Comparative evaluation of radionuclide therapy using 90Y and 177Lu.

OBJECTIVE: Both 90Y and 177Lu are attractive ß-emitters for radionuclide therapy and have been used in clinical practice. Nevertheless, comparative evaluation between 90Y- and 177Lu-labeled molecules has not been fully conducted. Thus, in this study, the features of 90Y and 177Lu for radionuclide therapy were assessed in tumor-bearing mice. METHODS: Two tumor cell lines with different growth rates were used. Biodistribution studies of 177Lu-labeled antibodies (177Lu-Abs) were conducted in each tumor-bearing mouse model. Subsequently, the therapeutic effect of 90Y- and 177Lu-Ab were assessed in tumor-bearing mice. The absorbed radiation dose for the tumor was estimated using the Monte Carlo simulation. RESULTS: 177Lu-Abs demonstrated high tumor accumulation in both tumor-xerograph. In the fast-growing tumor model, 90Y-Ab showed a better therapeutic effect than 177Lu-Ab, reflecting a higher absorbed radiation dose of 90Y-Ab than that of 177Lu-Ab. In the slow-growing tumor model, both 90Y- and 177Lu-Ab showed an excellent therapeutic effect; however, 177Lu-Ab had a longer efficacy period than 90Y-Ab, which could be attributed to the longer half-life and better dose uniformity of 177Lu than those of 90Y. CONCLUSIONS: To accomplish a maximum therapeutic effect, selecting 90Y or 177Lu, to depend on the growth rate of individual cancer, would be helpful.

Discovery of EP300/CBP histone acetyltransferase inhibitors through scaffold hopping of 1,4-oxazepane ring.

EP300 and its paralog CBP play an important role in post-translational modification as histone acetyltransferases (HATs). EP300/CBP inhibition has been gaining attention as an anticancer treatment target in recent years. Herein, we describe the identification of a novel, highly selective EP300/CBP inhibitor, compound 11 (DS17701585), by scaffold hopping and structure-based optimization of a high-throughput screening hit 1. Compound 11 (DS17701585) shows dose-dependent inhibition of SRY-box transcription factor 2 (SOX2) mRNA expression in a human lung squamous cell carcinoma cell line LK2-xenografted mouse model.

Biochemical profiles of rat primary cultured hepatocytes following treatment with rotenone, FCCP, or (+)-usnic acid.

The metabolomic profiles of rat primary hepatocytes following treatment with rotenone, FCCP, or (+)-usnic acid were determined using liquid chromatography-mass spectrometry/mass spectrometry and gas chromatography-mass spectrometry. Significant and similar changes in the levels of 283 biochemical metabolites were associated with the three treatments compared with solvent control samples. Overall, the three treatments generated similar global biochemical profiles, with some minor differences associated with rotenone treatment. All three treatments resulted in a shift in energy metabolism as demonstrated by decreased glycogen stores and glycolysis. A reduced antioxidant response was detected in cells following all treatments. In addition, bile acid biosynthesis decreased as a potential consequence of increased oxidative stress by all three treatments. Conversely, rotenone treatment induced a number of changes after 1 hr, which were not detected in FCCP- or (+)-usnic acid-treated samples; these changes were not sustained over time and included increased NAD+ salvage and lysine degradation. In conclusion, these biochemical profiles could provide new insights into the mechanism(s) of mitochondrial toxicity.

Fluoroquinolones suppress gluconeogenesis by inhibiting fructose 1,6-bisphosphatase in primary monkey hepatocytes.

Dysglycemia is one of the most serious adverse events associated with the clinical use of certain fluoroquinolones. The purpose of this study was to investigate the effects of the representative fluoroquinolones moxifloxacin and gatifloxacin on hepatic gluconeogenesis using primary monkey hepatocytes. Glucose production was induced after the cells were incubated for 4 h with 10 mM sodium lactate and 1 mM sodium pyruvate as gluconeogenic substrates. Under these conditions, moxifloxacin and gatifloxacin dose-dependently suppressed gluconeogenesis at concentrations of 100 µM or higher. Transcriptome analysis of rate-limiting enzymes involved in hepatic gluconeogenesis revealed that moxifloxacin and gatifloxacin at a concentration of 1000 µM did not affect the expression of key gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase, glucose 6-phosphatase, and fructose 1,6-bisphosphatase. Furthermore, metabolome analysis, in vitro glucose production assay using additional gluconeogenic substrates, and fructose 1,6-bisphosphatase assay using the cell extracts showed that fluoroquinolones enzymatically suppressed hepatic gluconeogenesis by inhibiting fructose 1,6-bisphosphatase. These inhibitory effects may involve in the clinically relevant dysglycemia associated with fluoroquinolones in human.

Electron-tracking Compton camera imaging of technetium-95m.

Imaging was conducted using an electron tracking-Compton camera (ETCC), which measures γ-rays with energies in the range of 200-900 keV from 95mTc. 95mTc was produced by the 95Mo(p, n)95mTc reaction on a 95Mo-enriched target. A method for recycling 95Mo-enriched molybdenum trioxide was employed, and the recycled yield of 95Mo was 70%-90%. Images were obtained with the gate of three energies. The results showed that the spatial resolution increases with increasing γ-ray energy, and suggested that the ETCC with high-energy γ-ray emitters such as 95mTc is useful for the medical imaging of deep tissue and organs in the human body.

Profile of patients submitted to gastroplasty in the university hospital of Maringá - Paraná State during 2008-2009 / Perfil dos pacientes obesos submetidos à gastroplastia no hospital universitário de Maringá ­ Estado do Paraná, 2008- 2009

This study evaluated the profile of patients submitted to gastroplasty in the University Hospital of Maringá during 2008-2009. Clinical charts were analyzed to obtain clinical and laboratory data of all patients with morbid obesity submitted to gastroplasty. During the study period, 28 surgeries were performed, 57% of the patients lived in Maringá, 82% female, 40 years average age, with mean body mass index (BMI) of 46.7kg m-2. It was verified that 39% of the patients maintained an usual non-hypocaloric diet, 25% had quoted dietary reeducation, 36% were sedentary, and 29% practiced some physical activity, and remaining 1/3 of patients presented no data about lifestyle. Regarding associated pathologies, 79% were hypertensive, and 71% of patients presented fasting glucose above 100 mg dL-1, but only 12 of them had diagnosis of Type 2 diabetes mellitus. The hospital stay length was 4 days for 89% of the patients, and 11% had surgery complications. In this group of subjects, there was a clear preponderance of females and high prevalence of other pathologies. Identifying the profile of obese patients contributes to more effective decision-making and emphasizes the crucial role of multidisciplinary approach to health promotion and prevention of early and late complications of morbid obesity and gastroplasty.

Conhecer o perfil dos pacientes submetidos à gastroplastia no Hospital Universitário de Maringá no período de 2008-2009. Foram analisados os prontuários verificando os dados clínicos e laboratoriais de todos os pacientes com obesidade mórbida submetidos à gastroplastia neste período. Foram realizadas 28 cirurgias, 57% dos pacientes eram residentes no município de Maringá, 82% eram do sexo feminino, a idade média foi de 40 anos e a média do índice de massa corporal foi de 46,7 kg m-2. Observou-se que 39% dos pacientes mantinham dieta habitual e não hipocalórica, 25% citaram re-educação alimentar, 29% realizavam atividade física e 36% eram sedentários, os demais pacientes não apresentavam estes dados. Em relação às doenças associadas, 79% eram hipertensos e 71% pacientes apresentavam glicemia de jejum maior que 100 mg dL-1, mas somente 12 deles referiam diagnóstico de diabetes mellitus do Tipo 2. Em 89% dos pacientes, o período de internação foi de quatro dias e 11% tiveram complicações após a cirurgia. Nos pacientes com obesidade mórbida submetidos à gastroplastia, houve predomínio do sexo feminino e alta prevalência de comorbidades. Identificar o perfil destes pacientes contribui para a tomada de decisões e reforça a importância da equipe multidisciplinar na promoção à saúde e prevenção de complicações imediatas e tardias da obesidade mórbida e gastroplastia.

Interlaboratory trial of the rat Pig-a mutation assay using an erythroid marker HIS49 antibody.

The peripheral blood Pig-a assay has shown promise as a tool for evaluating in vivo mutagenicity. In this study five laboratories participated in a collaborative trial that evaluated the transferability and reproducibility of a rat Pig-a assay that uses a HIS49 antibody reacts with an antigen found on erythrocytes and erythroid progenitors. In preliminary work, flow cytometry methods were established that enabled all laboratories to detect CD59-negative erythrocyte frequencies (Pig-a mutant frequencies) of <10×10(-6) in control rats. Four of the laboratories (the in-life labs) then treated male rats with a single oral dose of N-nitroso-N-ethylurea, 7,12-dimethylbenz[a]anthracene (DMBA), or 4-nitroquinoline-1-oxide (4NQO). Blood samples were collected up to 4 weeks after the treatments and analyzed by flow cytometry for the frequency of CD59-negative cells among total red blood cells (RBCs; RBC Pig-a assay). RBC Pig-a assays were conducted in the four in-life laboratories, plus a fifth laboratory that received blood samples from the other laboratories. In addition, three of the five laboratories performed a Pig-a assay on reticulocytes (RETs; PIGRET assay), using blood from the rats treated with DMBA and 4NQO. The four in-life laboratories detected consistent, time- and dose-related increases in RBC Pig-a mutant frequency (MF) for all three test articles. Furthermore, comparable results were obtained in the fifth laboratory that received blood samples from other laboratories. The three laboratories conducting the PIGRET assay also detected consistent, time- and dose-related increases in Pig-a MF, with the RET MFs increasing more rapidly with time than RBC MFs. These results indicate that rat Pig-a assays using a HIS49 antibody were transferable between laboratories and that data generated by the assays were reproducible. The findings also suggest that the PIGRET assay may detect the in vivo mutagenicity of test compounds earlier than the RBC Pig-a assay.

Enantioresolution of 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl oxide using inclusion complex with chiral 2,2'-dihydroxy-1,1'-binaphtyl.

An enantioresolution of 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl oxide (BINAPO) into its enantiomers was achieved using the inclusion complex with a commerciallyavailable chiral 2,2'-dihydroxy-1,1'-binaphthyl ((R)-BINOL), giving the two enantiomers with 99% ee and 72% ee, respectively.

Preparation and evaluation of 186/188Re-labeled antibody (A7) for radioimmunotherapy with rhenium(I) tricarbonyl core as a chelate site.

OBJECTIVE: Rhenium is one of the most valuable elements for internal radiotherapy because (186)Re and (188)Re have favorable physical characteristics. However, there are problems when proteins such as antibodies are used as carriers of (186/188)Re. Labeling methods that use bifunctional chelating agents such as MAG3 require the conjugation of the (186/188)Re complex to protein after radiolabeling with the bifunctional chelating agent. These processes are complicated. Therefore, we planned the preparation by a simple method and evaluation of a stable (186/188)Re-labeled antibody. For this purpose, we selected (186/188)Re(I) tricarbonyl complex as a chelating site. In this study, A7 (an IgG1 murine monoclonal antibody) was used as a model protein. (186/188)Re-labeled A7 was prepared by directly reacting a (186/188)Re(I) tricarbonyl precursor, [(186/188)Re(CO)(3)(H(2)O)(3)](+), with A7. We then compared the biodistribution of (186/188)Re-labeled A7 in tumor-bearing mice with (125)I-labeled A7. METHODS: For labeling A7, [(186/188)Re(CO)(3)(H(2)O)(3)](+) was prepared according to a published procedure. (186/188)Re-labeled A7 ((186/188)Re-(CO)(3)-A7) was prepared by reacting [(186/188)Re(CO)(3)(H(2)O)(3)](+) with A7 at 43 degrees C for 2 h. Biodistribution experiments were performed by the intravenous administration of (186/188)Re-(CO)(3)-A7 solution into tumor-bearing mice. RESULTS: (186)Re-(CO)(3)-A7 and (188)Re-(CO)(3)-A7 were prepared with radiochemical yields of 23 and 28%, respectively. After purification with a PD-10 column, (186/188)Re-(CO)(3)-A7 showed a radiochemical purity of over 95%. In biodistribution experiments, 13.1 and 13.2% of the injected dose/g of (186)Re-(CO)(3)-A7 and (188)Re-(CO)(3)-A7, respectively, accumulated in the tumor at 24-h postinjection, and the tumor-to-blood ratios were over 2.0 at the same time point. Meanwhile, uptake of (125)I-A7 in the tumor was almost the same as that of (186/188)Re-(CO)(3)-A7 at 24-h postinjection. Blood clearances of (186/188)Re-(CO)(3)-A7 were faster than those of (125)I-A7. CONCLUSION: (186/188)Re-labeled A7 showed high uptakes in the tumor. However, further modification of the labeling method would be necessary to improve radiochemical yields and their biodistribution.

Usefulness of competitive inhibitors of protein binding for improving the pharmacokinetics of 186Re-MAG3-conjugated bisphosphonate (186Re-MAG3-HBP), an agent for treatment of painful bone metastases.

PURPOSE: We have developed a (186)Re-mercaptoacetylglycylglycylglycine complex-conjugated bisphosphonate ((186)Re-MAG3-HBP) for the treatment of painful bone metastases. We assumed competitive inhibitors of protein binding to be useful for procuring a favorable biodistribution of (186)Re-MAG3-HBP for the palliation of bone pain because it has been reported that the concurrent administration of (99m)Tc-MAG3 and drugs with high affinity for serum protein produced competitive displacement at specific binding sites and enhanced total clearance and tissue distribution. METHODS: The displacement effects of several protein-binding inhibitors on the protein binding of (186)Re-MAG3-HBP were investigated. Biodistribution experiments were performed by intravenously administering (186)Re-MAG3-HBP into rats with ceftriaxone as a competitive protein-binding inhibitor or saline. RESULTS: The protein binding of (186)Re-MAG3-HBP in rat serum, human serum, and a human serum albumin solution was significantly decreased by the addition of ceftriaxone, which has high affinity for binding site I on serum albumin. In the biodistribution experiments, pretreatment with ceftriaxone enhanced the clearance of the radioactivity of (186)Re-MAG3-HBP in blood and nontarget tissues but had no effect on accumulation in bone. CONCLUSIONS: The findings suggested that the use of protein-binding competitive inhibitors would be effective in improving the pharmacokinetics of radiopharmaceuticals with high affinity for serum protein.

Effect of glutathione (GSH) depletion on DNA damage and blood chemistry in aged and young rats.

DNA is damaged by reactive oxygen species (ROS) and such damage is age-dependent. Blood chemical parameters also change age-dependently. Glutathione (GSH) plays an important role as an antioxidant. However, the effects of GSH on DNA damage and blood chemistry are unclear. Therefore, this study was aimed to evaluate GSH contribution to DNA damage and changes of blood chemical parameters in aged and young rats. The GSH content in the livers and kidneys of aged rats (20 months) were lower than that in young rats (9 weeks of age) with higher DNA damage detected by a comet assay. There was a negative correlation between the GSH content and the DNA damage in the liver and kidney. L-buthionine (S,R)-sulfoximine (BSO; 0, 5, 20 mM), which inhibits GSH synthesis, was administered in drinking water for 28 days to young and aged rats (8 weeks and 19 months of age at the start of the administration). The treatment significantly decreased GSH levels in the heart, liver, lung and kidney of either the young or aged rats without causing DNA damage in those organs. When compared with young rats, aged rats showed higher levels in aspartate aminotransferase, alanine aminotransferase, total bilirubin, total cholesterol, globulin, creatinine, sodium and chloride and lower levels in alkaline phosphatase, triglyceride, albumin/globulin and inorganic phosphorus. However, BSO did not change these parameters in young or aged rats. These results showed that there was a negative correlation between GSH and DNA damage during aging, but the BSO-induced GSH depletion did not affect DNA damage or blood chemistry levels in young and aged rats under these study conditions.

DNA damage measured by comet assay and 8-OH-dG formation related to blood chemical analyses in aged rats.

To evaluate the effects of aging on DNA damage, spontaneous and chemical-induced DNA damage and its repair were examined using comet assays at pH 9, 12.1 and 13, and an 8-OH-dG assay in the liver and kidney of young (9-week-old) and aged (20-month-old) rats. Additionally, blood chemistry was examined to investigate any correlation between vital functions and age-dependent DNA damage. DNA migration at pH 13 and 8-OH-dG levels increased in the liver and/or kidney of aged rats, but DNA migration did not increase at pH 9 or 12.1; that is, alkali-labile sites and 8-OH-dG were concomitantly accumulated in aged rats. These results suggest that 8-OH-dG production caused by reactive oxygen species exceeded glycosylation and that the glycosylation activity is far more than the AP endonucleation in aged rats. Methyl methanesulfonate (MMS, 80 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr after treatment in young and aged rats. The DNA damage in aged rats was less and decreased more slowly compared with young rats. The pictures of MMS-induced DNA migrations at pH 12.1 and 13 were very similar to each other. These results suggest that the adduct glycosylation and repair of the single-strand breaks (SSBs) of aged rats are less than those of young rats, although AP endonucleation is sufficient to remove the AP sites. N-nitrosodiethylamine (160 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr in young rats and at pH 12.1 and 13 in the kidney at 24 hr in aged rats. These results showed that SSBs were predominantly detected as chemical-induced DNA damage and DNA repairs such as N-glycosylase, DNA polymerase and DNA ligase, and that the metabolic activation declined in aged rats. Aspartate aminotransferase, alanine aminotransferase, total bilirubin, total cholesterol, total protein, globulin, creatinine and chloride age-dependently increased and alkaline phosphates, albumin/globulin ratio, inorganic phosphorus and potassium age-dependently decreased, and these changes were correlated with the DNA migration at pH 13 and/or 8-OH-dG. These results suggest that the activity of DNA repair and metabolic activation enzymes declines in aged rats and that the accumulation of spontaneous DNA damage may affect vital functions.

Assessment of 186Re chelate-conjugated bisphosphonate for the development of new radiopharmaceuticals for bones.

INTRODUCTION: The preferable pharmacokinetics of rhenium-186 (186Re)-monoaminemonoamidedithiol-conjugated or 186Re-mercaptoacetyltriglycine-conjugated bisphosphonates (BPs) suggested that the molecular design would be applicable to other radionuclides such as 68Ga, 99mTc, 153Sm and 177Lu. In this study, a key factor affecting the pharmacokinetics of a chelate-conjugated BP was investigated to estimate the validity and the applicability of molecular design. METHODS: Chemically inert and well-characterized tricarbonyl[186Re][(cyclopentadienylcarbonyl amino)-acetic acid]rhenium ([186Re]CpTR-Gly) was conjugated with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate and purified by high-performance liquid chromatography (HPLC) to prepare [186Re](1-{3-[tricarbonyl(cyclopentadienylcarbonyl amino)-acetylamido]-1-hydroxy-1-phosphono-propyl}-phosphonic acid)rhenium ([186Re]CpTR-Gly-APD). Plasma stability, plasma protein binding, hydroxyapatite (HA) binding and the pharmacokinetics of [186Re]CpTR-Gly-APD were compared with those of 186Re 1-hydroxyethylidene-1,1-diphosphonate (HEDP). The effect of HEDP coadministration and preadministration on the pharmacokinetics of [186Re]CpTR-Gly-APD was also determined. RESULTS: The HPLC-purified [186Re]CpTR-Gly-APD showed higher plasma stability, higher HA binding, higher bone accumulation and lower plasma protein binding than did 186Re-HEDP. However, HA binding of [186Re]CpTR-Gly-APD decreased to levels slightly higher than that of 186Re-HEDP at similar HEDP concentrations. Bone accumulation of [186Re]CpTR-Gly-APD also decreased to levels similar to that of 186Re-HEDP when [186Re]CpTR-Gly-APD was coinjected with HEDP equivalent to that in 186Re-HEDP. In contrast, HEDP pretreatment did not impair bone accumulation of the two 186Re-labeled compounds. However, a delay in blood clearance and an increase in renal radioactivity levels were observed particularly with 186Re-HEDP. CONCLUSIONS: Although 186Re-HEDP possessed HA binding and bone accumulation similar to those of [186Re]CpTR-Gly-APD, the specific activity of 186Re-labeled BPs was found to play a crucial role in bone accumulation and blood clearance. Thus, the molecular design of chelate-conjugated BP would be useful for the development of bone-seeking radiopharmaceuticals with a variety of radionuclides by selecting chelating molecules that provide high specific activities.

Therapeutic effects of a 186Re-complex-conjugated bisphosphonate for the palliation of metastatic bone pain in an animal model.

UNLABELLED: Previously, based on the concept of bifunctional radiopharmaceuticals, we developed a highly stable (186)Re-mercaptoacetylglycylglycylglycine (MAG3) complex-conjugated bisphosphonate, [[[[(4-hydroxy-4,4-diphosphonobutyl)carbamoylmethyl]carbamoylmethyl]carbamoylmethyl]carbamoylmethanethiolate] oxorhenium(V) ((186)Re-MAG3-HBP), for the treatment of painful bone metastases. This agent showed a superior biodistribution as a bone-seeking agent in normal mice when compared with (186)Re-1-hydroxyethylidene-1,1-diphosphonate ((186)Re-HEDP). In this study, we evaluated the therapeutic effects of (186)Re-MAG3-HBP using an animal model of bone metastasis. METHODS: The model was prepared by injecting syngeneic MRMT-1 mammary tumor cells into the left tibia of female Sprague-Dawley rats. (186)Re-MAG3-HBP (55.5, 111, or 222 MBq/kg) or (186)Re-HEDP (55.5 MBq/kg) was then administered intravenously 21 d later. To evaluate the therapeutic effects and side effects, tumor size and peripheral blood cell counts were determined. Palliation of bone pain was evaluated by a von Frey filament test. RESULTS: In the rats treated with (186)Re-HEDP, tumor growth was comparable with that in untreated rats. In contrast, when (186)Re-MAG3-HBP was administered, tumor growth was significantly inhibited. Allodynia induced by bone metastasis was attenuated by treatment with (186)Re-MAG3-HBP or (186)Re-HEDP, but (186)Re-MAG3-HBP tended to be more effective. CONCLUSION: These results indicate that (186)Re-MAG3-HBP could be useful as a therapeutic agent for the palliation of metastatic bone pain.

Design, synthesis, and evaluation of [188Re]organorhenium-labeled antibody fragments with renal enzyme-cleavable linkage for low renal radioactivity levels.

Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. We have reported that Fab fragments labeled with 3'-[131I]iodohippuryl Nepsilon-maleoyl-lysine (HML) showed markedly low renal radioactivity levels even shortly after injection, due to a rapid and selective release of m-[131I]iodohippuric acid by the action of brush border enzymes. To estimate the applicability of the molecular design to metallic radionuclides, [188Re]tricarbonyl(cyclopentadienylcarbonate)rhenium ([188Re]CpTR-COOH) was conjugated with Nepsilon-tert-butoxycarbonyl-glycyl-lysine or Nepsilon-maleoyl-glycyl-lysine to prepare [188Re]CpTR-GK-Boc or [188Re]CpTR-GK. The cleavage of the glycyl-lysine linkage of the two compounds generates a glycine conjugate of [188Re]CpTR-COOH ([188Re]CpTR-Gly), which possesses in vivo behaviors similar to those of m-iodohippuric acid. The hydrolysis rate of the peptide bond in [188Re]CpTR-GK-Boc was compared with that in 3'-[125I]iodohippuryl Nepsilon-Boc-lysine ([125I]HL-Boc) using brush border membrane vesicles (BBMVs) prepared from rat kidneys. [188Re]CpTR-GK was conjugated to thiolated Fab fragments to prepare [188Re]CpTR-GK-Fab. The biodistribution of radioactivity after injection of [188Re]CpTR-GK-Fab was compared with that of [125I]HML-Fab and [188Re]CpTR-Fab prepared by conjugating N-hydroxysuccinimidyl ester of [188Re]CpTR-COOH with antibody fragments. While [188Re]CpTR-GK-Boc liberated [188Re]CpTR-Gly in BBMVs, [125I]HL-Boc liberated m-[125I]iodohippuric acid at a much faster rate. In addition, although [125I]HL-Boc was hydrolyzed by both metalloenzymes and nonmetalloenzymes, metalloenzymes were responsible for the cleavage of the peptide linkage in [188Re]CpTR-GK-Boc. In biodistribution studies, [188Re]CpTR-GK-Fab exhibited significantly lower renal radioactivity levels than did [188Re]CpTR-Fab. However, the renal radioactivity levels of [188Re]CpTR-GK-Fab were slightly higher than those of [125I]HML-Fab. The analysis of urine samples collected for 6 h postinjection of [188Re]CpTR-GK-Fab showed that [188Re]CpTR-Gly was the major radiometabolite. In tumor-bearing mice, [188Re]CpTR-GK-Fab significantly reduced renal radioactivity levels without impairing the radioactivity levels in tumor. These findings indicate that the molecular design of HML can be applied to metallic radionuclides by using a radiometal chelate of high inertness and by designing a radiometabolite of high urinary excretion when released from antibody fragments following cleavage of a glycyl-lysine linkage. This study also indicates that a change in chemical structure of a radiolabel attached to a glycyl-lysine linkage significantly affected enzymes involved in the hydrolysis reaction. Since there are many kinds of enzymes that cleave a variety of peptide linkages on the renal brush border membrane, selection of a peptide linkage optimal to a radiometal chelate of interest may provide radiolabeled antibody fragments that exhibit renal radioactivity levels similar to those of [131I]HML-labeled ones. The in vitro system using BBMVs might be useful for selecting an appropriate peptide linkage.

Development of fluorescence-linked immunosorbent assay for high throughput screening of interferon-gamma.

BACKGROUND: Human interferon-gamma (hIFN-gamma) is produced by lymphocytes and has a variety of biological properties. Measurement of hIFN-gamma is widely used for various immunological responses for allergic or autoimmune diseases. Enzyme-linked immunosorbent assay (ELISA) is an established immunoassay used to quantify cellular metabolites or cytokines. ELISA requires many incubation and wash steps and is not practically suitable for screening large numbers of samples. METHODS: We have developed a fluorescence-linked immunosorbent assay (FLISA) method for the detection of hIFN-gamma. We measured the 50% inhibitory concentration (IC50) value of the hIFN-gamma production by interleukin (IL)-18 binding protein and anti-IL-18 monoclonal antibody. The IC50 described by FLISA was compared with that by ELISA. RESULTS: We developed a new system for measuring hIFN-gamma using Allophycocyanine (APC) fluorescent protein and compared it with the previous method using Cy5.5. The proposed FLISA had a smaller coefficient of variation than ELISA, and the means of coefficient of variation using the same samples measured by ELISA and FLISA were, respectively, 11.1% and 3.8%, suggesting that the edge effect often giving non-specific results may be smaller in FLISA than in ELISA. CONCLUSIONS: The improved FLISA system proposed is ideally suited for efficient measurements of hIFN-gamma. This homogeneous and multiplex method will be a powerful tool for high throughput screening for drug discovery research.

Rhemium-186-monoaminemonoamidedithiol-conjugated bisphosphonate derivatives for bone pain palliation.

To develop a radiopharmaceutical for the palliation of painful bone metastases based on the concept of bifunctional radiopharmaceuticals, we synthesized a bisphosphonate derivative labeled with rhenium-186 (186Re) that contains a hydroxyl group at the central carbon of its bisphosphonate structure, we attached a stable 186Re-MAMA chelate to the amino group of a 4-amino butylidene-bisphosphonate derivative [N-[2-[[4-[(4-hydroxy-4,4-diphosphonobutyl)amino]-4-oxobutyl]-2-thioethylamino]acetyl]-2-aminoethanethiolate] oxorhenium (V) (186Re-MAMA-HBP) and we investigated the effect of a hydroxyl group at the central carbon of its bisphosphonate structure on affinity for hydroxyapatite and on biodistribution by conducting a comparative study with [N-[2-[[3-(3,3-diphosphonopropylcarbamoyl)propyl]-2-thioethylamino]acetyl]-2-aminoethanethiolate] oxorhenium (V) (186Re-MAMA-BP). The precursor of 186Re-MAMA-HBP, trityl (Tr)-MAMA-HBP, was obtained by coupling a Tr-MAMA derivative to 4-amino-1-hydroxybutylidene-1,1-bisphosphonate. 186Re-MAMA-HBP was prepared by a reaction with 186ReO(4-) and SnCl2 in citrate buffer after the deprotection of the Tr groups of Tr-MAMA-HBP. After reversed-phase high-performance liquid chromatography, 186Re-MAMA-HBP had a radiochemical purity of over 95%. Compared with 186Re-MAMA-BP, 186Re-MAMA-HBP showed a greater affinity for hydroxyapatite beads in vitro and accumulated a significantly higher level in the femur in vivo. Thus, the introduction of a hydroxyl group into 186Re complex-conjugated bisphosphonates would be effective in enhancing accumulation in bones. These findings provide useful information on the design of bone-seeking therapeutic radiopharmaceuticals.

Development of a rhenium-186-labeled MAG3-conjugated bisphosphonate for the palliation of metastatic bone pain based on the concept of bifunctional radiopharmaceuticals.

Rhenium-186-1-hydroxyethylidene-1,1-diphosphonate (186Re-HEDP) has been used for the palliation of metastatic bone pain. Delayed blood clearance and high gastric uptake of radioactivity have been observed upon injection, due to the instability of (186)Re-HEDP in vivo. In this study, on the basis of the concept of bifunctional radiopharmaceuticals, we designed a stable 186Re-mercaptoacetylglycylglycylglycine (MAG3) complex-conjugated bisphosphonate, [[[[(4-hydroxy-4,4-diphosphonobutyl)carbamoylmethyl]carbamoylmethyl]carbamoylmethyl]carbamoylmethanethiolate]oxorhenium(V) (186Re-MAG3-HBP). As a precursor, [1-hydroxy-1-phosphono-4-[2-[2-[2-(2-tritylmercaptoacetylamino)acetylamino]acetylamino]acetylamino]butyl]phosphonic acid (Tr-MAG3-HBP) was synthesized by the conjugation of N-[(tritylmercapto)acetyl]glycylglycylglycine (Tr-MAG3) with the bisphosphonate analogue. After deprotection of the trityl group of Tr-MAG3-HBP, 186Re-labeling was performed by reacting 186ReO4- with SnCl2 in citrate buffer. After purification by HPLC, 186Re-MAG3-HBP showed a radiochemical purity of over 95%. To compare the stability of 186Re-MAG3-HBP and 186Re-HEDP, these (186)Re complexes were incubated in phosphate buffer. No measurable decomposition of 186Re-MAG3-HBP occurred over a 24-h period, while only approximately 30% of 186Re-HEDP remained intact 24 h postincubation. In biodistribution experiments, the radioactivity level of 186Re-MAG3-HBP in bone was significantly higher than that of (186)Re-HEDP. Blood clearance of 186Re-MAG3-HBP was faster than that of 186Re-HEDP. In addition, the gastric accumulation of 186Re-MAG3-HBP radioactivity was lower than that of 186Re-HEDP. In conclusion, 186Re-MAG3-HBP is expected to be a useful radiopharmaceutical for the palliation of metastatic bone pain.

Molecular mechanism of a temperature-sensitive phenotype in peroxisomal biogenesis disorder.

Peroxisomal biogenesis disorders include Zellweger syndrome and milder phenotypes, such as neonatal adrenoleukodystrophy (NALD). Our previous study of a NALD patient with a marked deterioration by a fever revealed a mutation (Ile326Thr) within a SH3 domain of PEX13 protein (Pex13p), showing a temperature-sensitive (TS) phenotype in peroxisomal biogenesis. Clinical TS phenotypes also have been reported in several genetic diseases, but the molecular mechanisms still remain to be clarified. The immunofluorescent staining with anti-Pex13p antibody also revealed TS phenotype of the I326T mutant protein itself in the patient cells. Protease digestion of the recombinant Pex13p-SH3 domain showed an increase of protease susceptibility, suggesting a problem of mutant protein fold. Conformational analyses against urea denaturation using urea gradient gel electrophoresis or fluorescence emission from tryptophan residue revealed that the mutant protein should be easily unfolded. Far-UV circular dichroism (CD) spectra demonstrated that both wild-type and the mutant protein have antiparallel beta-sheets as their secondary structure with slightly different extent. The thermal unfolding profiles measured by CD showed a marked lower melting temperature for I326T protein compared with that of wild-type protein. Analysis of the protein 3D-structure indicated that the Ile326 should be a core residue for folding kinetics and the substitution of Ile326 by threonine should directly alter the kinetic equilibrium, suggesting a marked increase of the unfolded molecules when the patient had a high fever. Structural analyses of the protein in the other genetic diseases could provide an avenue for better understanding of genotype-phenotype correlations.

Systematic optimization of active protein expression using GFP as a folding reporter.

Many recombinant proteins have been used as drugs; however, human proteins expressed using heterologous hosts are often insoluble. To obtain correctly folded active proteins, many optimizations of expression have been attempted but usually are found to be applicable only for specific targets. Interleukin-18 (IL-18) has a key role in many severe disorders including autoimmune diseases, and therapeutic approaches using IL-18 have been reported. However, production of IL-18 in Escherichia coli resulted in extensive inclusion body formation and previous conventional screenings of expression conditions could obtain only a condition with a low yield. To address the problem, we applied a folding reporter system using green fluorescent protein (GFP) for screening of the expression conditions for hIL-18. The established system efficiently screened many conditions, and optimized conditions for the expression of hIL-18 significantly enhanced the final yield of the active protein. Systematic screening using a GFP reporter system could be applied for the production of other proteins and in other organisms.