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Biomedical subjects

D L Heene

Publications and source records attributed to D L Heene.

At least 37 records · Page 2Linked to original sources
PubMed1999

A 96-well microfiltration assay for measurement of total clottable fibrinogen.

In clinical routine use, fibrinogen is measured by clotting-time methods, or by clot turbidity in photometric prothrombin time determination. For calibration of these assays measurement of total thrombin-clottable protein has been recommended. We have now developed a microfiltration assay for total thrombin-clottable protein. Plasma samples were mixed with thrombin in a 96-well microfiltration device. After clot formation, the fluid was extracted by vacuum suction, and fibrin adherent to the filter membranes washed with buffer. Membrane segments with adherent fibrin were recovered from the 96-well manifold with a punch and transferred to tubes containing denaturing buffer solution. After dissolution of fibrin, protein concentration was determined by optical absorption at 280 nm. The microfiltration assay displayed a high correlation with the total clottable protein method (R = 0.95), and fibrinogen antigen (r = 0.96). Correlation with clotting time assays, and PT-derived fibrinogen in 150 clinical plasma samples was in the range of r = 0.84 to r = 0.97. Intraassay and day-to-day variability of the assay was comparable to the conventional total clottable fibrinogen assay. The novel microfiltration assay appears to be well suited for measurement of large series of samples for calibration, screening purposes, and clinical trials.

Blood Coagulation↗
PubMed1999

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.

Adolescent↗
PubMed1999

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.

Animals↗
PubMed1998

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).

Albumins↗
PubMed1998

Evaluation and validation of a Crohn's disease inflammatory activity index reflecting pattern of endoscopic severity.

BACKGROUND: This study was designed to investigate objective variables assessing the inflammatory activity of Crohn's disease accessible for routine management and their suitability to act as surrogate indicators for endoscopic alterations. METHODS: Thirty-six patients were included in a prospective study and underwent endoscopic investigation, 18 with clinically exacerbated disease and 18 after acute-phase conservative therapy. The endoscopic findings were classified to define the dependent variable, yielding two categoric levels: acute active disease and remission. The extent of affected mucosal area was not taken into consideration. The serum variables alpha1-antitrypsin, acid alpha1-glycoprotein (AGP), C-reactive protein (CRP), sialic acids, prealbumin (PAB), and albumin were used as independent variables to predict the dependent variable. To compare the results with common indices, the Crohn's Disease Activity Index (CDAI) and van Hees Index were calculated. RESULTS: The following model was calculated by stepwise logistic regression analysis: if AGP (mg/dl) -4.2 x PAB (mg/dl) > or =0.8, then endoscopically active disease will be predicted with a sensitivity of 100% and a specificity of 95% (P < 0.001). The predictive values of the single variables, CDAI, and van Hees Index were lower. For validation of results an analogous study was performed including 44 patients, 29 with active disease and 15 controls. The existence of the model was confirmed, again showing high values for sensitivity (86%) and specificity (100%). CONCLUSIONS: On a qualitative level focusing on clinical relevance, the endoscopic and biologic findings of Crohn's disease are highly associated. In addition to clinical assessment, usage of the developed index as a rationale contributing to therapeutic decisions in the short- and long-term management might be reasonable.

Biomarkers↗
PubMed1997

Evaluation of signal-averaged cardiokymography for the detection of ischaemic left ventricular dysfunction.

UNLABELLED: Cardiokymography (CKG) is a non-invasive method for the detection of patients with coronary artery disease (CAD). Issues of the present study were to evaluate the feasibility, sensitivity and specificity of a recently developed signal-averaged CKG system for detecting patients with pharmacologically induced ischaemic left ventricular wall motion abnormalities (WMA) during pharmacologic stress echocardiography (SE). Precordial CKG curves were recorded in 100 consecutive patients who underwent dobutamine-SE for suspected CAD. For interpretation, CKG curves were classified into three different types, depending on the degree of systolic outward motion. CKG test results were regarded as positive (indicating myocardial ischaemia) if there was a change of the baseline CKG type at peak pharmacologic stress. The CKG test results were positive in 18 of 27 patients with a pathologic dobutamine-SE (sensitivity 67%), but did not show any change of the prior CKG type in 57 of 69 patients with a normal SE (specificity 83%). Patients with a true positive CKG test had significantly (P<0.05) more echocardiographic segments with WMA than patients with a false negative CKG test. CONCLUSIONS: Signal-averaged CKG can detect patients with ischaemic ventricular dysfunction. Sensitivity of CKG in detecting patients with WMA depends on the extent of left ventricular ischaemia. Further studies are needed to define the diagnostic value of signal-averaged CKG in the non-invasive detection of patients with suspected CAD.

Adult↗
PubMed1997

Detection of patients with restenosis after PTCA by dipyridamole-atropine-stress-echocardiography.

Stress-echocardiography (SE) has been proven to be a valuable method for the diagnosis of coronary artery disease. For patients who cannot exercise, pharmacological stress-echocardiography represents an alternative method for the induction of cardiovascular stress. Few studies exist concerning the value of dipyridamole-SE for the detection of restenosis in patients after primary successful PTCA. It has been demonstrated that the addition of atropine can significantly increase the diagnostic potential of dipyridamole-SE, especially in patients with 1- or 2-vessel disease. The purpose of our study was to investigate the diagnostic value of high-dose dipyridamole-SE plus atropine (DASE) for the detection of restenosis after primary successful PTCA. We investigated 65 patients 3-6 months after PTCA before a control angiography was performed. Restenosis was defined as > 70% lumen narrowing, determined by quantitative coronary angiography. In 20/27 patients with restenosis, the DASE was pathological (sensitivity 74%); in 34/38 patients without restenosis the DASE was normal or showed no induced WMA (specificity 89%). Patients with tight restenosis (> 90%) were always correctly detected by DASE. Concerning the different vessels, restenosis of the LAD was correctly predicted by DASE in 11/12 patients, restenosis of the LCX in 6/9 patients and restenosis of the RCA in 8/11 patients. From the results of our study we conclude that DASE is a reliable diagnostic method for the non-invasive evaluation of patients after PTCA. DASE can identify patients with relevant restenosis after PTCA and help to select those patients who will probably benefit from further coronary interventions, for repeat angiography.

Angioplasty, Balloon, Coronary↗
PubMed1997

Monoclonal antibodies against heparin and heparinoids.

The antigenicity of glycosaminoglycans and galactosaminoglycans is very weak. Accordingly, only a few reports on the successful production of monoclonal antibodies against glycosaminoglycans and galactosaminoglycans have been published. Antibodies have been raised against the heparinlike compounds heparan sulfate, chondroitin sulfate, and keratan sulfate. The production of heparin antibodies was reported. But further analysis showed that the antibodies were not directed against native heparin but against heparin conjugates or chemically modified heparins. After immunization with a heparin-bovine serum albumin conjugate, prepared by reductive amination, a monoclonal antibody against heparin and heparin fractions was obtained (Huhle et al: Semin Thromb Hemostas 20:193-204, 1995). For further analysis, tyramine, which was covalently bound to low-molecular-mass heparin (LMMH) by endpoint attachment (Malsch et al: Anal Biochem 217:255-264, 1994), was labeled with iodine-125 at the aryl residue. The tracer antibody complex was immunoprecipitated by goat anti-mouse immunoglobulin IgG. H1.18 recognized specifically intact heparin and heparin fractions. The lower detection limit for heparin preparations was 100 ng/mL. The H1.18 antibody was purified by ammonium sulfate precipitation. H1.18 remained biologically and functionally active after purification. The smallest disaccharide that was detected by H1.18 was found to be iduronic acid-anhydromannose, obtained by nitrous acid degradation of LMMH. Endpoint attachment of tyramine to anhydromannose did not modify the binding of the disaccharide to H1.18, indicating the importance of iduronic acid for binding of glycosaminoglycans to the antibody.

Animals↗
PubMed1997

Measurement of fluorescent-labeled LMM-heparin in biological fluids using protamine-linked microbeads.

A quantitative assay for fluorescent heparin in a purified system and in plasma was developed (Piazolo et al: Semin Thromb Hemostas 20:227-235, 1994). The protamine microbeads (1.6 microns) showed a broad size distribution and a large standard variation in low concentrations. Our aim was to optimize these protamine microbeads for the measurement of fluorescent heparin. The following results were obtained: Paramagnetic protamine microbeads of different average diameters (0.8, 1.6, 2.8, and 4.5 microns) were synthesized by cyclocarbodiimide and tosyl activation. These microbeads bind heparin and are assayed using flow cytometry. The protamine concentration on the surface of the beads ranged between 2.0 and 61 mg/mL. The protamine microbeads bound fluorescent heparin and were analyzed by flow cytometry. The protamine microbeads bound LMM-heparin-tyramine-FITC dose dependently in saline solution, plasma, and blood. There are substantial differences between the microbeads of different origins with regard to the amount of protamine bound, the sensitivity of the detection, and the reliability for the determination of heparins in plasma and blood. The minimal sensitivity of the final method was 0.001 U/mL LMMH-tyramine-FITC in saline solution and in plasma. Human blood cells were not bound to protamine microbeads. The half-maximal binding of LMMH-tyramine-FITC of the different protamine-coated microbeads ranged from 1.7 to 8.0 micrograms/mL in saline solution, 2.3 to 8.7 micrograms/mL in plasma, and 3.1 to 6.4 micrograms/mL in blood. We conclude that all protamine microbeads can be used to quantify the concentration of LMMH-tyramine. Protamine Dynabeads M-450 (diameter 4.5 microns) have advantages over other microbeads because of their more homogeneous size distribution, a higher selectivity, and they can be measured together with leukocytes. They are currently used to develop a competitive binding assay for heparin in plasma.

Binding, Competitive↗
PubMed1997

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.

Animals↗
PubMed1997

Dosage, anticoagulant, and antithrombotic effects of heparin and low-molecular-weight heparin in the treatment of deep vein thrombosis.

We have performed a prospective, randomized, controlled trial comparing continuous intravenous unfractionated heparin with twice-daily subcutaneous (s.c.) high-dose low-molecular-weight (LMW) heparin in the initial treatment of 50 patients with acute proximal deep vein thrombosis. In this article we analyze the relationship between the dosage of the heparins, the anticoagulant effects on aPTT, and thrombin and factor Xa inhibition to the improvement of the Marder score after a 10-day treatment period. Improvement of the Marder score was observed in about 70% of patients without regard to administration of unfractionated or LMW heparin. Patients in both treatment categories were divided into two groups, namely, those who showed an improvement of the Marder score and those who did not. In the group of patients with unfractionated heparin and regression of thrombus size the mean dosage was 33,000 U/day, whereas the mean dosage was 37,000 U/day in the patients with status idem of the Marder score after the 10-day treatment period. Thrombin clotting time values were in contrast to the dosage. Patients with regression of thrombosis showed higher thrombin clotting time values compared with those with status idem. These results were also seen with aPTT and the Heptest coagulation assay, but the differences between the two groups were less pronounced. No differences between these two groups of patients were seen or detected with the S2222 chromogenic anti-factor Xa method. Patients receiving 2 x 12,000 IU s.c./day LMW heparin did not show these differences, the dosage being adjusted by the anti-Xa levels, ranging from 0.6 to 1.0 U/mL 4 hours after the s.c. injection. The groups of patients categorized as to improvement or not of the Marder score did not show differences in the daily dose. The anti-Xa activity was higher in patients with regression of thrombosis compared with patients without regression. The other coagulation parameters did not show any relation to the clinical outcome of thrombus regression. The relationship between the change of the Marder score at day 10 and the anticoagulant effect on the different coagulation systems correlated weakly for patients receiving unfractionated heparin. The highest correlation was found for the improvement of Marder score and thrombin inhibition in the heparin group with r = 0.42. For LMW heparin no correlation could be detected. Heptest coagulation values were in the same range for patients receiving unfractionated and LMW heparin. In contrast to the chromogenic anti-Xa assay, aPTT, thrombin clotting time, and prothrombin time values differed substantially in the two treatment regimens. Treatment of recent deep vein thrombosis with unfractionated heparin profits from laboratory monitoring, whereas monitoring of the anticoagulant effect during the treatment with s.c. LMW heparin does not influence the outcome on thrombus regression.

Adult↗
PubMed1997

Comparison of the pharmacodynamic and pharmacokinetic profiles of two low-molecular-mass heparins in rats.

The pharmacodynamic and pharmacokinetic properties of certoparin and dalteparin were analyzed after intravenous and subcutaneous administration. The two different LMMHs exhibited different molecular mass and in vitro activities. The aim of the present study was to show the extent to which these in vitro differences influenced the biological activity and pharmacokinetics in vivo. It was possible to measure the plasma concentrations of the LMMHs by using a competitive assay with protamine-coated latex beads. Both LMMHs showed a biphasic aXa and aIIa activity curve after intravenous injection. Certoparin and dalteparin showed comparable aXa pharmacodynamics after IV and SC injection. By measuring the aIIa activities after IV administration of the LMMHs, T1/2, Amax, and AUC were comparable but tPC differed. After SC injection the aIIa activities of the LMMHs exhibited comparable T1/2 and Amax but different AUC and tPC. The plasma concentrations of the LMMHs showed comparable T1/2 but certoparin exhibited a higher Amax and AUC after IV and SC administration. The relative bioavailability of the aXa activity, aIIa activity, and the plasma concentrations ranged between 70 and 100%. The differences in the aIIa pharmacodynamic and pharmacokinetic profiles of certoparin and dalteparin may be caused by the differences in the in vitro activities and the different molecular mass. Clinical relevance of the different pharmacologic profiles is only expected of the aIIa activity-related biological effects of the LMMHs.

Animals↗
PubMed1997

Long-term anticoagulation of outpatients with adverse events to oral anticoagulants using low-molecular-weight heparin.

Bleeding complications are one of the major risks during oral anticoagulation. If further anticoagulation is indicated, low-molecular-weight heparin (LMWH) may offer an alternative treatment in those patients. In a prospective, nonrandomized study, 120 patients have been switched from oral anticoagulants to LMWH because of bleeding complications or other severe side effects during treatment with vitamin K antagonists. Indication for further anticoagulation was prophylaxis of recurrent thromboembolism, artificial heart valve replacement, atrial fibrillation with embolism and cardiomyopathy. The treatment period ranged from 2 months to 10.8 years. No fatal embolism occurred. One major but not severe episode of gastrointestinal bleeding occurred in a patient with an as yet unknown colon carcinoma. The cumulative treatment period amounts to 250 years. No drop in platelet count occurred in any patient. No other side effects were observed. LMWH was injected subcutaneously at doses ranging from 2500 to 15,000 anti-factor Xa units per day by the patient himself. The dose was adjusted on the basis of body weight, bleeding risk and thromboembolic risk. The results indicate that LMWH may be effectively and safely used as alternative anticoagulant regimen in patients with side effects or other complications on oral anticoagulants.

Administration, Oral↗
PubMed1997

Anticoagulation in patients with heparin-induced thrombocytopenia type II.

Heparin-induced thrombocytopenia (HIT) together with simultaneously occurring thromboembolism is a serious complication of heparin treatment. At present an immunologic cause of this side effect of heparin is equivocally accepted. However, further anticoagulation of these patients is still debated. The present article summarizes the treatment of 20 patients with such complications. Two of these patients did not develop thrombocytopenia but presented cutaneous allergy or necrosis. All patients were treated either with intravenous heparinoid (Orgaran) or with low-molecular-weight heparin without/with simultaneous intravenous high-dose immunoglobulins or with intravenous r-hirudin. Based on these experiences the treatment of choice depends at present on the availability of the anticoagulants and on the local experience with the different anticoagulants. In the future r-hirudins and other nonheparin thrombin inhibitors may become the drugs of choice in this indication. Surgical intervention has to be considered additionally.

Adult↗