Dieter L. Heene

Subcutaneous Low-Molecular-Weight Heparin versus Standard Heparin and the Prevention of Thromboembolism in Medical Inpatients

In a multicenter, double-blind clinical trial in 1,968 inpatients 1 daily subcutaneous administration of LMW heparin plus 2 placebo injections or 3 x 5,000 IU unfractionated (UF) heparin was given for 10 (8-11) days. The primary end point was the incidence of proximal deep-vein thrombosis or pulmonary embolism. Patients were assessed during the study period for development of proximal deep-vein thrombosis by compression sonography at days 1 and 10 and for pulmonary embolism by scintigraphy in symptomatic patients. Aim of the study was to demonstrate the equivalence of both treatment regimens. A total of 1,968 patients were randomized to receive UF or LMW heparin. Of these, 378 patients were excluded during the study period, so that 780 patients on UF and 810 on LMW heparin were included in the efficacy analysis. Four primary end points were observed with UF and 6 with LMW heparin, demonstrating the equivalence of treatments (p = 0.012). Additionally, pulmonary embolism was suspected as the cause of death in 6 patients who died during the study (3 per treatment group). A higher frequency of death (n = 32) was observed in the LMW-heparin group (p = 0.02) particularly documented in a part of the centers. Safety analysis showed a higher frequency of local pruritus, local erythema and subcutaneous hematoma, a higher increase in plasma levels of triglycerides, total cholesterol, alanine aminotransferase and aspartate aminotransferase, and a decrease of antithrombin III in patients receiving UF heparin. A decrease in platelet count (values ranging between 40,000 and 80,000/microliter) was observed in 4 patients with UF and in none with LMW heparin. No severe thrombocytopenia was observed. Subcutaneous LMW heparin is as effective as UF heparin for prophylaxis of thromboembolism in bedridden, hospitalized medical patients.

Randomized controlled study of heparin and low molecular weight heparin for prevention of deep-vein thrombosis in medical patients.

166 patients aged 40-80 years were included in a controlled, randomized, double-blind study to determine the efficacy and safety of a single daily injection of a low molecular weight (LMW) heparin for prevention of deep-vein thrombosis compared to low dose conventional heparin. Patients received 1 x 1.500 aPTT units of a LMW heparin fraction (plus 2 x placebo injection) or 3 x 5.000 IU of an unfractionated heparin. During 10 days of treatment, patients underwent repeated clinical investigation, serial impedance plethysmography, and Doppler sonography for detection of thrombosis of the lower limbs. Combined application of these methods revealed evidence of thrombosis in 4.5% of patients on unfractionated heparin and 3.6% of patients on LMW heparin. Subcutaneous hematomas were significantly smaller in diameter upon treatment with LMW heparin (p less than 0.001). Antithrombin III levels were significantly higher at the end of the observation period in the LMW heparin group (p less than 0.005). Thrombocyte count, transaminases, creatinine, and haemoglobin did not change in either group. The results indicate that LMW heparin administered by a single s.c. dose daily may be as effective as low dose heparin in prevention of deep venous thrombosis in medical inpatients.

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.

Therapeutic Application of Subcutaneous Low-Molecular-Weight Heparin in Acute Venous Thrombosis

Fifty patients presenting with acute deep-vein thrombosis were randomized in a prospective, controlled study to determine the safety and efficacy of a treatment with low-molecular-weight (LMW) heparin compared with unfractionated heparin. LMW heparin (n = 24) was administered twice daily subcutaneously at a dose of 2 X 150 anti-Xa units/kg body weight, and unfractionated heparin (n = 26) was given intravenously by continuous infusion at a dose of 450 anti-Xa units/kg body weight daily for 10 days. Doses were adjusted to maintain peak anti-Xa levels between 0.5 and 1.0 anti-Xa units per milliliter. One patient in the unfractionated heparin group and 2 patients in the LMW heparin group suffered from bleeding complications. Two patients on LMW heparin and on unfractionated heparin had high evidence of pulmonary embolism based on defects on ventilation-perfusion scintigraphy. Control phlebography and duplex sonography demonstrated a significant improvement during both treatment regimens. Reperfusion of the deep-vein system was 70% with LMW heparin and 75% with unfractionated heparin. The anti-Xa levels were significantly higher in the LMW heparin group, and activated partial thromboplastin and thrombin clotting times were significantly higher in the group receiving unfractionated heparin. Thrombin-antithrombin III complexes and D-dimer concentration decreased during the treatment, but did not differ between the two regimens. At the end of the treatment period with LMW heparin, protein C and antithrombin III were significantly higher.

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.

Chromatographic and electrophoretic applications for the analysis of heparin and dermatan sulfate

Heparin and dermatan sulfate are highly sulfated polydisperse glycosaminoglycans. The methods to determine such compounds include chromatographic and electrophoretic techniques. Here we report on the performances of various analytical methods for the characterization and the determination of GAGs. Heparin, low-molecular-mass heparins, dermatan sulfate and low-molecular-mass dermatan sulfate were analyzed. High-performance size exclusion chromatography was used to determine the molecular mass, polydispersity, absorbance and the area under the absorbance-time curve. Polyacrylamide gel electrophoresis was used to determine the average molecular mass and the polydispersity. Heparin and dermatan sulfate preparations were analyzed by capillary electrophoresis using reversed polarity. The results obtained reflect different performances of various analytical methods used to characterize GAGs.

Continuous Measurement of Hemodynamic Alterations During Pharmacologic Cardiovascular Stress Using Automated Impedance Cardiography

The contribution of computerized impedance cardiography in monitoring and differentiating cardiovascular responses to pharmacologic stress after the administration of dipyridamole (group 1, n = 24) or dobutamine (group 2, n = 26) was investigated during stress echocardiography. Heart rate, stroke volume index, cardiac index and systemic vascular resistance index were evaluated continuously with an automated, computerized, signal‐averaged impedance cardiography system. Dipyridamole had little average effect on heart rate, stroke volume index, and cardiac index. The responses were similar in patients with positive (n = 9) or negative (n = 15) stress echocardiography test results (as characterized by echocardiographic wall‐motion abnormalities). Dobutamine induced a similar mean increase in heart rate in patients with negative (n = 13) or positive (n = 13) results on stress echocardiography. The mean increase in stroke volume index induced by dobutamine was greater in patients with negative stress echocardiography test results than in patients with stress‐induced wall‐motion abnormalities. This distinction was also seen in the cardiac index; the mean change in patients with negative stress echocardiography test results was larger than in patients with positive results. It is concluded that automated computerized impedance cardiography not only allows surveying and monitoring hemodynamic changes during pharmacologic stress echocardiography but also contributes to differentiation of pathologic stress responses.

High-resolution capillary electrophoresis and polycrylamide gel electrophoresis of heparins

Abstract Heparin-oligosaccharides, low-molecular-mass heparins (LMMHs) and heparins were determined using high-resolution capillary electrophoresis (HPCE) and polycrylamide gel electrophoresis (PAGE). The conditions for HPCE were 20 mM phosphate buffer (pH3.5) and 20°C. The method was equivalent to that used previously with a borate-sodium dodecyl sulfate buffer (pH8.8). Six LMMHs were determined using PAGE. The method showed a standard deviation of the average molecular mass from 6.4 to 19.8% and of their polydispersity from 0.7 to 10.2%. HPCE and PAGE revealed different important structural and compositional differences of heparins.

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.