Magnetic resonance imaging of nude mice with heterotransplanted high-grade squamous cell carcinomas: use of a low-loaded, covalently bound Gd-Hsa conjugate as contrast agent with high tumor affinity.

RATIONALE: Malignant tumors often show an increased uptake and metabolism of plasma proteins, especially albumin. OBJECTIVES: Determine whether the accumulation of low loaded Gd-albumin improves visualization of malignant tumors by MRI. METHODS: Twelve nude mice with heterotransplanted squamous cell carcinomas were studied. The signal intensity of tumor, blood, liver, kidney and muscle tissue was studied in MR images after application of Gd-albumin during a period of 144 hours. MRI results were histologically correlated after simultaneously injection of Gd- and fluorescein-labeled albumins in 9 nude mice. RESULTS: Although liver and kidney had a maximum increase in signal intensity within 30 minutes, tumors showed a delayed 51% increase in the 24 hours after application. Histologic and fluorescence evaluation demonstrated albumin localization in tumors predominantly in stroma and necroses. CONCLUSIONS: Gd-albumin is efficiently accumulated in SCC transplants. MRI with low loaded Gd-albumin may offer relevant opportunities for recognizing tumors sensitive to a therapy with cyostic drug-labeled albumins.

Mode of action of methotrexate-albumin in a human T-cell leukemia line and activity against an MTX-resistant clone.

Limitations of low mol. wt anticancer drugs are short tumor exposure times and toxicity to normal tissue. Methotrexate (MTX) covalently linked to human serum albumin (HSA) as a macromolecular carrier caused tumor regressions concomitant with a favorable toxicity profile in a clinical phase I trial (Hartung et aL, Clin. Cancer Res., 1999, 5, 753). We examined the uptake, intracellular degradation, metabolism and thymidylate synthase (TS) inhibition of MTX-HSA in the T-cell leukemia line CCRF-CEM and the MTX transport resistant clone CCRF-CEM/MTX. The number of MTX molecules per albumin molecule was determined by electrospray mass spectrometry. A loading ratio (LR) of approximately 1.4 mol MTX/albumin revealed intact complexes with one and two MTX molecules/albumin. In the complex with an LR of 5.7, albumin with up to 16 MTX molecules was seen. MTX-HSA was taken up by CCRF-CEM cells via endocytosis and cleaved by lysosomal enzymes. Liberated MTX was a poor substrate of folylpolyglutamate synthetase and was exported into the medium. TS was inhibited and cell survival was impaired by MTX-HSA in a time- and concentration-dependent manner. CCRF-CEM/MTX cells exhibited a growth inhibition of 30-40% after MTX-HSA treatment, but no TS inhibition. The alternative uptake route via endocytosis enables MTX-HSA to overcome transport resistance to MTX.

Albumin-based drug carriers: comparison between serum albumins of different species on pharmacokinetics and tumor uptake of the conjugate.

Albumin-based drug carrier systems have been developed in the field of chemotherapy to improve the passive tumor targeting properties of anti-cancer drugs. Usually, serum albumins of different species are used as carrier proteins, mostly of bovine (BSA), human (HSA) or rat (RSA) origin. The resulting albumin conjugates are often tested for anticancer activity in heterologous tumor models. No data is available whether the choice of the albumin species might influence the pharmacokinetics or the tumor uptake rates of the conjugates in vivo. Residualizingly ([111In]DTPA) radiolabeled RSA, BSA or HSA were administered to Walker-256 carcinoma-bearing rats. No significant difference was found in the absolute or the weight-adjusted tumor uptake rates of the three albumin tracers. The tumors were the major catabolic sites accumulating 14-18% of the injected dose (ID). Low hepatic uptake rates were determined for all albumins (below 100% ID). Minor differences were found for hepatic uptake in favor of the autologous RSA (5.8% ID) versus HSA (6.9%) and BSA (8.0%). These differences might have occurred during the commercial preparation or the radiolabeling of the different batches. In addition, there are structural differences between the three albumins, which might have contributed, despite high sequence homologies above 70% for RSA, HSA and BSA. These minor differences in the distribution patterns of RSA, HSA or BSA might not decisively influence the results of drug targeting experiments in rats. For further studies with albumin conjugates, HSA was chosen as drug carrier in rodent animal models when considering later human use. In rats or nude mice multiple injections of various HSA-drug conjugates were well tolerated without signs of allergy or anaphylaxis.

Methotrexate-albumin conjugate causes tumor growth delay in Dunning R3327 HI prostate cancer-bearing rats.

Based on the rationale of a preferred albumin uptake by tumors, conjugates comprising of rat serum albumin (RSA) as a drug carrier and of methotrexate (MTX) as chemotherapeutic drug were prepared. For a comparative study of MTX-RSA and MTX we chose a slow growing Dunning R3327 HI prostate cancer model. In a radiopharmacologic study blood kinetics and the tumor and organ distribution pattern of residualizingly labeled MTX-RSA were determined, and were found to be similar to that of residualizingly labeled RSA. The MTD was established for Copenhagen rats at a total four injections of 2 mg/kg MTX or MTX-RSA administered at days 0, 4, 8 and 12. Tumor volume measurements and tumor removal showed a small non-significant growth delay in the MTX treatment group, suggesting MTX resistance for the Dunning R3327 HI prostate carcinoma. In contrast, treatment with MTX-RSA resulted in a significant (50%) growth inhibition of the Dunning R3327 HI tumor. The cellular mechanisms responsible for MTX resistance in Dunning HI tumor cells is not known. The improved therapeutic effects seen during MTX-RSA treatment in this slow growing adenocarcinoma might be a result of prolonged tumor exposure time and an altered cellular uptake by a lysosomal route. MTX-albumin conjugates have shown antitumor activity exceeding that of MTX in several tumor xenografts in nude mice, including human prostate cancer. The recently initiated clinical development of MTX-human serum albumin will be continued and cancer of the prostate will be included as a potential target tumor during further clinical phase II testing.

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.

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.

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.

Serum albumin (SA) accumulation by bronchogenic tumours: a tracer technique may help with patient selection for SA-delivered chemotherapy.

Systemic toxicity and inadequate tumour uptake of chemotherapeutic agents limit effective therapy of disseminated malignant disease. We seek to use macromolecules for improved delivery of therapeutic agents to tumours, and hope to use radiotracer procedures to identify those malignancies able to accumulate the transport molecule. A literature search identified in vitro and animal experimental data which indicated that serum albumin is taken up in malignancies. Selected cytostatic drugs can be bound to albumin, which suggests the suitability of the molecule as a potential transport vehicle. We therefore evaluated indium-111 labelled human serum albumin (HSA) to determine the frequency of its accumulation in bronchogenic tumours. Single-photon emission tomographic (SPET) images were obtained in 23 patients 48 h after intravenous injection of 1.5 mCi 111In diethylenetriamine penta-acetic acid (DTPA)-HSA. SPET imaging with technetium-99m labelled erythrocytes was included in the protocol to assess the influence which vascularity has on the HSA-based images. All patients went on to surgery. We documented the histological diagnosis, T-stage and differentiation grade. The scintigraphic examination demonstrated HSA uptake in three squamous cell carcinomas and four adenocarcinomas. Of these, six malignancies accumulating HSA had 2.2-5.4 times, the tracer concentrations observed in comparable background regions. Small cell carcinoma failed to accumulate the labelled HSA during the 2-day scintigraphic evaluation. The HSA images did not appear to represent tumour vascularity. T-stage and differentiation grade failed to predict which tumours would demonstrate HSA uptake.(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.

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.

Design of compounds having enhanced tumour uptake, using serum albumin as a carrier--Part II. In vivo studies.

In the present in vivo study the uptake kinetics of radioiodinated albumin were determined in normal organs, and tumours of rats using sequential scintigraphy. Rat serum (RSA) was radioiodinated either directly at a tyrosine residue (d-RSA), or indirectly at a residualizing marker tagged to the albumin (rm-RSA). These labelling procedures did not alter the kinetics of labelled albumin, as shown by blood disappearance curves. Directly labelled albumin was shown to have tumour uptake. Residualizing markers like tyramine-cellobiose (TCB), tyramine-deoxysorbitol (TDS) and aminonaphthaltyrimide-deoxysorbitol (ANTDS) are metabolically inert. After the intracellular degradation of the albumin carrier the TCB-, TDS- and ATNDS-residues accumulate in the lysosomes, particularly those of tumour cells. It was able to be demonstrated that residualizing-marker tagged albumin-bound radioactivity was five times higher after 72 h than the tumour radioactivity after use of directly labelled RSA. These data found support when whole-body retention of directly labelled RSA, and residualizing marker-RSAs, were determined. After 72 h, 60% of 131I bound to RSA directly had been excreted, compared to only 25% of the activity attached indirectly to RSA with a residualizing marker. Whole-body autoradiography of rats injected with directly labelled RSA, or residualizing marker-RSA, support these results. Most of the radioactivity of directly labelled RSA was excreted within 24 h, whereas labelled residualizing marker-RSAs were also stored in tumour and liver tissue. ANTDS bound to RSA allows fluorescence microscopy. Cryosections of tumours from rats preinjected 10 min and 24 h with ANTDS-RSA before dissection, demonstrated that the fluorescence is localized on and in tumour cells. This indicates that cellular uptake of the marker takes place. Fluorescence was not observed in muscle tissue. This appears to suggest that the albumin uptake is greater in tumours than in normal tissue, and that it is metabolized in the tumour cells.

Design of compounds having an enhanced tumour uptake, using serum albumin as a carrier. Part I.

The search for a radioiodinated "cumulative" protein label, stored within cells following intracellular protein degradation, suggested that plasma protein turnover of tumours might be of use. While earlier investigators were primarily interested in metabolism and utilization of plasma proteins by tumours, we tried to utilize the tumour protein turnover to channel radioiodine labelled compounds, covalently bound to serum albumin, into neoplastic tissues. To identify those parameters which influence the tumour uptake and storage, we investigated a series of compounds having different chemical and physicochemical properties. Unbound, small molecular weight compounds were rapidly eliminated from the circulatory system. They had a prolonged biological half life if linked to serum albumin (SA), especially when derivatized with deoxysorbitol. Parallel with the prolongation of the biological half-life we noted a remarkable increase in tumour uptake, which was not accompanied by increased liver activity. Furthermore, without thyroid blockade, we failed to detect significant radioiodine uptake in this organ after 24 or 72 h. This is due to the particular coupling mechanism, which may be relevant for other (radio)iodinated pharmaceuticals used in medicine. Glucose and aromatic amines, as well as aromatic aldehydes and glucamine react to form deoxysorbitol derivates, which then have similar biokinetics after linkage to serum albumin. This indicates that a new approach in tumour detection and possibly in tumour therapy may be possible when SA is used as a carrier molecule, using the described labelling procedure.

Radioiodination of proteins and lipoproteins using N-bromosuccinimide as oxidizing agent.

In an attempt to improve conditions for radioiodination of sensitive proteins we used N-bromosuccinimide as a mild oxidizing agent. Under gentle conditions we increased the average labeling efficiency of a wide variety of proteins to above 97%. There was no loss of binding activity of low density lipoprotein particles, which are most sensitive to oxidation. Depending on high labeling efficiency, our method reduces preparation time as well as radioactive waste, costs, and irradiation exposure to personnel.

A new radioiodine exchange labeling technique.

A new technique of radioiodine exchange labeling is presented. The method was developed with respect to the short physical half lives of 123I and 121I, the altered activity concentrations and varying impurities, which hamper the previously introduced labeling methods. The described "one step reaction" is applicable to a series of organic compounds with different physicochemical and physiological behaviour as well as to all radioisotopes of iodine.