Hannsjörg Sinn

Albumin-Based Drug Delivery as Novel Therapeutic Approach for Rheumatoid Arthritis

We reported recently that albumin is a suitable drug carrier for targeted delivery of methotrexate (MTX) to tumors. Due to pathophysiological conditions in neoplastic tissue, high amounts of albumin accumulate in tumors and are metabolized by malignant cells. MTX, covalently coupled to human serum albumin (MTX-HSA) for cancer treatment, is currently being evaluated in phase II clinical trials. Because synovium of patients with rheumatoid arthritis (RA) shares various features observed also in tumors, albumin-based drug targeting of inflamed joints might be an attractive therapeutic approach. Therefore, the pharmacokinetics of albumin and MTX in a mouse model of arthritis was examined. Additionally, uptake of albumin by synovial fibroblasts of RA patients and the efficacy of MTX and MTX-HSA in arthritic mice were studied. The results show that when compared with MTX, significantly higher amounts of albumin accumulate in inflamed paws, and significantly lower amounts of albumin are found in the liver and the kidneys. The protein is metabolized by human synovial fibroblasts in vitro and in vivo. MTX-HSA was significantly more effective in suppression of the onset of arthritis in mice than was MTX. In conclusion, albumin appears to be a suitable drug carrier in RA, most likely due to effects on synovial fibroblasts, which might increase therapeutic efficacy and reduce side effects of MTX.

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

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 [111ln]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.

Pre-clinical evaluation of a methotrexate–albumin conjugate (MTX-HSA) in human tumor xenografts in vivo†

Methotrexate covalently bound to human serum albumin in a 1:1 molar ratio (MTX‐HSA) is a new macromolecular drug which is currently being studied in phase I clinical trials by the German Association for Medical Oncology (AIO) Phase I/II study group. Previous studies have shown that MTX‐HSA differs favorably from unbound MTX in terms of plasma half‐life time, tumor accumulation of albumin and uptake mechanisms into cancer cells. To achieve optimal drug efficacy, repeated treatment cycles were necessary. To evaluate the anti‐tumor activity of MTX‐HSA and MTX in pre‐clinical in vivo models, we selected 7 solid human tumor xenografts growing s.c. in nude mice and administered drug either i.p. or i.v. weekly for 3 weeks. The maximal tolerated dose (MTD) of MTX‐HSA in nude mice was 12.5 mg/kg given i.p. on days 1, 8 and 15, whereas the MTD for free MTX was 100 mg/kg given i.v. MTX‐HSA was significantly more active (p > 0.01) than MTX in 3 models. In the soft tissue sarcoma SXF 1301, MTX‐HSA effected complete remission/cure after a single injection, whereas free MTX resulted in short‐lasting, partial tumor regression. In the prostate‐cancer model PRXF PC3M, MTX‐HSA produced growth inhibition of 92.8% of control or an optimal test/control (T/C) of 7.2% compared to a T/C of 20.8% for MTX (p = 0.05). In the osteosarcoma model SXF 1410, optimal T/C values were 10.2% and 14.5%, respectively (p = 0.025). In lung cancers LXFE 409 and LXFL 529, bladder cancer BXF 1258 and breast cancer MAXF 449, both compounds were inactive. The improved therapeutic effects seen in 3 xenograft models under MTX‐HSA treatment are promising and might be due to specific accumulation of the compound in solid tumors owing to their enhanced permeability and retention effect. Thus, clinical development of MTX‐HSA will continue and sarcomas as well as prostate cancers will be included as potential target tumors for upcoming clinical phase II trials.

Albumin targeting of damaged muscle fibres in the mdx mouse can be monitored by MRI

Increased sarcolemmal permeability has been implicated as a major pathological event in muscular dystrophies. In our study, we evaluated whether damaged muscle fibres can be specifically targeted using albumin as a carrier. We tagged human serum albumin (HSA) with Gadolinium (Gd) and systemically applied this compound (Gd-DTPA-HSA) to wildtype and dystrophin-deficient mdx mice. We performed magnetic resonance imaging before and after intravenous administration of Gd-DTPA-HSA and found localised signal enhancement only in mdx skeletal muscle. We also examined skeletal muscle after contrast enhanced magnetic resonance imaging using anti-human albumin antibodies and demonstrated intracellular accumulation of Gd-DTPA-HSA in clusters of damaged mdx muscle fibres. Comparison of magnetic resonance imaging and histological data emphasised the value of contrast agent enhanced magnetic resonance imaging for the in vivo assessment of fibre damage in muscular dystrophies. Furthermore, our data provide evidence that albumin can be used as a carrier to target covalently bound molecules to degenerating muscle fibres.

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.

Tumour targeting with antibody-coupled liposomes: Failure to achieve accumulation in xenografts and spontaneous liver metastases

SummaryThe potential of small unilamellar liposomes coupled to anti-tumour monoclonal antibodies (immunoliposomes) to accumulate in solid tumour tissue was tested in two systems, i.e. a human malignant melanoma xenografted into nude mice and a syngeneic murine lymphoma ESb.Mp exhibiting spontaneous metastasis to the liver. Both monoclonal antibodies tested were partly released from immunoliposomes within a few hours, thus generating a seemingly constant level of circulating antibody. Nevertheless it was possible to follow the biodistribution of intact immunoliposomes by virtue of a radioiodine label incorporated into the lipid moiety. It was found that in both tumor systems, though they differed with respect to the size of lesions and maybe also to the vascular architecture of surrounding tissue, immunoliposome uptake was virtually nil. The blockade of uptake into solid tumour tissue was caused by the limited availability of immunoliposomes due to their moderate stability, but especially by the inability of the particulate carrier to extravasate.

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(1)‐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 in Sprague‐Dawley rats bearing a Walker‐256 carcinoma. The maximum tolerated dose (MTD) for MTX and MTX(1)‐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). Int. J. Cancer76:884–890, 1998.© 1998 Wiley‐Liss, Inc.

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.

Efficacy and tolerability of an aminopterin-albumin conjugate in tumor-bearing rats.

The antifolate aminopterin (AMPT) was developed before methotrexate (MTX), but was not clinically established or generally used due its increased toxicity compared to MTX. Recently, we reported on the increased metabolism of albumin conjugates such as methotrexate–albumin (MTX–SA) in malignant tumors and the feasibility of using albumin as a carrier for drug targeting. Consequently, AMPT was covalently bound to serum albumin (AMPT–SA) at a 1:1 molar ratio. Biodistribution, tolerability and efficacy of this novel conjugate were studied in Walker-256 (W-256) carcinoma-bearing rats. As compared to native albumin, the same biodistribution and plasma clearance were found for AMPT–SA, which achieved 20.1% tumor uptake (estimated uptake per g tumor 6.4%) within 24 h after i.v. administration in rats. In a randomized study, AMPT–SA, repeatedly i.v. injected, was compared with low-molecular-weight AMPT. Depending on the molar concentration, the maximum tolerated dose (MTD) of AMPT covalently bound to SA was twice that of unbound AMPT (three repeated injections of 1.0 mg AMPT–SA/kg body weight versus three repeated injections of 0.5 mg AMPT/kg body weight;p =0.0006). Efficacy was studied at the level of the MTD and MTD/2, and demonstrated that AMPT–SA was significantly more active. At the MTD/2 in W-256 carinoma-bearing rats, AMPT–SA achieved a 100% volume reduction and an optimal volume reduction during treatment/control (T/C) of 8.3% compared to a 53% volume reduction of AMPT and a T/C of 16.5% (p =0.032). Tumor relapses were reduced and occurred later in the AMPT–SA group (two tumor recurrences for AMPT–SA versus seven for AMPT;p =0.05). In this comparative study, the AMPT–SA conjugate showed high antitumor activity in vivo and a favorable toxicity compared to low-molecular-weight AMPT. These effects are attributed to the albumin carrier which seems to be an effective tool for selective tumor drug targeting.