The Radioprotective Effects of Caffeic Acid Phenethyl Ester and Thymoquinone on Oxidative and Nitrosative Stress in Liver Tissue of Rats Exposed to Total Head Irradiation.

OBJECTIVE: The aim of this study is to investigate the effects of addition of caffeic acid phenethyl ester (CAPE) and thymoquinone (TQ) on oxidative and nitrosative stress in the liver tissue of irradiated rats. METHODS: Forty Sprague-Dawley rats were divided into five groups to test the radioprotective effectiveness of TQ and CAPE administered by intraperitoneal injection. Appropriate control groups were also studied. RESULTS: Liver antioxidant capacity, as measured by levels of total superoxide scavenger activity (TSSA), non-enzymatic superoxide scavenger activity (NSSA) and glutathione-S-transferase (GST) activity except superoxide dismutase (SOD) activity, were statistically lower in the irradiation (IR) group compared to all other groups. Total superoxide scavenger activity and NSSA were statistically higher in the IR plus TQ and IR plus CAPE groups compared to all other groups. In contrast, glutathione peroxidase (GSH-Px) activity was significantly found to increase in the IR plus CAPE group compared to control groups. The xanthine oxidase (XO), nitric oxide synthase (NOS) activities, nitric oxide (NO●) and malondialdehyde (MDA) levels in the IR group were statistically higher than in the other groups. Moreover, XO activity in the IR plus TQ group was statistically lower than all other groups including the IR plus CAPE group. In addition, NO● level was found to increase in all groups when compared to the normal control group. CONCLUSIONS: Thymoquinone and CAPE decrease oxidative and nitrosative stress markers and have antioxidant effects, which also increase antioxidant capacity in the liver tissue of irradiated rats.

The processing and fate of antibodies and their radiolabels bound to the surface of tumor cells in vitro: a comparison of nine radiolabels.

UNLABELLED: Processing radiolabeled degradation products is the key factor affecting retention of antibodies within the cell. In this study, we have analyzed the processing of antibodies labeled in nine different ways. METHODS: Antibodies were labeled with three different radioisotopes and seven different forms of 125I. Eight of the radiolabels (except 188Re) were conjugated to the same antibody, MA103, and tested on the renal carcinoma cell line SK-RC-18 and/or the ovarian carcinoma cell line SK-OV-6. Rhenium conjugation utilized the antibody RS7, the target cell line ME180 and three of the other radiolabels were also tested with this antibody-target cell combination for comparison. RESULTS: Iodine conjugated to antibodies by conventional methods was rapidly released from the cell after antibody catabolism. In contrast, iodinated moieties, such as dilactitol-tyramine and inulin-tyramine were retained within cells four to five times longer. CONCLUSIONS: The use of radiolabels that are trapped within cells after antibody catabolism can potentially increase the dose of radiation delivered to the tumor, from the same amount of radioactivity deposited by a factor of four or five. The prolonged retention of 111In relative to 125I is not due to deiodination of iodine conjugates, but rather to intracellular retention of catabolic products containing 111In, perhaps within lysosomes.

In vivo comparison of copper blood-pool agents: potential radiopharmaceuticals for use with copper-62.

Two techniques for labeling of albumin with copper-67 (67Cu) and 62Cu were investigated; one using the native Cu(II) binding site of the protein and the other employing a bifunctional chelate, 6-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradecane- N,N'N",N"'-tetraacetic acid (Br-benzyl-TETA or BAT), conjugated to the protein. Rat biodistribution experiments with 67Cu demonstrated retention of i.v. 67Cu-benzyl-TETA-albumin in the blood pool identical to co-injected 125I-albumin. By contrast, i.v. administration of either [67Cu]-Cu-acetate or [67Cu]-Cu-acetate pre-mixed with albumin results in relatively rapid clearance of blood-pool radioactivity as the tracer is excreted into the urine. The 62Cu-benzyl-TETA-albumin radiopharmaceutical was obtained in ca. 17% radiochemical yield (end of synthesis, without decay correction) following a procedure that can be completed in 15-18 min. In PET experiments with a baboon, myocardial blood volume images with 62Cu-benzyl-TETA-albumin were identical to those obtained with C15O. Use of the 62Cu-benzyl-TETA-albumin image for blood-pool subtraction of a 62Cu-PTSM myocardial perfusion image is illustrated. Copper-62-benzyl-TETA-HSA should be a useful, generator-produced radiotracer for the detection of the vascular pool at PET facilities without cyclotrons.

Simplified method for conjugating macrocyclic bifunctional chelating agents to antibodies via 2-iminothiolane.

A one-step method for conjugating macrocyclic chelators to antibodies using the protein modification reagent 2-iminothiolane controls aggregation, maintains immunoreactivity, and produces consistent chelate/antibody ratios. Conjugation conditions have been investigated with the macrocyclic chelates 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N ',N",N"'-tetraacetic acid and 2-[p-(bromoacetamido)benzyl]-1,4,7,10-tetraazacyclododecane-N,N',N ",N"'-tetraacetic acid, with three different monoclonal antibodies. The bifunctional chelating agents are prepared by bromoacetylation of their amine precursors using a two-phase H2O/CHCl3 system, which improves product purity.

Strategies for enhancement of radioimmunotherapy.

Two new strategies for increasing tumor uptake have been investigated. First the effect of interleukin-2 (IL-2) on tumor uptake of 125I-Lym-1 antibody in nude mice was investigated. Secondly, the use of 67Cu-labeled Lym-1 was evaluated in patients. In nude mice implanted with Raji human lymphoma, a greater than 2-fold enhancement of tumor uptake of 125I-Lym-1 was observed after administration of PEG-interleukin-2 (PEG-IL-2). The macrocycle 1,4,8,11-tetraazacylcotetradecane-N,N',N",N"'-tetraacetic acid (TETA), synthesized specifically for copper chelation, has been conjugated to Lym-1 for 67Cu labeling of the monoclonal antibody (MoAb). There was no evidence for bone or normal marrow uptake and the residence time in the tumor was prolonged. Surprisingly, a dose of 4.4 mCi that was intended for imaging induced substantial tumor regression in a patient.

Technetium-99m, rhenium-186, and rhenium-188 direct-labeled antibodies.

BACKGROUND: Antibody sulfhydryl groups can act as effective carriers of reduced technetium and rhenium species for radioimmunodetection and radioimmunotherapy. METHODS: Intact immunoglobulin G and fragments were labeled with the isotopes and examined in vitro and in vivo. RESULTS: Technetium bound to intact immunoglobulin G was found to be the most stable species in vitro, but in vivo, clearances of technetium and rhenium bound to intact antibody were similar. Serum clearances were faster than those seen for the corresponding radioiodinated antibodies. In vivo clearance rates of the radiolabeled fragments were similar, with kidney uptake and retention seen. Rhenium-labeled antibodies, despite a greater tendency toward in vitro reoxidation than technetium-labeled antibodies, did not show enhanced kidney clearance in animal models. Rhenium-188 and technetium-99m were obtained from similar generator systems in carrier-free form. Using rhenium-188 spiked with cold rhenium, it was determined that approximately one rhenium atom per molecule of antibody can be conjugated directly. Rhenium-186 also was coupled at almost a 1:1 ratio to antibody. CONCLUSIONS: Only radiolysis concerns will limit the amount of rhenium-188 conjugated to antibody. Large doses of antibody will be necessary to deliver rhenium-186 at this isotope's currently available specific activity. Otherwise, higher specific activity rhenium-186, and/or greater loading capacity of rhenium-186 onto antibody, will be needed to generate the type of product that will be usable at a clinical dose of several hundred millicuries.

Processing of antibody-radioisotope conjugates after binding to the surface of tumor cells.

BACKGROUND: Previous experiments indicated that most antibodies binding to cell surface antigens are internalized gradually and degraded within lysosomes, with a half-life of degradation of approximately 1 day, for most antibodies. The research discussed in this article extended our studies to eight additional antibodies reacting with six different antigens, including three antigens anchored in the membrane by glycosyl-phosphatidylinositol. The authors also tested antibodies labeled with 111indium, as well as 125iodine, to determine whether different radiolabels would be processed differently. METHODS: Antibodies were radiolabeled with 125I or with 111In bound to benzyl-DTPA. After binding to the surface of tumor cells in vitro, excess antibody was washed away, and the fate of the radiolabel was investigated over periods of 3-7 days. Radiolabel released into the supernatant or retained by the cells was analyzed to determine whether it was still on intact antibody. RESULTS: In 13 of the 15 antibodies that were tested, a similar pattern of irreversible binding and gradual catabolism was observed. Iodine conjugated to antibodies was released rapidly from the cell after antibody catabolism. In contrast, the 111In was retained within the cell much longer than 125I, with the rate of degradation and release into the medium being at least fivefold slower. More than 50% of the bound 111In was still present on the cells after 7 days. Biochemical analysis of the retained 111In extracted cells after 4-6 days demonstrated that it was no longer associated with antibodies and was in a low molecular weight form, probably still associated with the chelator benzyl-DTPA. CONCLUSIONS: Different radiolabels are processed by tumor cells differently, after catabolism of the antibody to which they originally were conjugated. The data suggest that the prolonged retention of 111In, relative to that of 125I, is due not to deiodination of iodine conjugates, but rather to intracellular retention of catabolic products containing 111In, perhaps within lysosomes. The use of radioisotopes that are retained within cells after antibody internalization and degradation may improve both radioimmunodetection and radioimmunotherapy of cancer.

Internalization and catabolism of radiolabelled antibodies to the MHC class-II invariant chain by B-cell lymphomas.

The fate of antibody (Ab) LL1, which reacts with the invariant chain (Ii) subunit of the immature MHC class-II antigen (CD74) after binding to the surface of B-cell lymphomas was investigated. This Ab was internalized and catabolized very rapidly, much faster than other Abs that are considered to be rapidly internalized, such as CD19, CD22 and anti-(transferrin receptor). Such internalization did not depend on Ab cross-linking. The capacity of this uptake process was determined in long-term experiments by increasing the Ab concentration: in 1 day, approx. 8 x 10(5) Ab molecules per cell were catabolized. This analysis was facilitated by the use of radiolabels that are trapped within cells after catabolism of the Abs to which they were conjugated. If the Ab is a reliable marker for the Ii antigen, which is likely, we can conclude that Ii directed to the cell surface appears to be sufficient, indeed more than sufficient, to account for the cell content of mature class-II molecules.

Macrocyclic chelates of radiometals for diagnosis and therapy.

Monoclonal antibody technology allows the specificity of an antibody for its antigen to be used in targeting cancer cells. Stable attachment of metal ions to antibodies by means of 'bifunctional' chelating agents can add the nuclear, physical and chemical properties of the metallic elements to these target-selective proteins. The conjugation of metals--particularly radionuclides--to monoclonal antibodies results in agents for radioimmunotherapy and other medical applications. Chelators that can hold radiometals with high stability under physiological conditions are essential to avoid excessive radiation damage to non-target cells. Derivatives of polyazamacrocycles (bearing a C-substituted functional group for antibody attachment) can exhibit remarkable kinetic inertness. We have developed a new synthetic route these macrocycles via peptide synthesis and intramolecular tosylamide ring closure. Incubation of the yttrium complex of 2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane-N,N1N",N'"-te traacetic acid (nitrobenzyl-DOTA) for 18 days in serum results in loss of so little yttrium from the complex (less than 0.5%) that the rate of loss cannot be measured under these conditions. In animal models, conjugates of this chelate with monoclonal antibodies show much lower levels of yttrium in the bone than are found with DTPA chelates prepared from the cyclic anhydride. The rates of loss of indium and cobalt from nitrobenzyl-DOTA in serum are slower than from previously studied chelates. Preliminary clinical imaging studies of 111In-labeled monoclonal antibody conjugates of DOTA show highly encouraging results.

A comparative study of copper-67 radiolabeling and kinetic stabilities of antibody-macrocycle chelate conjugates.

BACKGROUND: The development of new chelating agents and radiolabeling protocols is essential to progress in radioimmunotherapy with antibody-chelate conjugates. METHODS: Immunoconjugates of four polyazamacrocycles with N-bonded acetate groups were prepared by conjugation via 2-iminothiolane to Lym-1, a murine antilymphoma immunoglobulin G2a MoAb. To optimize 67Cu radiolabeling, complexation conditions were explored. The kinetic stabilities in vitro in human serum of four 67Cu labeled immunoconjugates were investigated. RESULTS: Lym-1-2IT-6-BAT-67Cu, the chelate conjugate of 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane- N,N',N''N'''-tetraacetic acid, exhibited excellent kinetic stability in human serum, while Lym-1-2IT-2-BAT-67Cu, prepared from the structural isomer 2-[p-(bromoacetamido)benzyl]-1,4,8,11- tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid, exhibited a markedly higher rate of loss of radiometal. It was observed that the radiolabeling ratio of Lym-1-2IT-6-BAT-67Cu, in mCi per mg immunoconjugate, was limited solely by the specific activity of the radiometal, which varied significantly from lot to lot. This ratio for a given lot of 67Cu can be predicted by a preliminary titration. CONCLUSIONS: The preparation of 67Cu labeled immunoconjugates of therapeutic quality has been improved by the determination of optimum radiolabeling conditions, and by development of a titration protocol which rapidly and accurately predicts the radiolabeling ratio in mCi per mg immunoconjugate. The surprising difference in the properties of 6-BAT and 2-BAT shows the exquisite dependence of kinetic stability on structure.