186Re radioimmunotherapy of small cell lung carcinoma xenografts in nude mice.

A 186Re-labeled monoclonal antibody (MAb), NR-LU-10, was used for the radioimmunotherapy of a subcutaneous human small cell lung carcinoma xenograft, SHT-1, in nude mice. Biodistribution with specific and irrelevant labeled MAb demonstrated peak tumor uptake of 8% and 3% of the injected dose/g at 2 days, respectively. Dosimetry analysis predicted tumor:whole-body radiation-absorbed dose ratios of 2.43:1 for NR-LU-10 and 0.62:1 for irrelevant MAb. Single-dose toxicity screening estimated a 50% lethal dose within 30 days of 600 microCi (880 cGy of whole-body radiation). As anticipated, a multiple-dose regimen of 490 microCi in four doses over 10 days (720 cGy of whole-body radiation, eight of eight surviving greater than 30 days) was less toxic than a single bolus dose of 430 microCi (644 cGy of whole-body radiation), six of eight surviving greater than 30 days). A multidose radioimmunotherapy regimen was initiated in nude mice bearing 66-mm3 tumors (total dose, 500 to 600 microCi). Complete remissions (greater than 140 days) were achieved in three of 16 mice, and the remainder showed a mean tumor growth delay of 53 days. Matched doses with irrelevant MAb produced one remission, one treatment-related death, and a mean growth delay of only 20 days in six of eight mice. Thus, in this nonoptimal radioimmunotherapy model, significant antitumor responses were observed using a mildly toxic multiple dosing regimen.

Biodistribution, pharmacokinetic, and imaging studies with 186Re-labeled NR-LU-10 whole antibody in LS174T colonic tumor-bearing mice.

Biodistribution, pharmacokinetic, and radioimaging studies were performed with 186Re-labeled NR-LU-10 whole antibody in athymic nude mice bearing the LS174T tumor growing either s.c. or in an experimental hepatic metastasis model. NR-LU-10 is an IgG2b murine monoclonal antibody (MAb) that reacts with virtually all human tumors of epithelial origin. NR-BC-1, a IgG2b murine MAb that reacts with normal human B-cell and B malignancies, was used as an isotype-matched control. These MAbs were radiolabeled with 186Re (3.7-day physical half-life; 1.07-MeV beta particle and 137-keV gamma, 9% abundance) by a preformed chelate approach by using the triamide thiolate ligand system. 186Re-labeled NR-LU-10 (50 microCi) was injected into nude mice bearing LS174T tumors growing s.c. Biodistribution studies revealed that the LS174T tumor retained the highest concentration of 186Re-labeled NR-LU-10 (5.3% injected dose/g) at day 6. The tumor:blood ratio ranged from 0.1:1 to 10.8:1 by day 6, the last day of analysis. In contrast the tumor:blood ratio of 186Re-labeled NR-BC-1, the isotype-matched MAb control, was 1:1 on day 6. Pharmacokinetic analysis indicated that the t1/2 beta of NR-LU-10 for blood and other tissues ranged from 21 to 25 h, while the t1/2 beta for the LS174T tumor averaged 52 h. The area under the curve for tumor compared to blood was 2.8- to 5.7-fold higher than the area under the curve for all other tissues and organs. The mean residence time for NR-LU-10 in blood and all other organs ranged from 23 to 26 h, while the mean residence time for NR-LU-10 in the LS174T tumor was 72 h. Scintigraphic images revealed selective uptake of the 186Re-labeled NR-LU-10, but not of the 186Re-labeled NR-BC-1, at the LS174T tumor site. Studies in an experimental model of hepatic metastasis revealed a similar selective pattern of 186Re-labeled NR-LU-10 accumulation. Scintigraphic images of the LS174T tumor growing within the athymic nude mouse liver were obtained. The biodistribution, pharmacokinetic, and scintigraphic image results suggest that 186Re-labeled NR-LU-10 shows promise as a therapeutic agent for gastrointestinal cancer.

Tissue distribution properties of technetium-99m-diamide-dimercaptide complexes and potential use as renal radiopharmaceuticals.

A series of new ligands and the corresponding technetium-99m chelates based on diamide dimercaptide donor groups were synthesized as derivatives of technetium-99m 1,2-bis(2-thioacetamido)ethane, a complex shown to be excreted by renal tubular secretion. Chelation with 99mTc resulted in single radiochemical products or the expected numbers of stereoisomers. They were purified by high-performance liquid chromatography (HPLC) and evaluated in mice as potential renal tubular function agents. The in vivo properties were sensitive to the presence of functional groups, the positional isomerism of the carboxylate group functionality, and the chelate ring stereochemistry of the ligand. The presence of methyl groups slowed renal transit and decreased renal specificity. Cyclohexyl rings fused to the ethylene bridge of the center chelate ring decreased renal excretion while aromatic rings essentially abolished renal excretion. Slow hepatobiliary clearance was observed as an alternate mode of excretion. Polar groups, such as hydroxyl, carboxylate, and carboxamide, increased renal excretion rates and specificity in a stereochemically dependent manner. 99mTc chelates of 1,3-bis(2-thioacetamido)-2-hydroxypropane, 3,4-bis(2-thioacetamido)butanoate and 1,8-dimercapto-2,7-dioxo-3,6-diazanonanoate were identified as promising new renal radiopharmaceuticals.

Targeted proteins for diagnostic imaging: does chemistry make a difference?

The Oyen et al. study is valuable in that it systematically evaluates several of the factors involved in radiolabeled protein uptake and retention in infectious foci. The role of particular proteins and their receptor specific interactions seems to be inconsequential in agreement with the findings of other. However, the role of the radiolabel was shown to be important and significant differences were delineated from comparisons of the radionuclides and their associated chemistries. The conclusion implicating radionuclide chemistry and associated linkages underscores the need to optimize the attachment and labeling chemical modifications of protein carriers. Evaluation criteria should include serum stability, determination and assessment of the effect of molar substitution ratio, and potential for improving blood clearance without reducing the target-to-non-target ratio. Important areas for future study include characterization of radioactive metabolites and the design and synthesis of new ligands which direct the disposition of metabolites reducing retention in normal organs or accelerating renal excretion. Additionally, intracellular processing of radiolabel, compartmental distribution and strategies for augmenting internalization and retention within the target cell merit detailed exploration. For each radionuclide of interest, 111In, radioiodines, 99mTc and others, improved chemical moieties exist for controlling radiolabel fate. When carrying out mechanistic and evaluative studies, clear-cut conclusions will only be reached when defined and controlled chemistry is used. Having established a "gold standard," simplifications in radiolabeling and other chemical refinements can then be pursued with a quantitative understanding of the trade-offs in targeting agent performance versus other considerations such as cost reduction, simplicity, and convenience.

HPLC analysis of Tc-99m iminodiacetate hepatobiliary agents and a question of multiple peaks: concise communication.

The application of high-performance liquid chromatography (HPLC) to Tc-99m-labeled iminodiacetate hepatobiliary agents is described. The method is sensitive to variations of phenyl ring structure and give different retention times for the different agents. Technetium-99m-labeled N-(2, 6-diethylacetanilide)iminodiacetate from different sources had two components in differing amounts. It was determined that the second component, which is minor at pH above 6, is major when prepared at pH 4.5 or less. Raising the pH results in rapid conversion of the second component into the first. This observation was demonstrated also in plasma and would be expected to occur when the radiopharmaceutical is injected intravenously. The kinetics of appearance of the preparations in the bile when prepared at pH 4 and pH 6 were essentially the same.

Synthesis and biological evaluation of technetium-99m MAG3 as a hippuran replacement.

A new technetium-chelating agent based on a triamide monomercaptide tetradentate set of donor groups, mercaptoacetylglycylglycylglycine (MAG3), was synthesized and evaluated. Chelation with 99mTc resulted in a single radiochemical product as expected. Studies in mice of [99mTc]MAG3 indicated excretion rates faster than omicron-iodohippurate (OIH) both in normal and in probenecid treated animals. Specificity for renal excretion was essentially complete. Clearance studies in rats resulted in 2.84 ml/min/100 g for [99mTc]MAG3, 2.17 for OIH, and 1.29 for [125I]iothalamate. Extraction efficiencies were 85% for [99mTc]MAG3, 69% for OIH and 39% for [125I]iothalamate. Probenicid depressed the clearance both of [99mTc]MAG3 and OIH at 25 and 50 mg/kg/hr, but to a greater extent with [99mTc]MAG3. The greater effect is offset, however, by the larger fraction secreted by the renal tubular cells. The animal results suggest that [99mTc]MAG3 may be a useful alternative to [131I]OIH.

99mTc-bioquin-7CA, a potential new hepatobiliary scanning agent.

The complex 99mTc-Sn-8-hydroxyquinoline-7-carboxylate (99mTc-bioquin-7CA) was investigated in animals as a potential hepatobiliary scanning agent. Following intravenous injection, the complex was found to localize in the liver rapidly and in the gallbladder in 5--15 min; after 15 min it was excreted into the biliary tract with a high degree of specificity. Urinary excretion was negligible, reducing scan interference from the kidneys. Distribution studies in mice compared well withe localization of activity observed in rabbit scans. Preparation of the complex was rapid and simple; stannous ion was used as the reducing agent.

Evaluation of formamidine sulfinic acid and other reducing agents for use in the preparation of Tc-99m labeled radiopharmaceuticals.

Various reducing agents have been evaluated for their potential usefulness in the preparation of 99mTc labeled radiopharmaceuticals for use in nuclear medicine. Adequate labeling of various radiopharmaceuticals was accomplished using formamidine sulfinic acid. Nitrogen-purging of solutions is not required, which is an advantage for in-house preparation. Tagging requires heating, however, so heat-labile material cannot be used. Various compounds that could not be labeled when stannous chloride was used, could be tagged with 99mTc when formanidine sulfinic acid was used as the reducing agent.

Comparison of iodine-131 OIH and technetium-99m MAG3 renal imaging in volunteers.

Animal studies have suggested that the nonisomeric N3S triamide mercaptide ligand, 99mTc mercaptoacetyltriglycine (MAG3), may provide a satisfactory 99mTc-labeled replacement for 131I hippurate (OIH). Sequential 30-min [99mTc]MAG3 (5-10 mCi) and [131I]OIH (300 microCi) imaging studies were performed in ten normal volunteers in order to compare the image quality, renal excretion, blood clearance, and time to peak height of the renogram curve. In addition, [99mTc] MAG3 (5 mCi) and [131I]OIH (150 microCi) were administered simultaneously in eight volunteers for comparison of 180-min blood and plasma clearances and urine excretion. In the sequential imaging studies, the blood clearance of [99mTc]MAG3 was more rapid than [131I]OIH with a mean clearance of 1.30 l/min compared with 0.88 l/min for [131I]OIH (p less than 0.05). Seventy-three percent of the injected dose of the MAG3 was excreted by 30 min compared with 66.8% for [131I]OIH. Whole kidney and cortical renogram curves showed no significant difference in the time to peak height for MAG3 and [131I]OIH. In all subjects, the quality of the [99mTc]MAG3 images were clearly superior to [131I]OIH. Following simultaneous injection, blood and plasma clearances for [131I]OIH were more rapid than MAG3 when determined for multiple time intervals from 0-30 to 0-180 min (p less than or equal to 0.05). The 0-30-min clearances of MAG3 and [131I]OIH were only slightly greater than the 0-180-min clearances and can be used to obtain valid comparisons of the two agents. As in the sequential study, 30-min urine excretion was greater for MAG3 than [131I]OIH (73.1 compared with 69.6%) but the difference was not statistically significant. Although the differences in the MAG3 clearances following sequential and simultaneous administration are not satisfactorily explained, the fact that both clearances were rapid, the MAG3 and OIH renogram curves were quite similar, and 30-min urine excretions of MAG3 and OIH were essentially identical suggests that MAG3 may become a 99mTc replacement for [13I]OIH and further clinical evaluation is warranted.

Animal evaluation of technetium-99m triamide mercaptide complexes as potential renal imaging agents.

Technetium-99m mercaptoacetylglycylglycylglycine (MAG3), a [99mTc]triamide mercaptide (N3S) compound has been synthesized in an attempt to obviate the stereochemistry problems associated with the diamide dimercaptide (N2S2) ligands. Because initial studies have been promising, the terminal glycine on the MAG3 compound has been varied to create a new series of N3S compounds. Twelve new N3S complexes were initially screened in mice and the more promising complexes, 99mTc mercaptoacetylgylcylglycyl-glycine [( 99mTc]MAG3), 99mTc mercaptoacetylgylcylglycyl-L-alanine [( 99mTc]MAG2-Ala), and both complexes of 99mTc mercaptoeacetylglycylglycyl-L-asparagine [( 99mTc]MAG2-Asn) and 99mTc mercaptoacetylglycylglycyl-L-glutamine [( 99mTc]MAG2-Gln), were further evaluated in rats utilizing constant infusion blood clearances, extraction efficiencies and protein binding assays. The renal excretion of all these complexes compared favorably with simultaneously administered [131I]OIH and [125I]iothalamate. The triamide mercaptide complexes represent a new ligand class for 99mTc, which may provide a variety of complexes for the evaluation of renal tubular function.

Normal appearance and reproducibility of liver-spleen studies with Tc-99m sulfur colloid and Tc-99m microalbumin colloid.

In order to determine the normal biodistribution and reproducibility of Tc-99m sulfur colloid and Tc-99m microalbumin colloid, each of ten normal subjects was studied twice with each of the two agents. In overexposed delayed analog images of the anterior chest, some thyroid radioactivity was demonstrated in all of 20 microalbumin colloid studies and in none of 20 sulfur colloid studies; lung radioactivity was seen in 19 of 20 (95%) sulfur colloid studies and in none of the microalbumin colloid studies (both p less than 0.001). Delayed digital images showed that the sulfur colloid gave a higher lung-to-heart ratio, a higher lung-to-liver ratio, and a lower bone marrow-to-liver ratio compared with microalbumin colloid (all p less than 0.05). In general, reproducibility was poor for both agents. We conclude that while there are some differences between these two radiocolloids, the differences do not indicate a superiority of the newer Tc-99m microalbumin colloid over the established Tc-99m sulfur colloid for liver-spleen imaging.

Synthesis and biological evaluation of Tc-99m N,N'-bis(mercaptoacetyl)-2,3-diaminopropanoate: a potential replacement for [131I]o-iodohippurate.

A new technetium-chelating agent based on amide and mercaptide donor groups, N,N'-bis(mercaptoacetyl)-2,3-diaminopropanoate, was synthesized as an analog of previously described N,N'-bis(mercaptoacetyl)ethylenediamine (DADS). Complexation of Tc-99m with the new chelating group resulted in two components that were separable by high-performance liquid chromatography. The component that eluted first demonstrated high specificity for renal excretion, with over 90% in the urine of rabbits at 35 min, 87% in the urine of mice at 2 hr, and 1.6% or less in the intestines of mice. Excretion was rapid, with the first component equaling or exceeding [131I]o-iodohippurate in the urine of rabbits at all times. The second or latter component demonstrated comparable specificity but slightly slower renal excretion kinetics. Clinical trials with the first component are probably warranted.

Chemical and biological studies of Tc-99m N,N'-bis(mercaptoacetamido)-ethylenediamine: a potential replacement for I-131 iodohippurate.

The tetradentate chelating agent N,N'-bis(benzoylmercaptoacetamido)ethylenediamine was synthesized for evaluation as a potential technetium-99m renal-function radiopharmaceutical. Complexes were prepared using different reducing agents and analyzed by high-performance liquid chromatography. Biological studies were performed in mice, rats, and rabbits and indicated that the new agent is cleared by the kidneys significantly faster than Tc-99m DTPA (p less than 0.01) and slightly slower than I-131 o-iodohippuric acid (p greater than 0.05). There was no evidence of significant renal retention. Renal excretion in all species studied was 70--75% of the injected dose in 30 min; biliary excretion in rats was 7% in animals with normal renal function and 18% in 90 min in the absence of renal function. We conclude that limited clinical trials are warranted.

Development and biologic evaluation of a kit for preformed chelate technetium-99m radiolabeling of an antibody Fab fragment using a diamide dimercaptide chelating agent.

A kit has been developed for 99mTc antibody radiolabeling via defined chemistry using an N2S2 diamide dimercaptide bifunctional chelating agent and the performed chelate method. The process involved efficient transchelation of 99mTc from gluconate to 2,3,5,6-tetrafluorophenyl 4,5-bis-S-(1-ethoxyethyl) mercaptoacetamidopentanoate as an active ester ligand and subsequent conjugation to antibody lysine amine functional groups. The use of the ethoxyethyl group for sulfur protection allowed optimum yields of 99mTc N2S2 chelate formation with complete retention of the active ester. Subsequent addition of antibody Fab fragment gave 99mTc chelate conjugates indistinguishable from the stepwise in situ esterification and purification of the 99mTc N2S2 complex followed by conjugation as previously shown to give stable 99mTc antibody fragments with retained immunoreactivity and tumor-targeting properties.

Development of a stable radioiodinating reagent to label monoclonal antibodies for radiotherapy of cancer.

A method of radioiodinating monoclonal antibodies such that the labeled antibodies do not undergo in vivo deiodination has been studied. The method utilizes conjugation of succinimidyl para-iodobenzoate to the antibody. The iodobenzoate was radiolabeled by using an organometallic intermediate to facilitate the reaction. Thus, succinimidyl para-tri-n-butylstannylbenzoate was radiolabeled in 60-90% radiochemical yield and subsequently conjugated to the antibody in 80-90% yield. Animal biodistribution studies were carried out with two separate anti-melanoma antibodies (9.2.27 and NR-M1-05) labeled by this method, and examined in nude mice bearing human melanoma tumor xenografts. Very large differences in the localization of radioactivity were observed in the thyroids and stomachs of mice when the iodobenzoyl-labeled antibodies were compared with the same antibodies labeled using the chloramine-T method of radioiodination. Few other significant differences in the tissue distribution of the radioiodinated antibodies were seen.

The investigation of radiopharmaceutical components by fast atom bombardment mass spectrometry: the identification of Tc-HIDA and the epimers of Tc-CO2DADS.

The nature of two technetium-labeled radiopharmaceutical components has been established by means of fast-atom-bombardment mass spectrometry (FABMS) in combination with carrier-added (CA) and no-carrier-added (NCA) reversed-phase high-pressure liquid chromatography (HPLC). Negative-ion FABMS was used to determine that the epimers of Tc-CO2DADS are the oxo[N,N'-(1-carboxyethylene)-bis-(2-mercaptoacetimido)]technetate(V) ions; positive-ion FABMS showed that Tc-HIDA is bis[N-(2,6-dimethylphenyl-carbamoylmethyliminodiaceto]technetate(III).

Clinical evaluation of Tc-99m N,N'-bis(mercaptoacetyl)-2,3-diaminopropanoate as a replacement for I-131 hippurate: concise communication.

A clinical comparison of Tc-99m N,N'-bis( mercaptoacetyl )-2,3- diaminopropanoate (Component A) (Tc-99m CO2-DADS-A) and I-131 hippurate was conducted in a series of five normal volunteers and 18 patients. Each subject was studied in one session with Tc-99m CO2-DADS-A and I-131-hippurate; digital and analog images were recorded for 30 min and after voiding. In the normal volunteers, digital images with Tc-99m CO2-DADS-A gave a kidney-to-background ratio at 3 min that was greater relative to I-131 hippurate, a leading-edge parenchymal transit time that was similar to I-131 hippurate, and a percent injected dose in the urine at 30 min that was slightly less than I-131 hippurate (p less than 0.05). In patients (serum creatinine 1.0 to 14.3 mg/dl), decreasing renal function impaired excretion of Tc-99m CO2-DADS-A more than that of I-131 hippurate (p less than 0.01). In analog images, Tc-99m CO2-DADS-A always gave superior spatial resolution. No evidence of hepatobiliary excretion was detected with either radiopharmaceutical. We conclude that Tc-99m CO2-DADS-A and similar compounds should be pursued as possible replacements for I-131 hippurate.

Clinical comparison of Tc-99m N,N'-bis(mercaptoacetamido)ethylenediamine and [131I]ortho-iodohippurate for evaluation of renal tubular function: concise communication.

Recent experiments in normal animals have shown that Tc-99m N,N'-bis(mercaptoacetamido)ethylenediamine (Tc-99m DADS) is a potential technetium-99m-labeled replacement for [131I]ortho-iodohippurate(I-131 Hipp). We compared the two agents in eleven patients with renal transplants (serum creatinine range: 0.9--5.7 mg/dl). Digital and analog images were recorded for 30 min and after voiding. The results indicate that the relative extraction efficiency index of Tc-99m DADS is 76% +/- 3 (mean +/- s.e.m.), that the leading-edge parenchymal transit time of Tc-99m DADS is 137% +/- 6, and that the percentage of injected Tc-99m DADS in the bladder at 30 min is 25% +/- 4 of that of I-131 Hipp (all p less than 0.05). Forty percent of patients showed some hepatobiliary excretion of Tc-99m DADS. We conclude that the biologic properties of Tc-99m DADS are inferior to those of I-131 Hipp and that analogs of Tc-99m DADS should be evaluated in an attempt to find a more suitable technetium-99m-labeled replacement for I-131 Hipp.

Effect of hydration and dehydration on technetium-99m CO2 DADS renal studies in normal volunteers.

Ten normal volunteers were studied in the hydrated and dehydrated states with the new renal radiopharmaceutical technetium-99m N,N'-bis(mercapto acetyl)-2, 3-diaminopropanoate [( 99mTc]CO2 DADS). The data were used to determine the effect of hydration and dehydration and to determine the normal range in each state. Visual evaluation of the images indicated that the first appearance of tracer in the collecting system was approximately the same in either state, that the concentration of tracer in the collecting system was always higher in the dehydrated state (p less than 0.01), and that the ureters always appeared more segmented in the dehydrated state (p less than 0.01). Quantitative analysis of the images indicated that the kidney to background ratio 1-2 min after injection was somewhat greater in the dehydrated state (13.5 +/- 4.0) than in the hydrated state (9.8 +/- 2.2) (p less than 0.05), that the size of the bladder was always greater in the hydrated state (p less than 0.05), and there was no difference in the amount of tracer in the bladder at 30 min after injection. The results define the normal hydrated and dehydrated [99mTc]CO2 DADS renal study and identify several differences between the two states which can be explained primarily by differences in urine flow rates.

Rhenium-186-labeled chimeric antibody NR-LU-13: pharmacokinetics, biodistribution and immunogenicity relative to murine analog NR-LU-10.

A mouse-human chimeric monoclonal antibody (NR-LU-13), with the same pancarcinoma antigen recognition site as a previously studied murine monoclonal antibody (NR-LU-10), was radiolabeled with 186Re using a bifunctional chelate. Nine patients (ages 31-81 yr) with metastatic adenocarcinoma received 186Re NR-LU-13. A single intravenous dose of 42 mg NR-LU-13 labeled with 25 mCi/m2 (two patients) or 60 mCi/m2 (seven patients) was administered. Mean serum disappearance half-time values for the chimeric 186Re NR-LU-10). Fifty percent of the radiolabel was excreted in the urine by 6 days. Tumor localization was demonstrated by gamma camera imaging in seven of nine patients. The percent injected dose per gram in a single tumor biopsy specimen was 0.003% at 72 hr postinjection. Absorbed dose to bone marrow was 1.5 +/- 0.7 rads/mCi and resulted in reversible myelosuppression in five of six evaluable patients who received 60 mCi/m2: median WBC nadir = 2500/microliters; median platelet nadir = 85,500/microliters. Low grade fever, nausea, slight elevations of liver function tests and mild allergic reactions were seen in some patients. The chimeric antibody elicited low levels of anti-NR-LU-13 antibody in six of eight evaluable patients (75%), in contrast to NR-LU-10 which elicited higher levels of human anti-mouse antibody in all patients. This pilot study demonstrates the ability of the chimeric antibody to target tumors with reduced (but not absent) immunogenicity and delayed clearance relative to the murine antibody.