The effects of MDCO-2010, a serine protease inhibitor, on activated clotting time in blood obtained from volunteers and cardiac surgical patients.

BACKGROUND: The activated clotting time (ACT) is widely used for monitoring heparin anticoagulation during cardiac surgery. Celite-based ACT values are prolonged when aprotinin is administered. MDCO-2010, a novel serine protease inhibitor, is currently being evaluated as a possible alternative to aprotinin. Therefore, we evaluated the in vitro effects of this novel agent on ACT values using 3 different point-of-care instruments with kaolin or celite as an activator. METHODS: The study was performed in 2 parts. In the first part, blood samples were obtained from 15 healthy volunteers. Samples were pipetted into small Eppendorf tubes and 2 concentrations of the MDCO-2010 (100 and 500 nM, final concentration) alone or with heparin (1.2 or 2.4 U/mL) were added. ACTs were measured using Helena (celite), Hemochron (kaolin), and Medtronic (kaolin) devices. In the second part of the study, blood samples were obtained intraoperatively, at 5 time points, from 15 patients undergoing cardiopulmonary bypass. MDCO-2010 at a final concentration of 100 or 500 nM was added and ACT testing was performed as before. Additional coagulation tests included prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin, prothrombin, and anti-Xa levels. RESULTS: Addition of MDCO-2010 concentration-dependently prolonged ACTs in volunteers' and patients' blood samples regardless of the ACT activator or device used. In volunteer samples (no heparin) and in patient samples (baseline and intensive care unit) percent changes in ACTs due to MDCO-2010 were on average 3.1 ± 1.8 times higher (95% confidence interval 2.6-3.6; P < 0.001) for the celite-based Helena device compared with either Hemochron or Medtronic devices. CONCLUSION: MDCO-2010 causes less ACT prolongation with kaolin than with celite activation.

Analysis of highly potent amidine containing inhibitors of serine proteases and their N-hydroxylated prodrugs (amidoximes).

The development of serine protease inhibitors often results in the discovery of new lead compounds containing strong basic amidine functions that usually suffer from poor absorption from the intestine. In order to improve oral bioavailability of these drugs, prodrug principles such as the conversion of amidines into amidoximes may be applied. In this work, two HPLC-based separation methods of serine protease inhibitors (amidines) and their N-hydroxylated prodrugs have been developed and characterised. This was performed by evaluating 11 distinct amidine-amidoxime pairs with different physicochemical parameters (clogP: -3 to 5.1). The HPLC methods developed allowed excellent separation of the compound pairs examined. Also, the possible selection of different separation techniques (i.e. adsorption- and ion-pair-chromatography) permits universal application. Moreover, both techniques are compatible with mass spectrometry and are superior to the previously described methods. In summary, both HPLC methods are suitable for the separation of most amidoxime-prodrugs currently in clinical or preclinical development.

Time-domain in vivo near infrared fluorescence imaging for evaluation of matriptase as a potential target for the development of novel, inhibitor-based tumor therapies.

Proteolytic enzymes expressed on the surface of tumor cells, and thus easily accessible to external interventions, represent useful targets for anticancer and antimetastatic therapies. In our study, we thoroughly evaluated matriptase, a trypsin-like transmembrane serine protease, as potential target for novel inhibitor-based tumor therapies. We applied time-domain near infrared fluorescence (NIRF) imaging to characterize expression and activity of matriptase in vivo in an orthotopic AsPC-1 pancreatic tumor model in nude mice. We show strong and tumor-specific binding of intravenously injected Cy5.5 labeled antimatriptase antibody (MT-Ab*Cy5.5) only to primary AsPC-1 tumors and their metastases over time within living mice, taking into account fluorescence intensities and fluorescence lifetimes of the applied probes. Specific binding of MT-Ab*Cy5.5 to tumor sites was confirmed by ex vivo NIRF imaging of tumor tissue, NIRF microscopy and by coregistration of the in vivo acquired NIRF intensity maps to anatomical structures visualized by flat-panel volume computed tomography (fpVCT) in living mice. Moreover, using an activatable synthetic substrate S*DY-681 we could clearly demonstrate that matriptase is proteolytically active in vitro as well as in vivo in tumor-bearing mice, and that application of synthetic active-site inhibitors having high affinity and selectivity toward matriptase can efficiently inhibit its proteolytic activity for at least 24 hr. We thus successfully applied NIRF imaging in combination with fpVCT to characterize matriptase as a promising molecular target for inhibitor-based cancer therapies.

The novel synthetic serine protease inhibitor CU-2010 dose-dependently reduces postoperative blood loss and improves postischemic recovery after cardiac surgery in a canine model.

BACKGROUND: Serine protease inhibitors such as aprotinin reduce perioperative blood loss and may improve postpump cardiac performance owing to their anti-inflammatory properties. After the "aprotinin era," we investigated the efficacy of the novel synthetic serine protease inhibitors CU-2010 with improved coagulatory and anti-inflammatory profile on blood loss and reperfusion injury in a canine model. METHODS: Thirty-six dogs were divided into 6 groups: control, aprotinin (n = 8; Hammersmith scheme), and CU-2010 (0.5, 0.83, 1.25, and 1.66 mg/kg). All animals underwent 90 minutes of cardiopulmonary bypass with 60 minutes of hypothermic cardioplegic arrest. End points were blood loss during the first 2 hours after application of protamine, as well as recovery of myocardial contractility (slope of the end-systolic pressure-volume relationship, coronary blood flow, and vascular reactivity. RESULTS: CU-2010 dose-dependently reduced blood loss to a degree comparable with that of aprotinin at lower doses and even further improved at higher doses (control/aprotinin/CU-2010 in increasing doses: 142 +/- 13, 66 +/- 17, 95 +/- 16, 57 +/- 17, 46 +/- 3, and 13 +/- 4 mL; P < .05). Whereas aprotinin did not influence myocardial function, CU-2010 improved the recovery of end-systolic pressure-volume relationship (control 60 +/- 6 mg kg vs aprotinin 73 +/- 7 mg/kg vs CU-2010 1.66 mg/kg; 102% +/- 8%; P < .05). Coronary blood flow (52 +/- 4 vs 88 +/- 7 vs 96 +/- 7; P < .05) and response to acetylcholine (44% +/- 6% vs 77% +/- 7% vs 81% +/- 6%; P < .05) were improved by both aprotinin and CU-2010. CONCLUSIONS: The novel serine protease inhibitor CU-2010 significantly reduced blood loss after cardiac surgery comparable with aprotinin. Furthermore, an additionally improved anti-inflammatory profile led to a significantly improved postischemic recovery of myocardial and endothelial function.

Effects of novel synthetic serine protease inhibitors on postoperative blood loss, coagulation parameters, and vascular relaxation after cardiac surgery.

OBJECTIVE: Although aprotinin has been widely used to reduce perioperative blood loss after cardiopulmonary bypass, recent concerns have led to its withdrawal. This study investigated effects of the novel synthetic serine protease inhibitors CU-2010 and CU-2020 on blood loss, coagulation parameters, and coronary relaxation in a canine model. METHODS: Thirty-seven dogs were divided into 5 groups: control (n = 5), aprotinin (n = 8, Hammersmith scheme of intravenous bolus, prime, and continuous infusion), Hammersmith CU-2010 (n = 8, 1.6 mg/kg Hammersmith scheme), continuous CU-2010 (n = 8, 1.6 mg/kg continuous infusion), and CU-2020 (n = 8, 8.9 mg/kg Hammersmith scheme). All animals underwent 90-minute cardiopulmonary bypass. End points were blood loss during first 2 hours after protamine and activated clotting, partial thromboplastin, and prothrombin times. At end of experiments, coronary rings were removed for in vitro testing of relaxation to acetylcholine and sodium nitroprusside. RESULTS: Hammersmith and continuous CU-2010, CU-2020, and aprotinin groups all had reduced blood loss (43 + or - 4, 43 + or - 8, 52 + or - 7, 61 + or - 7, respectively, vs control 149 + or - 24 mL, P < .05). After protamine, activated clotting time and partial thromboplastin time normalized in control, aprotinin, and Hammersmith CU-2010 groups but remained elevated in continuous CU-2010 and CU-2020 groups. Prothrombin time and vascular relaxation did not differ between groups. CONCLUSIONS: CU-2010 and CU-2020 significantly reduced blood loss after cardiac surgery, with prolonged partial thromboplastin and activated clotting times, demonstrating improved antithrombotic profile. Neither aprotinin nor the novel serine protease inhibitors influenced vascular relaxation.

Use of IHC and newly designed matriptase inhibitors to elucidate the role of matriptase in pancreatic ductal adenocarcinoma.

Matriptase, also known as MT-SP1, is a type II transmembrane serine protease strongly implicated in both the development and progression of a variety of epithelial cancers. Evidence comes from studies of its expression in human cancers and from mouse models of spontaneous cancer. Matriptase is considered to be a major activator of two key stimulators of invasive growth, namely hepatocyte growth factor/scatter factor and urokinase-type plasminogen activator. The aim of this study was to examine the role of matriptase in pancreatic ductal adenocarcinoma by expression analysis and functional assays in vitro. Immunohistochemical analysis of matriptase performed on microtissue arrays and large samples of 55 pancreatic ductal adenocarcinomas and on 31 samples of normal pancreatic ducts revealed that although matriptase expression differed greatly in both malignant and normal ductal pancreatic tissue, matriptase scores were significantly (p=0.02) elevated in pancreatic ductal adenocarcinoma compared to normal pancreatic ducts. To evaluate the role of matriptase during development of pancreatic cancer, we studied the effects of newly designed matriptase inhibitors on the processing of the zymogen of urokinase-type plasminogen activator in the human adenocarcinoma cell lines AsPC-1 and BxPC-3. In both cell lines, at 1 microM, all matriptase inhibitors completely prevented zymogen activation. At lower inhibitor concentrations, the degree of inhibition of zymogen processing correlated with the affinities of the inhibitors towards matriptase indicating that this is a specific result of matriptase inhibition. Furthermore, matriptase inhibitors reduced the phosphorylation of the HGF receptor/cMet and the overall cellular invasiveness of the human pancreatic adenocarcinoma cell line AsPC-1. Our findings demonstrate for the first time that matriptase may be involved in the progression of pancreatic ductal adenocarcinoma and that matriptase inhibition may contribute to preventing the progression of this devastating disease.

CU-2010--a novel small molecule protease inhibitor with antifibrinolytic and anticoagulant properties.

BACKGROUND: In cardiac surgery, the contact of blood with the artificial surfaces of the cardiopulmonary bypass results in activation of coagulation, fibrinolysis, and platelets, which is recognized as reason for increased bleeding tendency. Antifibrinolytics like tranexamic acid or the broad-spectrum protease inhibitor aprotinin attenuate this response. The marketing of aprotinin has been suspended after a recent clinical trial suggested increased risks associated with aprotinin. Moreover, aprotinin is a protein of animal origin and has antigenic properties. As a result, alternative antifibrinolytic compounds are desirable. METHODS: This in vitro study compared the antifibrinolytic efficacy of the synthetic small molecule CU-2010 with aprotinin and tranexamic acid. Antifibrinolytic activity in plasma and whole blood of ten healthy volunteers was examined with a turbidometric method and with tissue factor-activated thromboelastometry (ROTEM; Pentapharm, Munich, Germany). In addition, anticoagulant effects were assessed through measurement of plasma and whole blood clotting times and thrombin generation. RESULTS: With its high affinity for plasmin (Ki, 2 nM), CU-2010 inhibited fibrinolysis comparable to aprotinin (Ki, 4 nM) and was ten times more potent than tranexamic acid. CU-2010 also inhibited plasma kallikrein (Ki < 1 nM) and factors Xa (Ki, 45 nM) and XIa (Ki, 18nM), which was reflected in prolongation of coagulation times and an attenuation of thrombin generation. CONCLUSION: These findings suggest that CU-2010 has similar antifibrinolytic potency compared to aprotinin, is more potent than tranexamic acid, and possesses some anticoagulant effects.