Simple quantitative method for determining the amount of blood-borne tumor cells: initial in vitro results.

In metastasis research it would be useful to determine the number of blood borne tumor cells which are released from a primary tumor into the blood circulation. One way to quantify the number of released tumor cells could be to take blood from a vessel which is located close to a primary tumor and is draining the tumor. The number and viability of tumor cells released into the blood stream at any given time could be measured in cancer patients, especially those known to bear a primary, hematogenous metastasizing tumor. Plating efficiency is a precise method for the quantitative determination of the number of colony-forming cells in an adherent cell population. We performed initial in vitro experiments using plating efficiency to count adherent tumor cells within whole human blood. Exploiting the difference in adherence properties of colon carcinoma cells and blood cells in standard cell culture medium, these initial investigations show that it is possible to determine the plating efficiency of colon carcinoma cells within fresh whole human blood.

Phase I trial of methotrexate-albumin in a weekly intravenous bolus regimen in cancer patients. Phase I Study Group of the Association for Medical Oncology of the German Cancer Society.

Methotrexate-albumin conjugate (MTX-HSA) is a novel human albumin-based prodrug conjugate of methotrexate (MTX). A low MTX loading rate provided optimal tumor targeting and therapeutic efficacy during preclinical testing. The objectives of this first Phase I study of MTX-HSA were to determine dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) in a weekly regimen. Seventeen cancer patients who were no longer amenable to standard treatment were enrolled and were evaluable for DLT. Up to eight injections were performed in weekly intervals. Dose escalation was as follows: 20, 40, 50, and then 60 mg/m2 MTX-HSA (based on the amount of MTX bound to albumin). Additional MTX-HSA courses were feasible in case of tumor response. DLT (mainly stomatitis, Common Toxicity Criteria grade 3) occurred, beginning at the 50 mg/m2 dose level after repeated administrations; in one case, thrombocytopenia was dose-limiting. Two events of DLT occurred at the 60 mg/m2 dose level within the first two administrations. Mild anemia, transaminitis, and one case of skin toxicity were found. No significant leukopenia, nausea, renal toxicity, or other toxicities were observed. MTX-HSA was well tolerated. Drug accumulation occurred on the weekly schedule. The half-life of the drug was estimated to be up to 3 weeks. Tumor responses were seen in three patients: (a) a partial response was seen in one patient with renal cell carcinoma (response duration, 30 months, ongoing); (b) a minor response was seen in one patient with pleural mesothelioma (response duration, 31 months, ongoing); and (c) a minor response was seen in one patient with renal cell carcinoma (response duration, 14 months until progression). Poststudy treatment was administered at 2-4-week intervals. No signs of toxicity or drug accumulation were seen. Altered pharmacological properties of MTX-HSA such as plasma half-life, tumor targeting, or intracellular metabolism might have contributed to these responses. The MTD for weekly administration was 4 x 50 mg/m2 MTX-HSA during short-term treatment. A regimen with MTX-HSA injections of 50 mg/m2 every 2 weeks was recommended for a further clinical Phase I study.

Blood cell damage after in vitro irradiation of fresh whole blood with 630 nm laser light.

A study on blood cell damage after irradiation of fresh whole blood with 630 nm laser light was carried out in vitro. Various fluence rates of laser light were used with and without cooling of blood. Damage to the blood was assessed by blood cell counts, osmotic fragility measurements and examination of blood films. Exposure of a 1 mm blood layer to 630 nm laser light without cooling led to changes in blood counts first detected at fluence rates of 130 mW/cm2. Changes in osmotic fragility first became evident at 210 mW/cm2. Increasing cell damage with increasing fluence rates was evident in blood films. Using the cooling device changes in whole blood after irradiation first occurred at a fluence rate of 293 mW/cm2. Measurement of the fluence rates at which cell damage begins is important in laser induced fluorescence diagnostics and photodynamic therapy applications in blood or blood products using photosensitizers.

Gallium-68 chelate imaging of human colon carcinoma xenografts pretargeted with bispecific anti-CD44V6/anti-gallium chelate antibodies.

UNLABELLED: Recently, we demonstrated the feasibility of combining improved tumor-to-tissue contrasts and PET imaging for immunoscintigraphic tumor localization using a multistep targeting technique that consists of the administration of an antitumor/antihapten bispecific monoclonal antibody (BS-MAb), a blocker to saturate the antihapten binding sites of the BS-MAb that are still present in the circulation, and a low molecular weight Ga chelate, labeled with positron emitter 68Ga, serving as the hapten. Due to this technique, the biodistribution of the radiolabeled hapten is governed mainly by the binding characteristics of both the antitumor and the antihapten part of the BS-MAb. For a future clinical implementation of the method, we investigated MAb VFF18, which is reactive with the adhesion molecule CD44V6, a tumor-associated antigen, and up-regulated in colon, squamous cell and pancreas carcinoma, and two anti-Ga chelate MAbs, which are highly selective for only one of the two enantiomers (optical isomers) of the inherently racemic Ga chelate. METHODS: From the VFF18 MAb and the anti-Ga chelate MAbs, two BS-MAbs containing the same antitumor parts, but different antihapten parts, were prepared and tested for multistep targeting in human colon carcinoma-bearing nude mice. RESULTS: Despite identical biodistributions of both BS-MAbs and their very similar affinities for the corresponding Ga chelate enantiomers, tumor uptake of the two enantiomers 1 hr postinjection was significantly different [8.7 +/- 1.9% versus 5.8% +/- 1.6% of the injected dose/g (%i.d./g)], with tumor-to-blood ratios being higher for the BS-MAb showing the lower tumor uptake (7.6 +/- 1.6 versus 4.7 +/- 0.6). From data obtained with each BS-MAb, a similar initial tumor binding of approximately 15.5%i.d./g, but different in vivo half-lives of the corresponding BS-MAb-enantiomer immune complexes, could be estimated. Pretargeting with a mixture of both BS-MAbs followed by the administration of the racemic Ga chelate resulted in the lowest tumor uptake (3.9% +/- 1.5%i.d./g). PET imaging of nude mice with the enantiomeric, as well as with the racemic, 68Ga chelate demonstrated a clear delineation of tumors against blood pool background. CONCLUSION: Multistep immunoscintigraphy with BS-MAbs markedly increases tumor-to-tissue ratios in nude mice and enables PET imaging. Using a BS-MAb containing MAb VFF18, a more sensitive localization of CD44V6-positive tumors in patients should also be obtained.

A chemical dosimeter for the determination of the photodynamic activity of photosensitizers.

Uric acid a known singlet oxygen scavenger, was investigated as a chemical dosimeter in physiological aqueous solution for use in photodynamic therapy. The uric acid test takes the decrease in uric acid (UA) absorbance at 293 nm after laser light irradiation of a solution containing UA and a photosensitizer as a rapid evaluation of relative photodynamic activities of the photosensitizer. A uric acid test standard procedure was defined. To compare photodynamic activity of different photosensitizers or irradiation conditions a proposal for a photodynamic activity scale based on the uric acid test is given. Examples of uric acid test operation are given by comparing the changes in UA absorbance decrease with respect to irradiation wavelength and to photosensitizer concentration of Photofrin II with that of two other photosensitizers (5,10,15,20-tetrakis-[4-hydroxyphenyl]-21H,23H-porphyrin (TOP) and 5,10,15,20-tetrakis-[4-carboxyphenyl)-21H, 23H-porphyrin (TCPP), both derivatized with methoxypolyethyleneglycol (TOP-MPV and TCPP-AMP) as a macromolecular carrier). The photodynamic activity of the three photosensitizers using the proposed photodynamic activity scale is given.

Photodynamic therapy as a tool for suppressing the haematogenous dissemination of tumour cells.

The chance of most cancer patients surviving their disease is to a high degree dependent on the status of the metastatic processes. One general route of cancer-cell dissemination is passive transport in the blood stream, i.e., haematogenous dissemination. In this study we try to find an answer to the following question: is it possible to use photodynamic therapy for suppressing the haematogenous dissemination of cancer cells? In first in vitro experiments we incubated CX1 cells (colon carcinoma cells) with two photosensitizers, Photofrin II and mesotetra(hydroxyphenyl)chlorin (mTHPC). We added the cells to fresh whole blood and irradiated the blood with suitable laser light in a flow-through irradiation system. The tumour-cell survival fraction (SF) was determined with plating efficiency. Using Photofrin II we observed a minimal tumour-cell survival in blood of SF = 3.5% and using mTHPC we measured SF = 0.02%. These results encourage further investigations concerning the use of photodynamic therapy for suppressing haematogenous dissemination.

Antitumor activity of methotrexate-albumin conjugates in rats bearing a Walker-256 carcinoma.

We have recently reported that albumin accumulates in solid tumors and serves there as a source of nitrogen and energy. Methotrexate-albumin conjugates [MTX(I)-RSA] derivatized at a molar ratio of 1:1 differ favorably from original MTX in terms of plasma presence and tumor uptake. The purpose of this study was to evaluate the therapeutic efficacy of these novel conjugates in a comparative study with low m.w. MTX is Sprague-Dawley rats bearing a Walker-256 carcinoma. The maximum tolerated dose (MTD) for MTX and MTX(I)-RSA was determined (2 mg/kg based on MTX injected on days 1, 3 and 8). The tumor-bearing rats received injections of either the MTD or MTD/2 of MTX, MTX-albumin or mixtures containing the MTD/2 or MTD/4 of both MTX and MTX-albumin. No toxic side effects were observed. Cure rate and tumor growth retardation were slightly better for the conjugate compared with MTX alone in the MTD group (16 complete remissions vs. 14 of 20 rats). The best results were achieved for the combination treatment with MTX and MTX-albumin, with complete remission in all 20 rats. In conclusion, MTX-albumin conjugates show therapeutic activity in vivo without toxic side effects. Additive effects were observed for a combination of MTX-albumin and MTX. These effects might be caused by the much longer tumor exposition time of the conjugate in conjunction with a different route of uptake (pinocytosis for MTX-albumin vs. folate receptors for MTX).

Pharmacokinetics of methotrexate-albumin conjugates in tumor-bearing rats.

Linking chemotherapeutic drugs to a macromolecular carrier system may enhance tumor targeting, reduce toxicity and overcome drug resistance mechanisms. As an elementary model to evaluate the pharmacological properties of macromolecular drug carrier systems we chose rat serum albumin (RSA) for carrier and methotrexate (MTX) as antineoplastic drug. The conjugation procedure yielded conjugates with an approximate 1:1 molar loading rate (MTX(1)-RSA). In the first part of the study a residualizing [111In]DTPA protein label was used for mapping in vivo the catabolic sites of the native carrier protein and of the MTX(1)-RSA drug conjugate in Walker 256 carcinosarcoma bearing rats. The tumor accumulation was about 14% of the injected dose for the RSA and MTX(1)-RSA tracers after 24 h. Tracer entrapment by organs with an active mononuclear phagocyte system was low (liver below 7% and spleen below 1.5% of the injected dose after 24 h). The 1:1 conjugation of MTX to RSA did not decisively alter the pharmacokinetic properties nor the tumor or tissue distribution of the native carrier protein RSA. In the second part of the study the different properties of the MTX(1)-RSA conjugate were compared with MTX in vivo. About 2 mg MTX/kg body weight either of the drug conjugate or of the original drug were injected after being additionally spiked with radiolabeled tracers. Plasma concentrations were simultaneously determined by immunological and radioactive means. After 24 h about 12% MTX(1)-RSA was found in circulation compared to 0.03% MTX. Favorable tumor accumulation rates of about 14% were achieved for MTX(1)-RSA versus 0.04% for MTX. About 45-fold more of the injected dose of [3H]MTX accumulated in the liver as compared to the tumor (1.5 versus 0.03%) after 24 h. Conjugation of MTX to RSA reversed this ratio in favor of the tumor to 1:1.4 (13.6 versus 9.6%). In conclusion, the potential therapeutic benefit of the MTX(1)-RSA conjugate lies in its very long tumor exposure time and its improved tumor accumulation rate compared to conventional MTX. In addition the conjugation to albumin might enhance the therapeutic effects over those achieved by long-term continuous infusion of MTX, as MTX(1)-RSA enters the cells by a different uptake mechanism. This might also help to circumvent MTX resistance mechanisms, such as a reduction in folate receptor numbers or impaired MTX polyglutamylation.

The loading rate determines tumor targeting properties of methotrexate-albumin conjugates in rats.

Albumin dominates the plasma proteins in man. Following our observation that albumin turnover in rodent tumors is markedly increased, we will present evidence that albumin can be employed as an efficient carrier for targeting cytostatic agents like methotrexate (MTX) into tumors. The considerable discrepancy in the molecular weight of MTX (454 Da) and albumin (about 67,000 Da) tempted researchers to load multiple drug molecules on one carrier molecule. It was supposed that the optimal therapeutic efficacy of MTX protein conjugates could be achieved by increasing the number of the molecules of MTX attached to the carrier. In this paper we will show that only loading rates of close to 1 mol of the cytostatic drug MTX/mol of albumin offer optimal conditions for targeting MTX-albumin conjugates into rodent tumors. Conjugates bearing 5, 7, 10 and 20 molecules of MTX on average showed considerable alterations in the HPLC profiles of the conjugates compared to albumin. Conjugates carrying 5-20 mol MTX, tagged with a residualizing radiolabel, were efficiently trapped by the liver before reaching the tumor. The tumor uptake rates of these conjugates declined dramatically with an increasing molecular load of the cytotoxic drug linked to albumin. Competition experiments with maleylated bovine serum albumin and fucoidan revealed that scavenger receptors present on the cells of the liver monocyte macrophage system were involved in this process. For further preclinical and clinical studies, we chose MTX-albumin conjugates, derivatized at a molar ratio of 1:1. These conjugates enjoy the same favorable tumor targeting properties like albumin, e.g. high tumor uptake rates, low liver uptake rates and a very long biological half-life.

A new fast-neutron source for radiobiological research.

A biomedical cyclotron facility primarily dedicated to radionuclide production has been extended by the addition of an experimental fast-neutron source for radiobiological and biophysical studies. Several beams of fast-neutrons with different average energies and LET distributions can now be provided. The neutrons are produced by bombarding berryllium targets with 18-32 McV protons or 8.7-15 MeV deuterons from our K = 32 negative-ion cyclotron. Average neutron energies range from approximately 4 to 15 MeV. Doses at maximum build-up vary from 0.24 to 1.85 cGy microA-1 min-1 at 1 m SSD, i.e. approximately 55 cGy min-1 at 30 microA of proton current at maximum energy. The design of the facility and some dosimetric results are presented.

Pharmacokinetic properties of LMW-heparin-tyramine fractions with high or low affinity to antithrombin III in the rat.

We have recently presented evidence that a macrophage scavenger receptor-mediated pathway is responsible for the hepatic uptake of unfractionated heparins and low-molecular-weight heparins (LMWHs) in the rat. The same receptor-mediated pathway was partially responsible for the removal of oxidized low-density lipoprotein. Unfractionated and fractionated LMWHs exert their anticoagulatory effects predominately by reversibly binding to the plasma glycoprotein antithrombin III. In this study LMWHs modified by endpoint attachment of tyramine were radiolabeled and their fractions with low or high affinity to AT-III studied in vivo in rats. The high-affinity fraction was predominately cleared from the circulation by a hepatic uptake mechanism. About 25% of the injected high-affinity tracer material was recovered, whereas only about 8% of the low-affinity material was found in the liver after 180 minutes. Blocking the scavenger receptor-mediated liver RES uptake mechanism by maleylated bovine serum albumin led to a considerable decline in liver uptake (9 versus 25%). The low-affinity material was rapidly eliminated into the urine after 180 minutes. About 45% of the low-affinity material was excreted versus 23% of the high-affinity material. Tight binding to AT-III prevented LMWH-tyramine from being rapidly cleared from the circulation via the kidneys into the urine; instead, the scavenger receptor-mediated hepatic uptake mechanism seemed to be more dominant in removing material with high affinity to AT-III from blood.

A variety of fast neutron beams for radiobiological research.

The design and construction of a new fast neutron facility and first dosimetric results obtained from seven neutron beams are presented. The neutrons are produced by bombarding beryllium targets with protons and deuterons from our K = 32 negative ion cyclotron. The dose rate in air 1 m distance from the thick target within a 13 x 13 cm2 field amounts to about 50 cGy/min at 30 microA of 32 MeV protons.

Evaluation of tumour metabolism and multidrug resistance in patients with treated malignant lymphomas.

The management of patients with treated malignant lymphomas requires functional methods to differentiate a residual soft tissue mass. Patients with treated Hodgkin's lymphoma (HL, n = 20, 68 malignant lesions, three benign lesions) or non-Hodgkin's lymphoma (NHL, n = 26, 46 malignant lesions, one benign lesion) were studied with positron emission tomography (PET) and fluorine-18 deoxyglucose (FDG). Oxygen-15 labelled water was used (n = 14, 25 lesions) in addition to FDG in order to obtain information on the tissue perfusion. Long-term follow-up studies with PET and FDG were performed in nine patients up to 511 days after the initiation of second-line therapy. Fourteen patients underwent single-photon emission tomography (SPET) with technetium-99m sestamibi immediately prior to the first PET examination. PET with FDG displays a high sensitivity for the detection of viable tumour tissue, all the malignant lesions being correctly classified in this study. The possible limitations are inflammatory processes, which may obscure tumour detection due to increased FDG uptake, and malignant lesions with low FDG uptake due to reduced perfusion. Difficulties exist in the prognosis of long-term response, since the change in FDG uptake may be variable. Long-term therapy outcome was correlated with the slope values obtained from the standardized integral uptake (SIU) data, which provides a new approach for the evaluation of PET follow-up studies. 99mTc-sestamibi, which should reflect the multidrug resistance, was evaluated with respect to therapy outcome. A high uptake of 99mTc-sestamibi was observed in patients with stable disease or better. The data support the hypothesis that sestamibi may reflect multidrug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

The injection of heparin prolongs the plasma clearance of oxidized low density lipoprotein in the rat.

There is evidence that oxidized-LDL plays an important role in atherogenesis. We now report on the in vivo interaction between unfractionated heparin and oxidized LDL in rats. The recovery rates of the native LDL particles ranged between 75% and 85% of the injected dose. Heparin did not interfere with the clearance rates of native LDL. After administration of radioactive labeled oxidized-LDL particles, 26% of the material was measured in circulation after 5 minutes, 8% after 20 minutes, and 3% after 60 minutes. After injection of heparin 2 minutes prior to oxidized-LDL tracer particles, 44% of the tracer was found in blood after 5 minutes, 23% after 20 minutes, and 9% after 60 minutes. Oxidized-LDL tracer particles disappeared from blood with an alpha half-life of 5 minutes and a beta half-life of 7.5 minutes. After receptor blocking with unfractionated heparin the alpha half-life of the oxidized-LDL tracer was prolonged to 17.5 minutes and the beta half-life to 27.5 minutes. These results indicate that heparin molecules of a comparatively small molecular weight competed the scavenger receptor mediated uptake of oxidized-LDL particles in vivo. Oxidized-LDL particles are known to mediate their pro-atherosclerotic activity in part by stimulating smooth muscle cell proliferation by a scavenger receptor-mediated pathway. It can be speculated, if heparins interfere with the uptake of oxidized-LDL, heparins might thus in part exert their known antiatherosclerotic properties.

Complexes of a modified low-molecular-weight heparin with protamine are predominantly cleared by a macrophage scavenger receptor-mediated process in rats.

Neutralization of heparin with its antidote protamine is associated with side effects such as pulmonary hypertension. The pharmacodynamic effects of protamine treatment are well documented. However, little is known about metabolic fate of heparin-protamine complexes. Twenty Wistar rats received a 131I-labeled low-molecular-weight heparin tracer intravenously. Four groups of animals were formed: a control group receiving the tracer, a second group receiving protamine after tracer application, a third group receiving maleylated bovine serum albumin (mal-BSA) prior to tracer and protamine injections to inhibit scavenger receptors of the reticuloendothelial system, and the last group receiving preformed heparin-protamine aggregates. All animals were examined by scintigraphy. Blood and tissue samples were analyzed for radioactivity. Protamine injection 2 min after heparin tracer application lead to a rapid decline in blood radioactivity. The radioactivity in the liver increased from 17% for the control to 43% after protamine application. Injection of mal-BSA prior to protamine prevented tracer accumulation in the liver and increased urine excretion (34% versus 20%). In vitro preformed heparin-protamine precipitates were rapidly trapped in the liver. We present evidence that, like the polyanionic heparin, polyanionic heparin-protamine complexes are phagocytosed by a scavenger receptor-mediated mechanism by macrophages, predominantly in the liver. The amount, the size, and the charge density of the complexes might trigger a mediator release from macrophages leading to phenomena such as pulmonary hypertension.

Multistep tumor targeting in nude mice using bispecific antibodies and a gallium chelate suitable for immunoscintigraphy with positron emission tomography.

To improve tumor:tissue ratios in immunoscintigraphy, a three-step targeting method has been developed. The reagents used were (a) a radioactive, low molecular weight chelate prepared from ionic gallium and a phenolic polyaminocarboxylic acid, which can be labeled either with the single-photon emitter 67Ga or with the short-lived positron emitter 68Ga (t1/2 = 68 min); (b) a bispecific monoclonal antibody (bs-mAb) synthesized from the F(ab)2 fragment of the 1.1ASML antibody specific for the glycoprotein CD44v associated with a rat pancreas carcinoma cell line and the F(ab') fragment of an antibody specific for the gallium chelate; and (c) the nonradioactive gallium chelate covalently coupled to transferrin, which served as a high molecular weight blocker to prevent binding of the radioactive gallium chelate to bs-mAbs in the circulation. Targeting experiments in tumor-bearing nude mice with different doses of bs-mAbs, blocker, and 67Ga chelate were adjusted to maximize tumor to tissue contrasts and tumor uptake. Compared with the biodistribution of the 131I-labeled, native 1.1ASML antibody 24 h postinjection, a schedule using 100 pmol bs-mab 24 h later 100 pmol blocker, 15 min later 16 pmol 67Ga chelate, 1 h later examination, increased tumor:blood and tumor: liver ratios by a factor of 5 while keeping the localization of radioactivity in the tumor constant (10.1% injected dose/g). High-contrast images using either 67Ga or 68Ga were obtained within 1 h. The targeting method described enables the use of the short-lived positron emitter 68Ga and thus allows the combination of an improved immunoscintigraphy and positron emission tomography.

Altered pharmacokinetic properties of a lipophilically derivatized low-molecular-weight heparin in rats.

A new generation of lipophilic heparins has been developed that show longer-lasting inhibitory effects on the coagulation system. We have studied the radiopharmacokinetics of a derivatized low-molecular-weight heparin (LMWH) with a residualizing lipophilic tyramine-deoxysorbitol label in comparison with conventional LMWH after intravenous application into Wistar rats. Whole body scintigraphy and analysis of the blood and organ distribution of different tracer preparations revealed that the lipophilically derivatized LMWH substance was predominantly trapped in the liver RES by a scavenger receptor-mediated mechanism. After the saturable uptake mechanism was blocked by maleylated bovine serum albumin, 41.4% of the lipophilic LMWH tracer circulated in blood, as compared with 18.4% of the control and 1% of conventional LMWH. The same results were attained by a competition experiment with an excess of unfractionated heparin. Urinary excretion of the lipophilic tracer among the rats in this competition experiment was considerably lower (13.7%) as compared with conventional LMWH (53.0%). Experiments with lipophilic LMWH tracer bound nonspecifically to rat serum albumin confirmed that the prolonged half-life might in part be due to an increased affinity for albumin. About 59% of the activity of the lipophilic tracer bound to albumin was found in the liver reticuloendothelial system, and only 3.3% were excreted to urine 3 hours after injection. Further studies are necessary to evaluate the accumulation rates and the metabolic fate of lipophilically derivatized heparins in the case of an impeded reticuloendothelial system uptake before attempts are made to therapeutically apply these compounds.

Stereotactically guided fractionated radiotherapy: technical aspects.

A system for high precision radiotherapy in the head and neck region has been developed. The components of the system are a head mask connected to a stereotactic frame, a localization unit that can be used during CT- and MR-imaging and a stereotactic target positioner. Conformal precision radiotherapy is planned with a new treatment planning system (Voxelplan-Heidelberg). Three different multi-leaf collimator systems are used. An evaluation of the precision and accuracy of the head fixation system, which was performed with a photogrammetry system, is presented.

A bifunctional HBED-derivative for labeling of antibodies with 67Ga, 111In and 59Fe. Comparative biodistribution with 111In-DPTA and 131I-labeled antibodies in mice bearing antibody internalizing and non-internalizing tumors.

To investigate whether bifunctional ligands containing chelating structures other than EDTA and DTPA and metallic radiotracers other than 111In will reduce the non-specific radioactivity uptake in the liver during immunoscintigraphy, we synthetized an isothiocyanato-substituted phenolic polyaminocarboxylic acid (HBED-CI) for labeling of MAbs with 67Ga, 111In and 59Fe. Biodistribution of HBED-CI-labeled MAbs was compared to that of 131I and 111In-DTPA labeled MAbs in nude mice bearing tumors, which differ with regard to intracellular internalization and catabolism of the corresponding MAb-antigen complex. In the liver a continuous radioactivity excretion for 67Ga-HBED-CI-labeled MAbs was observed with kinetics that parallel 131I clearance after administration of 131I-MAbs, while 111In-HBED-CI-labeling led to a constant 111In liver level quite similar to that of 111In-DTPA-MAbs. In tumors, 67Ga-HBED-CI-MAb uptake again paralleled that of 131I-MAbs, showing continuous accumulation in tumor tissues when internalization of the MAb-antigen complex was not involved. A much lower uptake, which peaked between 24 and 48 h, was found in the case of MAb-antigen internalization. 111In of 111In-HBED-CI- and 111In-DTPA-labeled MAbs continuously accumulated in both types of tumors. Compared with 111In-DTPA-MAbs, an improvement in tumor-to-liver ratios, due to the reduced liver radioactivity associated with 67Ga-HBED-CI-labeled MAbs, could only be obtained with non-internalizing tumors. The time course of radioactivity distribution in the liver and in MAb-internalizing tumors after administration of 67Ga-HBED-CI-, 111In-HBED-CI- and 111In-DTPA-labeled MAbs further indicates a dominating influence of the metallic radiotracer rather than the ligand on retention or excretion of radioactivity in MAb-catabolizing tissues.