicin-loaded HSA nanoparticles with a size similar to doxorubicin-unloaded particles could be prepared by desolvating at a higher pH (8-9), and the size (100-150 nm) was optimum for delivery to tumor tissues. Using this procedure, HSA nanoparticles were loaded with other anthracycline derivatives, and all showed cytotoxicity in cancer cells. However, the efficiency of drug loading and dissolution rate were different among them possibly due to the differences in the type of association of the drugs on nanoparticles (doxorubicin and daunorubicin; covalently bound to nanoparticles, pirarubicin; both covalently bound to and adsorbed on nanoparticles, aclarubicin; adsorbed on nanoparticles). Since the formulation of such drug-loaded HSA nanoparticles should be modified for efficient delivery to tumors, the findings reported herein provide the useful information for optimizing the formulation and the production process for the HSA nanoparticles using a desolvation technique.

Development of radioiodinated acridine derivatives for in vivo imaging of prion deposits in the brain.

Prion diseases are caused by deposition of abnormal prion protein aggregates (PrPSc) in the central nervous system. This study aimed to develop in vivo imaging probes that can detect cerebral PrPSc deposits. We synthesized several quinacrine-based acridine (AC) derivatives with 2,9-substitution and radioiodinated them. The AC derivatives were evaluated as prion-imaging probes using recombinant mouse prion protein (rMoPrP) aggregates and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. The distribution of these compounds in mice was also evaluated. The 2-methoxy derivative [125I]2 exhibited the highest binding affinity for rMoPrP aggregates with an equilibrium dissociation constant (Kd) value of 43.4nM. Fluorescence imaging with 2 showed clear signals at the thioflavin T (ThT)-positive amyloid deposits in the mBSE-infected mouse brain. Although a discrepancy was observed between the in vitro binding of AC derivatives to the aggregates and in vivo distribution of these compounds in the brain and we failed to identify prospective prion-imaging probes in this study, the AC derivatives may be considered a useful scaffold for the development of in vivo imaging probes. Further chemical modification of these AC derivatives may discover clinically applicable prion imaging probes.

Characterization of Selenium Species in the Shijimi Clam.

Selenium is an essential trace element for humans and animals. Fish and shellfish are known to be rich in selenium and suppose to be an effective selenium source. In this study, we characterized the selenium species in the Shijimi clam (Corbicula japonica), which is a typical clam eaten in Japan. The Shijimi clam contains a relatively high concentration of selenium (3.5 µg-selenium/g-dry Shijimi). Approximately 30% of the total selenium in the Shijimi clam meat was extractable with water, while selenium in the Shijimi clam was hardly extracted with ethanol, chloroform and hexane. Based on an ultrafiltration study, the molecular mass of the major selenium species in the Shijimi water-extract was estimated to be less than 5000. Because amphoteric selenium species were contained in the Shijimi water-extract, which was indicated by ion-exchange chromatographic separation, an ion-pair reagent was utilized to extract the ionic selenium species into an organic solvent. A matrix assisted laser desorption ionization (MALDI) time of flight (TOF)-mass spectrometric analysis revealed the selenium isotopic pattern involving one selenium atom in a molecule with the 80Se molecular ion peak at m/z 534. This selenium species was mainly found in the visceral part of the Shijimi clam by imaging mass spectrometry.

Pronounced Cellular Uptake of Pirarubicin versus That of Other Anthracyclines: Comparison of HPMA Copolymer Conjugates of Pirarubicin and Doxorubicin.

Many conjugates of water-soluble polymers with biologically active molecules were developed during the last two decades. Although, therapeutic effects of these conjugates are affected by the properties of carriers, the properties of the attached drugs appear more important than the same carrier polymer in this case. Pirarubicin (THP), a tetrahydropyranyl derivative of doxorubicin (DOX), demonstrated more rapid cellular internalization and potent cytotoxicity than DOX. Here, we conjugated the THP or DOX to N-(2-hydroxypropyl)methacrylamide copolymer via a hydrazone bond. The polymeric prodrug conjugates, P-THP and P-DOX, respectively, had comparable hydrodynamic sizes and drug loading. Compared with P-DOX, P-THP showed approximately 10 times greater cellular uptake during a 240 min incubation and a cytotoxicity that was more than 10 times higher during a 72-h incubation. A marginal difference was seen in P-THP and P-DOX accumulation in the liver and kidney at 6 h after drug administration, but no significant difference occurred in the tumor drug concentration during 6-24 h after drug administration. Antitumor activity against xenograft human pancreatic tumor (SUIT2) in mice was greater for P-THP than for P-DOX. To sum up, the present study compared the biological behavior of two different drugs, each attached to an N-(2-hydroxypropyl)methacrylamide copolymer carrier, with regard to their uptake by tumor cells, body distribution, accumulation in tumors, cytotoxicity, and antitumor activity in vitro and in vivo. No differences in the tumor cell uptake of the polymer-drug conjugates, P-THP and P-DOX, were observed. In contrast, the intracellular uptake of free THP liberated from the P-THP was 25-30 times higher than that of DOX liberated from P-DOX. This finding indicates that proper selection of the carrier, and especially conjugated active pharmaceutical ingredient (API) are most critical for anticancer activity of the polymer-drug conjugates. THP, in this respect, was found to be a more preferable API for polymer conjugation than DOX. Hence the treatment based on enhanced permeability and retention (EPR) effect that targets more selectively to solid tumors can be best achieved with THP, although both polymer conjugates of DOX and THP exhibited the EPR effects and drug release profiles in acidic pH similarly.

Synthesis and evaluation of a radioiodinated 4,6-diaryl-3-cyano-2-pyridinone derivative as a survivin targeting SPECT probe for tumor imaging.

Survivin is overexpressed in most of the cancerous tissues but not in terminally differentiated normal tissues, making it an attractive target for diagnosis and therapy of various types of cancers. In this study, we aimed to develop 4,6-diaryl-3-cyano-2-pyridinone (DCP) derivatives, as novel cancer imaging probes that target survivin. Chloro and iodo analogs of DCP (CDCP and IDCP, respectively) were successfully synthesized by using a previously unreported carbon monoxide-free procedure. IDCP exhibited a slightly higher binding affinity for recombinant human survivin (Kd=34 nM) than that of CDCP (Kd=44 nM). Fluorescence staining indicated that both CDCP and IDCP showed high signals in MDA-MB-231 cells with high levels of survivin expression. Significantly low fluorescent signals were observed in MCF-10A cells, which showed low levels of survivin expression. [(125)I]IDCP was synthesized for the application of IDCP to single photon emission computed tomography (SPECT) imaging. Quantitative in vitro binding of [(125)I]IDCP in cell cultures showed results consistent to those observed after fluorescent staining. In vivo biodistribution studies in tumor-bearing mice demonstrated that the tumor uptake of [(125)I]IDCP increased gradually with time and was 0.65% injected dose per gram (% ID/g) at 180 min. The maximum tumor/blood and tumor/muscle ratio at 60 min were 0.87 and 2.27, respectively, indicating inadequate [(125)I]IDCP accumulation in tumors necessary for in vivo imaging. Although further structural modifications are necessary to improve pharmacokinetic properties of IDCP, this study demonstrates the feasibility of using the DCP backbone as a scaffold for the development of survivin-targeting tumor imaging probes.

A Comprehensive Analysis of Selenium-Binding Proteins in the Brain Using Its Reactive Metabolite.

The intracellular metabolism of selenium in the brain currently remains unknown, although the antioxidant activity of this element is widely acknowledged to be important in maintaining brain functions. In this study, a comprehensive method for identifying the selenium-binding proteins using PenSSeSPen as a model of the selenium metabolite, selenotrisulfide (RSSeSR, STS), was applied to a complex cell lysate generated from the rat brain. Most of the selenium from L-penicillamine selenotrisulfide (PenSSeSPen) was captured by the cytosolic protein thiols in the form of STS through the thiol-exchange reaction (R-SH+PenSSeSPen→R-SSeSPen+PenSH). The cytosolic protein species, which reacted with the PenSSeSPen mainly had a molecular mass of less than 20 kDa. A thiol-containing protein at m/z 15155 in the brain cell lysate was identified as the cystatin-12 precursor (CST12) from a rat protein database search and a tryptic fragmentation experiment. CST12 belongs to the cysteine proteinase inhibitors of the cystatin superfamily that are of interest in mechanisms regulating the protein turnover and polypeptide production in the central nervous system and other tissues. Consequently, CST12 is suggested to be one of the cytosolic proteins responsible for the selenium metabolism in the brain.

An effective method for profiling the selenium-binding proteins using its reactive metabolic intermediate.

Currently, the intracellular reduction and/or transport of selenium still remain unknown. Certain reduced forms of selenium species are thought to be reactive with various endogenous molecules, particularly thiol-containing proteins. In this study, a profiling method for identifying the selenium-binding proteins using L-penicillamine selenotrisulfide (PenSSeSPen) as a model of the selenium metabolic intermediate was applied to the cell lysate generated from the rat liver. Several proteins with cysteine thiol were found to be reactive with PenSSeSPen through the thiol-exchange reaction by MALDI TOF-MS analysis. The most distinctive cysteine-containing protein at m/z 14,313 in the liver cell lysate was identified as the liver fatty acid-binding protein based on a rat protein database search and a tryptic fragmentation experiment. This methodology could be used for determining the selenium-binding proteins and/or selenium-interactive species and provide a better understanding of the selenium metabolism and utilization in biological systems.

Development of alkoxy styrylchromone derivatives for imaging of cerebral amyloid-ß plaques with SPECT.

We report here the development of radioiodinated styrylchromone derivatives with alkoxy groups as single photon emission computed tomography (SPECT) imaging probes for cerebral amyloid-ß (Aß) plaques. Among the derivatives, the methoxy derivative 14 and the dimethoxy derivative 15 displayed relatively high affinity for the Aß(1-42) aggregates with K(i) values of 22 and 46 nM, respectively. Fluorescent imaging demonstrated that 14 and 15 clearly labeled thioflavin-S positive Aß plaques in the brain sections of Tg2576 transgenic mice. In the in vivo studies, [(125)I]14 and [(125)I]15 showed high initial brain uptake expressed as the percentage of the injected dose per gram (2.25% and 2.49% ID/g at 2 min, respectively) with favorable clearance (0.12% and 0.20% ID/g at 180 min, respectively) from the brain tissue of normal mice. Furthermore, in vitro autoradiography confirmed that [(125)I]15 binds thioflavin-S positive regions in Tg2576 mouse brain sections. The derivative 15 may be a potential scaffold for the development of in vivo imaging probes targeting Aß plaques in the brain. In particular, further structural modifications are required to improve the compounds binding affinity for Aß.

Synthesis and evaluation of ethyleneoxylated and allyloxylated chalcone derivatives for imaging of amyloid ß plaques by SPECT.

We report radioiodinated chalcone derivatives as new SPECT imaging probes for amyloid ß (Aß) plaques. The monoethyleneoxy derivative 2 and allyloxy derivative 8 showed a high affinity for Aß(1-42) aggregates with Ki values of 24 and 4.5 nM, respectively. Fluorescent imaging demonstrated that 2 and 8 clearly stained thioflavin-S positive Aß plaques in the brain sections of Tg2576 transgenic mice. In vitro autoradiography revealed that [(125)I]2 displayed no clear accumulation toward Aß plaques in the brain sections of Tg2576 mice, whereas the accumulation pattern of [(125)I]8 matched with the presence of Aß plaques both in the brain sections of Tg2576 mice and an AD patient. In biodistribution studies using normal mice, [(125)I]2 showed preferable in vivo pharmacokinetics (4.82%ID/g at 2 min and 0.45%ID/g at 60 min), while [(125)I]8 showed only a modest brain uptake (1.62%ID/g at 2 min) with slow clearance (0.56%ID/g at 60 min). [(125)I]8 showed prospective binding properties for Aß plaques, although further structural modifications are needed to improve the blood brain barrier permeability and washout from brain.

Characterization of selenium species in extract from Niboshi (a processed Japanese anchovy).

Fish are selenium rich foodstuffs and a major selenium source for the Japanese population. Niboshi is processed from Japanese anchovy (Engraulis japonicus) and commonly used to prepare soup stock for Japanese dishes. In this study, we characterized selenium species in the Niboshi extract by ultrafiltration, ion-exchange chromatography and mass spectrometry. Selenium species in the Niboshi were more extractable by polar solvents (water and ethanol) than an apolar one (hexane) along with amino acids and proteinous species. Selenium in the water-extract from the Niboshi was mostly ascribed to organoselenium compounds with a molecular mass less than 5 kDa. Although selenoamino acids and selenoproteins and their fragments were involved in the extract, a large portion of the selenium species appeared to be low-molecular-mass organoselenium compounds other than selenoamino acids and their derivatives. Ion-exchange chromatographic separations revealed that most of the selenium species in the extract possess anionic and/or amphoteric characteristics. One of these selenium species from the Niboshi extract was detected at m/z 577 for 80Se by mass spectrometry subsequent to ion-pair extraction.

A strontium-90 sequestrant for first-aid treatment of radiation emergency.

In this study, hydrophilic porous polymer beads with phosphonic acid groups (PGMA-EGDMA-TTA-MP) were synthesized, and assessed as a radioactive strontium-90 sequestrant for the treatment of the radiation emergency. Strontium ions were rapidly absorbed into the blood from the gastrointestinal (GI) tract after oral administration to rats, and distributed to the target organ, i.e., bones. Over 40% of the administered strontium was absorbed into the blood, while the remainder was discharged in the feces within 48 h after the administration. When the PGMA-EGDMA-TTA-MP beads were administered to rats subsequent to the strontium solution, the strontium had accumulated less in the femur. Consequently, the oral administration of the PGMA-EGDMA-TTA-MP beads was effective in suppressing the absorption of strontium from the GI tract.

Diphenylpropynone derivatives as probes for imaging ß-amyloid plaques in Alzheimer's brains.

A new series of diphenylpropynone (DPP) derivatives for use in vivo to image ß-amyloid (Aß) plaques in the brain of patients with Alzheimer's disease (AD) were synthesized and characterized. Binding experiments in vitro revealed high affinity for Aß (1-42) aggregates at a K(i) value ranging from 6 to 326 nM. Furthermore, specific labeling of plaques was observed in sections of brain tissue from Tg2576 transgenic mice stained using one of the compounds, 1. In biodistribution experiments with normal mice, [(125)I]1 displayed moderate uptake (1.55%ID/g at 2 min) and clearance from the brain with time (0.76 ID/g at 60 min). Taken together, DPP can serve as a new molecular scaffold for developing novel Aß imaging agents by introducing appropriate substituted groups.

A dual fluorinated and iodinated radiotracer for PET and SPECT imaging of ß-amyloid plaques in the brain.

We report a fluorinated and iodinated radiotracer as a probe for PET/SPECT to detect of ß-amyloid (Aß) plaques in the brain of patients with Alzheimer's disease (AD). We successfully designed and synthesized the fluorinated and iodinated aurone derivative (3) and its radiolabels ([(125)I]3 and [(18)F]3). In binding experiments in vitro, 3 showed high affinity for Aß aggregates (K(i)=6.81nM). In brain sections of AD model mice, 3 intensely stained Aß plaques. Furthermore, a specific plaque labeling signal was observed on the autoradiography of postmortem AD brain sections using [(125)I]3. In biodistribution experiments using normal mice, [(125)I]3 and [(18)F]3 displayed good uptake into and a rapid washout from the brain, properties highly desirable for Aß imaging agents. These results suggest that 3 may function as a PET/SPECT dual imaging agent for detecting Aß plaques in AD brains.

Synthesis and characterization of [¹²5I]2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide as novel imaging probe for glycine transporter 1.

In this study, 2-iodo substituted 1-methylpiperidin-2-yl benzamide derivatives were synthesized and evaluated as candidate SPECT imaging agents for glycine transporter 1 (GlyT1). In JAR cells, which predominantly express GlyT1, 2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide (5) showed excellent inhibitory activity of [(3)H]glycine uptake (IC(50)=2.4 nM). Saturation assay in rat cortical membranes revealed that [(125)I]5 had a single high affinity binding site with a K(d) of 1.54 nM and a B(max) of 3.40 pmol/mg protein. In vitro autoradiography demonstrated that [(125)I]5 showed consistent accumulation with GlyT1 expression. The in vitro binding was greatly inhibited by GlyT1 inhibitors but not by other site ligands, which suggested the high specific binding of [(125)I]5 with GlyT1. In the biodistribution and ex vivo autoradiography studies using mice, [(125)I]5 showed high blood-brain barrier permeability (1.68-2.17% dose/g at 15-60 min) and similar regional brain distribution pattern with in vitro results. In addition, pre-treatment of GlyT1 ligands resulted in significant decrease of [(125)I]5 binding in the GlyT1-rich regions. This preliminary study demonstrated that radio-iodinated 5 is a promising SPECT imaging probe for GlyT1.

Preparation of Enzymatically Highly Active Pegylated-D-Amino Acid Oxidase and Its Application to Antitumor Therapy.

BACKGROUND: D-Amino acid oxidase (DAO) is an H2O2-generating enzyme, and tumor growth suppression by selective delivery of porcine DAO in tumors via the cytotoxic action of H2O2 has been reported. DAO isolated from Fusariumspp. (fDAO) shows much higher enzyme activity than porcine DAO, although the application of fDAO for antitumor treatment has not yet been determined. OBJECTIVE: The purpose of this study was to prepare enzymatically highly active pegylated-fDAO, and to determine whether it accumulates in tumors and exerts a potent antitumor effect in tumor- bearing mice. METHODS: Polyethylene glycol (PEG; Mw. 2000) was conjugated to fDAO to form PEGylated fDAO (PEG-fDAO). PEG-fDAO was intravenously administered into S180 tumor-bearing mice, and the body distribution and antitumor activity of PEG-fDAO was determined. RESULTS: The enzyme activity of PEG-fDAO was 26.1 U/mg, which was comparable to that of fDAO. Intravenously administered PEG-fDAO accumulated in tumors with less distribution in normal tissue except in the plasma. Enzyme activity in the tumor was 60-120 mU/g-tissue over 7-20 h after i.v. injection of 0.1 mg of PEG-fDAO. To generate the H2O2 in the tumor tissue, PEG-fDAO was intravenously administered, and then, D-phenylalanine was intraperitoneally administered after a lag time. No remarkable tumor suppression effect was observed under conditions used in this study, compared to the non-treated group. CONCLUSION: The results suggest that PEG-fDAO maintained high enzymatic activity after pegylation. Treatment with PEG-fDAO conferred high enzyme activity on tumor tissue; 3-6 fold higher than that of previously reported pDAO; however, high enzyme activity in the plasma limited repeated treatment owing to lethal toxicity, which seemingly led to poor therapeutic outcome. Overall, the use of PEG-fDAO is promising for antitumor therapy, although the suppression of DAO activity in the plasma would also be required rather than only the increase in DAO activity in the tumor for an antitumor effect.