Coadministration Of Heparin Research Articles

The Coronary ARteriogenesis with combined Heparin and EXercise therapy in chronic refractory Angina (CARHEXA) trial: A double-blind, randomized, placebo-controlled stress echocardiographic study.

Coronary collateral circulation exerts protective effects on myocardial ischaemia due to coronary artery disease and can be promoted by exercise with heparin co-administration. Whether this arteriogenetic effect is accompanied by functional improvement of left ventricle during stress and lessening of angina symptoms remains unknown. To evaluate the anti-ischaemic efficacy of heparin plus exercise in coronary artery disease. In a prospective, single-centre, randomized, double-blind study we recruited 32 'no-option' patients (27 males; mean age 61 ± 8 years) with stable angina, exercise-induced ischaemia and coronary artery disease not suitable for revascularization. All underwent a two-week cycle of exercise (two exercise sessions per day, five days per week) and were randomized (n = 16 per group) to intravenous placebo (0.9% saline) versus unfractionated heparin (5.000 IU intravenously), 10 min prior to exercise. We assessed Canadian Cardiovascular Society angina class, stress electrocardiogram and echo parameters (wall motion score index) and computed tomography angiography for collaterals. After two-week cycle, Canadian Cardiovascular Society class statistically decreased in both groups (heparin plus exercise group: 2.6 ± 0.7 to 1.9 ± 0.7, p < 0.001, exercise group: 2.4 ± 0.7 to 2.1 ± 0.9, p = 0.046). Only the heparin plus exercise group improved time-to-ST segment depression (before 270, 228-327 s vs. after 339, 280-360 s, p = 0.012) and wall motion score index (before 1.38 ± 0.25 vs. after 1.28 ± 0.18, p = 0.005). By multi-slice computed tomography angiography, collaterals improved in 12/15 (80%) in the heparin plus exercise group versus 2/16 (12.5%) in the exercise group (p < 0.001). A two-week, 10-test cycle of heparin plus exercise is better than exercise in improving angina class, myocardial ischaemia and collaterals by computed tomography angiography.

Procoagulant in vitro effects of clinical cellular therapeutics in a severely injured trauma population.

Clinical trials in trauma populations are exploring the use of clinical cellular therapeutics (CCTs) like human mesenchymal stromal cells (MSC) and mononuclear cells (MNC). Recent studies demonstrate a procoagulant effect of these CCTs related to their expression of tissue factor (TF). We sought to examine this relationship in blood from severely injured trauma patients and identify methods to reverse this procoagulant effect. Human MSCs from bone marrow, adipose, and amniotic tissues and freshly isolated bone marrow MNC samples were tested. TF expression and phenotype were quantified using flow cytometry. CCTs were mixed individually with trauma patients' whole blood, assayed with thromboelastography (TEG), and compared with healthy subjects mixed with the same cell sources. Heparin was added to samples at increasing concentrations until TEG parameters normalized. Clotting time or R time in TEG decreased relative to the TF expression of the CCT treatment in a logarithmic fashion for trauma patients and healthy subjects. Nonlinear regression curves were significantly different with healthy subjects demonstrating greater relative decreases in TEG clotting time. In vitro coadministration of heparin normalized the procoagulant effect and required dose escalation based on TF expression. TF expression in human MSC and MNC has a procoagulant effect in blood from trauma patients and healthy subjects. The procoagulant effect is lower in trauma patients possibly because their clotting time is already accelerated. The procoagulant effect due to MSC/MNC TF expression could be useful in the bleeding trauma patient; however, it may emerge as a safety release criterion due to thrombotic risk. The TF procoagulant effect is reversible with heparin.

Heterogeneity in Standard Operating Procedures for Catheter Directed Thrombolysis for Peripheral Arterial Occlusions in The Netherlands: A Nationwide Overview

Catheter directed thrombolysis (CDT) for acute arterial occlusions of the lower extremities is associated with a risk of major bleeding complications. Strict monitoring of vital functions is advised for timely adjustment or discontinuation of thrombolytic treatment. Nevertheless, current evidence on the optimal application of CDT and use of monitoring during CDT is limited. In this study the different standard operating procedures (SOPs) for CDT in Dutch hospitals were compared against a national guideline in a nationwide analysis. SOPs, landmark studies, and national and international guidelines for CDT for acute lower extremity arterial occlusions were compared. The protocols of 34 Dutch medical centres where CDT is performed were assessed. Parameters included contraindications to CDT, co-administration of heparin, thrombolytic agent administration, angiographic control, and patient monitoring. Thirty-four SOPs were included, covering 94% of medical centres performing CDT in the Netherlands. None of the SOPs had identical contraindications and a strong divergence in relative and absolute grading was found. Heparin and urokinase dosages differed by a factor of five. In 18% of the SOPs heparin co-administration was not mentioned. Angiographic control varied between once every 6h to once every 24h. In 76% of the SOPs plasma fibrinogen levels were used for CDT dose adjustments. However, plasma fibrinogen level threshold values for treatment adjustments varied between 2.0g/L and 0.5g/L. The SOPs for CDT for acute arterial occlusions of the lower extremities differ greatly on five major operating aspects among medical centres in the Netherlands. None of the SOPs exactly conforms to current national or international guidelines. This study provides direction on how to increase homogeneity in guideline recommendations and to improve guideline adherence in CDT.

Functional modulation of liver mitochondria in lipopolysaccharide/drug co-treated rat liver injury model.

Drug-induced liver injury is not readily detectable using conventional animal studies during pre-clinical drug development. To address this problem, other researchers have proposed the use of co-administration of lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, and a drug. Using this approach, liver injury that is otherwise not detected following drug administration alone can be successfully identified. Previous studies have demonstrated that such injury is suppressed by heparin; therefore, the mechanism may involve coagulation-dependent ischemia. However, it has not been established how LPS-induced ischemia might sensitize hepatocytes to a potentially hepatotoxic drug. In the present study, we aimed to determine the effect of LPS-induced ischemia on liver mitochondrial function and downstream toxicologic responses. Consistent with previous findings, plasma alanine transaminase (ALT) activity was higher in rats co-administered with LPS (1 mg/kg) and diclofenac (100 mg/kg), but reduced by heparin. Liver mRNA expression of Hmox1, encoding heme oxygenase-1, an oxidative stress indicator, was three times higher at 2 hr after LPS administration. Furthermore, respiratory activity via mitochondrial complex II, lipid peroxidation in mitochondria, and the susceptibility to mitochondrial permeability transition pore opening in response to diclofenac administration were significantly increased by LPS administration. The increase in plasma ALT activity and the sensitization to mitochondrial permeability transition pore opening were reduced by the co-administration of heparin. In conclusion, LPS-induced transient ischemia disrupts respiratory chain complex activities, enhances reactive oxygen species production, especially in mitochondria, and sensitizes mitochondria to permeability transition pore opening when testing a potentially hepatotoxic drug in vivo.

Bone Marrow-Derived Mesenchymal Stem Cells Have Innate Procoagulant Activity and Cause Microvascular Obstruction Following Intracoronary Delivery: Amelioration by Antithrombin Therapy.

Mesenchymal stem cells (MSCs) are currently under investigation as tools to preserve cardiac structure and function following acute myocardial infarction (AMI). However, concerns have emerged regarding safety of acute intracoronary (IC) MSC delivery. This study aimed to characterize innate prothrombotic activity of MSC and identify means of its mitigation toward safe and efficacious therapeutic IC MSC delivery post-AMI. Expression of the initiator of the coagulation cascade tissue factor (TF) on MSC was detected and quantified by immunofluorescence, FACS, and immunoblotting. MSC-derived TF antigen was catalytically active and capable of supporting thrombin generation in vitro. Addition of MSCs to whole citrated blood enhanced platelet thrombus deposition on collagen at arterial shear, an effect abolished by heparin coadministration. In a porcine AMI model, IC infusion of 25 × 10(6) MSC during reperfusion was associated with a decrease in coronary flow reserve but not when coadministered with an antithrombin agent (heparin). Heparin reduced MSC-associated thrombosis incorporating platelets and VWF within the microvasculature. Heparin-assisted therapeutic MSC delivery also reduced apoptosis in the infarct border zone at 24 hours, significantly improved infarct size, left ventricular (LV) ejection fraction, LV volumes, wall motion, and attenuated histologic evidence of scar formation at 6 weeks post-AMI. Heparin alone or heparin-assisted fibroblast control cell delivery had no such effect. Procoagulant TF activity of therapeutic MSCs is associated with reductions in myocardial perfusion when delivered IC may be successfully managed by heparin coadministration. This study highlights an important mechanistic insight into safety concerns associated with therapeutic IC MSC delivery for AMI.

Pharmacokinetic and pharmacodynamic interactions between palifermin and heparin.

Oral mucositis, a severe complication during chemo- and/or radiotherapy, is prevented with palifermin treatment, a recombinant human keratinocyte growth factor (KGF/FGF-7). The FGF family belongs to the larger family of heparin-binding growth factors. Because it has been shown that heparin modulates binding of KGF to the KGF receptor and subsequently affects cellular proliferation induced by the KGF mitogenic signal, it is critical to understand the drug-drug interactions between palifermin and heparin, particularly because of heparin's narrow therapeutic margin. Two studies were performed in healthy subjects to characterize the effect of palifermin on the pharmacodynamics of heparin (activated partial thromboplastin time) and evaluate the impact of heparin on the pharmacokinetics and pharmacodynamics (Ki67 staining of buccal mucosal tissue) of palifermin. Results demonstrated a pronounced pharmacokinetic interaction; heparin coadministration increased the palifermin AUC 4- to 5-fold and decreased its half-life by 40%-45%, suggesting an approximate 70%-80% decrease in palifermin clearance and volume of distribution. These changes in the pharmacokinetics of palifermin during coadministration of heparin, however, did not affect the pharmacodynamic effect of palifermin, or the anticoagulant activity of heparin, and did not lead to increased safety findings. Therefore, these results suggest that dose adjustments for heparin and palifermin are not warranted when administered concurrently.

Comprehensive meta-analysis of safety and efficacy of bivalirudin versus heparin with or without routine glycoprotein IIb/IIIa inhibitors in patients with acute coronary syndrome.

The aim of this meta-analysis was to compare the 30-day safety and efficacy of bivalirudin with those of heparin with or without routine administration of a glycoprotein IIb/IIIa inhibitor (GPI) in patients with acute coronary syndrome (ACS). Bivalirudin has been a mainstay of anticoagulation in patients with ACS compared with heparin. The extent to which trial results have been affected by the coadministration of heparin with a GPI, however, remains unclear. A total of 13 randomized, controlled trials involving 24,605 patients were included. There was no significant difference in 30-day mortality or myocardial infarction rate with bivalirudin compared with heparin with or without routine GPI administration. A reduction of 30-day major bleeding was observed with bivalirudin compared with heparin that was significant when GPI was routinely administered (odds ratio [OR]: 0.52, 95% confidence interval [CI]: 0.45 to 0.60), p < 0.001) but not with provisionally administered GPI (OR: 0.66, 95% CI: 0.33 to 1.32; p = 0.24). The occurrence of stent thrombosis (ST) at 30 days was significantly increased with bivalirudin compared with heparin plus routinely administered GPI (OR: 1.67, 95% CI: 1.13 to 2.45, p = 0.02), but not compared with heparin plus provisionally administered GPI (OR: 2.08, 95% CI: 0.35 to 12.32, p = 0.42). The rate of acute ST (≤ 24 h), however, was almost 4.5-fold higher with bivalirudin compared with heparin with or without GPI, whereas the rate of subacute ST (24 h to 30 days) did not differ significantly. Overall, bivalirudin in ACS patients is associated with a significant reduction of major bleeding compared with heparin plus routinely administered GPI, but with a marked increase in ST rates compared with heparin with or without GPI.

Efficacy and bleeding risk of antithrombin supplementation in septic disseminated intravascular coagulation: a secondary survey.

IntroductionIn a previous report, we demonstrated a favorable trend for supplementation with antithrombin (AT) concentrate at a dosage of 3,000 IU/day over 1,500 IU/day for the treatment of sepsis-associated disseminated intravascular coagulation (DIC) in patients with an AT activity of 70% or less. Since the survival difference did not reach statistical significance, we planned to examine the effects in a larger number of cases with severer disease.MethodsWe performed a non-randomized multi-institutional survey. In total, 307 septic DIC patients who had AT activity less than 40% and who had undergone AT substitution at a dose of either 1,500 IU/day or 3,000 IU/day for three consecutive days were analyzed. Of these, 259 patients received 1,500 IU/day (AT1500 group) and 48 patients received 3,000 IU/day (AT3000 group). The primary efficacy endpoints were recovery from DIC by day 7 and an all-cause mortality on day 28. Adverse bleeding events were also examined. A logistic regression analysis was conducted by using age, sex, body weight, initial AT activity, DIC score, platelet count, coadministration of heparin, recombinant thrombomodulin, suspected source of infection, surgery, and supplemented AT dose.ResultsSupplementation significantly decreased the DIC score in the AT3000 group, leading to the superior resolution of DIC, compared with the results in the AT1500 group (66.7% versus 45.2%, P = 0.007). In addition, the AT3000 group exhibited a better survival than the AT1500 group (77.1% versus 56.4%, P = 0.010). Bleeding events were observed in 6.96% (severe bleeding: 3.04%) in the AT1500 group and 6.52% (severe bleeding, 4.35%) in the AT3000 group (P = 1.000; severe bleeding, P = 0.648). A logistic regression analysis revealed that the use of AT3000 (odds ratio (OR), 2.419; P = 0.025), a higher initial platelet count (OR, 1.054; P = 0.027), and patient age (OR, 0.977; P = 0.045) were significantly correlated with an improved survival.ConclusionsThe AT3000 group exhibited significantly improved rates of survival and recovery from DIC without an increased risk of bleeding, compared with the AT1500 group, among the patients with sepsis-associated DIC and an AT activity of less than 40%.

Warfarin-Induced Skin Necrosis

Warfarin-induced skin necrosis is an infrequent complication occurring in individuals under warfarin treatment who have a thrombophilic history or after administration of large loading doses of warfarin particularly without simultaneous initial use of heparin. A 62-year-old lady developed skin necrosis 4 days after initiating warfarin therapy of 5 mg daily without initial co-administration of heparin. The patient had a normal clotting profile. Skin necrosis progressed to eschar formation after cessation of warfarin and heparinization stopped expanding. Warfarin was reintroduced at 2 mg daily, initially together with low molecular weight heparin. Autolytic debridement of the necrotic tissue was followed by healing of the cutaneous deficit by secondary intention. Prompt diagnosis and discontinuation of warfarin are crucial for the prognosis.

Abstract 19260: Prothrombotic Activity and Reduction in Coronary Flow Reserve Associated With Therapeutic Intracoronary Delivery of Bone Marrow-Derived Mesenchymal Stem Cells is Ameliorated by Heparin Co-Administration in a Porcine Myocardial Infarction Model

Cell therapy remains an evolving therapeutic option for cardiac repair in the case of acute myocardial infarction (AMI) associated with significant cardiomyocyte death. Mesenchymal stem cells (MSCs) are promising candidates for promoting myocardial salvage post AMI, but concerns regarding the safety of intracoronary MSC delivery remain. Surface expression of tissue factor (TF), a key initiator of the soluble coagulation cascade, has recently been implicated as a possible cause of MSC procoagulant activity. This study aimed to characterize the prothrombotic activity of MSC and identify means of managing any thrombotic side effects during therapeutic intracoronary delivery post AMI. Expression of TF on MSC was detected by immunoflourescence, flow cytometry and immunoblotting. TF antigen was catalytically active (252.4 ± 8.3 pM/2 x 105 MSC) and capable of supporting thrombin generation in vitro. Addition of MSCs to whole citrated blood enhanced platelet-driven thrombus deposition on collagen at arterial shear flow (12.67 ± 1.0% untreated vs 18.9 ± 0.9% surface coverage with MSC, p&gt;0.001), an effect abolished by heparin co-administration (p&lt;0.01 vs MSC alone). Infusion of 25 x 106 MSC via an intracoronary route in a porcine AMI model was associated with a decrease in coronary flow reserve when delivered during reperfusion (-0.7 ± 0.05) but not when coadministered with heparin (0.1 ± 0.02, p&lt;0.01). Heparin also reduced MSC-associated thrombosis incorporating platelets and vWF in the microvasculature (p&lt;0.05). Therapeutic MSC delivery was able to reduce apoptosis in the infarct border zone 24 post AMI (p&lt;0.01). Procoagulant activity associated with therapeutic MSCs is independently associated with reduction in myocardial perfusion when delivered I.C. which may be successfully managed with heparin co-administration. This study highlights an important mechanistic insight into safety concerns associated with therapeutic intracoronary MSC delivery for AMI.

Influence of coadministration of antithrombotic medicines, warfarin, and NSAIDs on heparin safety: data from a prospective observational study.

Utilization of heparins has been increasing in the last decade, thus, in-depth analysis is needed to assess heparins safety monitoring patterns, incidence rates of adverse drug reactions (ADR), and frequency of coadministration with other medicines. To investigate the safety monitoring of heparin in hospitals and the influence of coadministration of nonsteroidal anti-inflammatory drugs (NSAIDs), antithrombotic medicines, and warfarin on heparin safety. We reviewed hospital records of 339 patients who had orders for heparin or low molecular weight heparin from May 2009 to May 2010. IBM SPSS Statistics version 18.0 was used to perform statistical analysis. Dalteparin (n=238, 70.21%) was the most frequently prescribed heparin. The most frequent indications given were for prophylaxis of venous thrombosis (n=135, 39.82%) and treatment of unstable coronary artery disease and myocardial infarction (n=166, 48.97%). ADRs were reported for 75 patients (22.12%), including coagulation abnormalities in 25 patients (7.37%), renal dysfunctions in 24 patients (7.08%), and thrombocytopenia in 10 patients (2.95%). 256 patients (75.52%) had relative contraindications. ADRs were associated with the previously reported relative contraindications (Spearman's rank correlation coefficient [rS] = 0.261, Pearson's chi-squared test [χ2]= 45.5, P less than 0.0005) and with prolonged treatment with heparins (rS=0.279 and χ2=74.7, P less than 0.0005). ADRs were not related to heparin use but indicated increased risk for negative treatment outcomes. Coadministration of heparin with warfarin, acetylsalicylic acid, clopidogrel, ketorolac, and NSAIDs was associated with the increased risk of adverse drug reactions. The relationship was low but statistically significant. The strongest relationship was with coadministration of aspirin (rS=0.283, χ2=21.42, P less than 0.0005), while the coadministration of NSAIDs showed only a very weak relationship to the development of ADRs (rS=0.133, χ2=21.01, P less than 0.0005). For the development of thrombocytopenia, the strongest risk was calculated for coadministration of warfarin (rS=0.248, χ2=28.14, P less than 0.0005), while coadministration of medicines from the list did not have a relationship with the risk of thrombocytosis. Safety monitoring of heparin orders is essential, especially for patients with relative contraindications during long-term treatment and in case of coadministration of oral anticoagulants, platelet inhibitors, and NSAIDs.

Heparin Oligosaccharides Inhibit Chemokine (CXC Motif) Ligand 12 (CXCL12) Cardioprotection by Binding Orthogonal to the Dimerization Interface, Promoting Oligomerization, and Competing with the Chemokine (CXC Motif) Receptor 4 (CXCR4) N Terminus

The ability to interact with cell surface glycosaminoglycans (GAGs) is essential to the cell migration properties of chemokines, but association with soluble GAGs induces the oligomerization of most chemokines including CXCL12. Monomeric CXCL12, but not dimeric CXCL12, is cardioprotective in a number of experimental models of cardiac ischemia. We found that co-administration of heparin, a common treatment for myocardial infarction, abrogated the protective effect of CXCL12 in an ex vivo rat heart model for myocardial infarction. The interaction between CXCL12 and heparin oligosaccharides has previously been analyzed through mutagenesis, in vitro binding assays, and molecular modeling. However, complications from heparin-induced CXCL12 oligomerization and studies using very short oligosaccharides have led to inconsistent conclusions as to the residues involved, the orientation of the binding site, and whether it overlaps with the CXCR4 N-terminal site. We used a constitutively dimeric variant to simplify the NMR analysis of CXCL12-binding heparin oligosaccharides of varying length. Biophysical and mutagenic analyses reveal a CXCL12/heparin interaction surface that lies perpendicular to the dimer interface, does not involve the chemokine N terminus, and partially overlaps with the CXCR4-binding site. We further demonstrate that heparin-mediated enzymatic protection results from the promotion of dimerization rather than direct heparin binding to the CXCL12 N terminus. These results clarify the structural basis for GAG recognition by CXCL12 and lend insight into the development of CXCL12-based therapeutics.

Analysis of infusion-site reactions in renal transplant recipients receiving peripherally administered rabbit antithymocyte globulin as compared with basiliximab

Antithymocyte globulin rabbit (r-ATG) has been used for the treatment and prevention of acute rejection in renal transplant recipients (RTR). Current manufacturer recommendations for r-ATG dictate the need for administration through a high-flow vein (central line). Previous studies have shown peripheral administration of r-ATG to be safe; however, these studies suggest the co-administration of heparin and hydrocortisone and did not compare the infusion-site reaction rates to a control group. A retrospective analysis was conducted of adult RTR receiving r-ATG or basiliximab between January 2004 and October 2006. Each agent was administered through a dedicated peripheral line. The primary endpoint was the incidence of infusion-site reactions. Other endpoints included the need to replace the intravenous catheter and the incidence of systemic thrombosis within 1 month of transplantation. During the study period, 152 peripheral infusions of r-ATG and 92 peripheral infusions of basiliximab were administered. No difference in infusion-site reactions was noted between the groups. There was also no difference either in the need for peripheral line replacement or the rates of systemic thrombosis. Peripheral administration of r-ATG is safe and can be infused without concomitant heparin and hydrocortisone. This method of r-ATG infusion was shown to be as safe as peripherally administered basiliximab.

Recombinant Nematode Anticoagulant Protein c2 in non-ST Segment Elevation Acute Coronary Syndrome and Beyond

Patients with acute coronary syndromes (ACS) have high recurrent ischemic event rates despite management with current guideline-based therapies. Recombinant nematode anticoagulant protein (rNAP)c2 provides factor Xa-dependent inhibition of the tissue factor/factor VIIa complex acting proximally on the clotting cascade. It may be administered either intravenously or subcutaneously and has an elimination half-life of approximately 50-60 h. rNAPc2 reduces thrombin formation in patients undergoing elective percutaneous coronary interventions (PCI) and in patients with non-ST segment elevation ACS managed with an early invasive strategy, while bleeding rates are comparable with currently used anticoagulants. Patients receiving rNAPc2 undergoing emergent coronary artery bypass surgery within 96 h of dosing have increased rates of major bleeding. Some heparin coadministration may be necessary to avoid PCI-related thrombotic complications. Large-scale trials are needed to confirm these findings and to evaluate the impact of rNAPc2 on clinical events.

Convection-Enhanced Delivery of Adeno-Associated Virus Type 2 (AAV2) into the Striatum and Transport of AAV2 Within Monkey Brain

Adeno-associated virus type 2 (AAV2)-based vectors are promising transgene carriers for experimental gene therapy treatments of brain diseases. However, detailed evaluation of transgene distribution, trafficking, and transport within the brain is of the utmost importance before applying any type of gene therapy in humans. We examined the distribution of AAV2-thymidine kinase (AAV2-TK) and AAV2-aromatic L-amino acid decarboxylase (AAV2-AADC) in monkey brain after convection-enhanced delivery (CED). The AADC group consisted of two 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys that received unilateral infusions of AAV2-AADC into six sites in the right hemisphere. The TK group consisted of three monkeys that received bilateral CED infusion of AAV2-TK into the putamen; one side in all three monkeys was coinfused with heparin. Six weeks after AAV delivery, the brains were collected and processed for immunohistochemical staining. Volumetric measurement of TK distribution showed that at least 75% of the putamen could be covered by a single infusion of the vector; however, no effects of heparin coadministration were found, most likely because of the already robust gene transfer achieved by CED. Interestingly, TK- and AADCimmunoreactive cells were also present outside the striatum, in the globus pallidus, subthalamic nucleus, thalamus, and substantia nigra. CED proved to be an efficient method for delivery of the AAV2 vector. Detection of the transgenes in brain structures distant from the site of injection emphasizes the potential for gene transport, and the advantages and disadvantages of CED for gene therapy deserve further study.

Heparin facilitates glial cell line-derived neurotrophic factor signal transduction.

Glial cell-line neurotrophic factor (GDNF), a neurotrophic factor with heparin binding affinity, promotes the survival and differentiation of a variety of neuronal cells including dopaminergic neuron. The effect of heparin on GDNF signaling was investigated based on the expression of the tyrosine hydroxyrase (TH) gene in neurobalstoma cells. Up-regulation of TH gene mRNA by GDNF was enhanced by co-administration of heparin. This facilitation by heparin was particularly evident at suboptimal levels of GDNF, which was consistent with the luciferase assay using TH gene promoter. Pretreatment with heparitinase decreased TH promoter activity in the absence of heparin. Phosphorylation of extracellular regulated kinase was increased in the presence of heparin, although tyrosine phosphorylation of Ret receptor tyrosine kinase was not affected by heparin. Expression of early response genes such as c-fos or Egr1 increased and sustained in the presence of heparin more than that without heparin. These results indicate that interaction with glycosaminoglycans such as heparin affects GDNF signal transduction positively.

The role of liver and kidney on the pharmacokinetics of a recombinant amino terminal fragment of bactericidal/permeability-increasing protein in rats.

The pharmacokinetics of rBPI23, a recombinant amino terminal fragment of bactericidal/permeability-increasing protein that binds to and neutralizes endotoxin, was investigated. rBPI23 was administered to rats at doses 0.01-10 mg/kg and plasma rBPI23 levels were measured by ELISA. rBPI23 was also administered to bilaterally nephrectomized rats. In addition, rBPI23 was administered intra-hepatically via the pyloric vein to determine the first-pass effect by the liver. rBPI23 concentrations were also simultaneously measured in the right atrium and aorta to determine the removal of rBPI23 by the lungs. The concentration-time profile of rBPI23 was described by a 3-compartmental model with parallel first order and Michaelis-Menten (saturable) elimination. The clearance of rBPI23 was not altered by bilateral nephrectomy. Clearance of intra-hepatically administered rBPI23 was 4.5 fold lower than intra-femorally administered rBPI23. The concentration difference of rBPI23 between aortic and right atrial blood was no greater than 11%. Clearance of rBPI23 in rats could be reduced up to 10 fold by co-administration of heparin. Uptake by liver of intra-hepatically administered rBPI23 was prevented by co-administration of heparin. rBPI23 is not significantly cleared by the kidneys, and no more than 11% of the rBPI23 was removed by the lungs with each pass. The liver could remove 78% of the rBPI23 from the hepatic circulation. Studies with heparin suggest rBPI23 is cleared by binding to heparan sulfate sites in the liver.

The synthetic pentasaccharide SR 90107A/org 31540 enhances tissue-type plasminogen activator-induced thrombolysis in rabbits

Current thrombolytic strategies have a number of shortcomings including resistance to recanalization and development of reocclusion. The purpose of this study was to compare the effects of heparin and of the synthetic pentasaccharide SR 90107A, which represents the antithrombin III binding site of heparin, on rt-PA-induced thrombolysis. The activity of these compounds was evaluated by following rt-PA-induced lysis of standard-sized, [ 125 I]-fibrinogen-labelled thrombi produced in the external jugular vein of rabbits. rt-PA alone (0.5 mg/kg/h for 4 h) induced a 43% thrombolysis. The i.v. co-administration of heparin (3 mg/kg, i.v.) or SR 90107A (300 nmol/kg, i.v.) significantly enhanced rt-PA-induced thrombolysis (83 and 80%, respectively). The present results therefore suggest that SR 90107A may be considered as an adjunctive antithrombotic therapy during thrombolysis of occluded vessels.

Pharmacokinetics and Pharmacodynamics of TP-9201, a gpIIbIIIa Antagonist, Administered in Combination with Recombinant Tissue-Type Plasminogen Activator, Heparin, and Aspirin in Beagles

The effect of heparin, aspirin, and recombinat tissue-type plasminogen activator (rt-PA) on TP-9201 pharmacokinetics and pharmacodynamics was investigated in beagles. Animals received TP-9201, an Arginine-Glycine-Aspartic acid (RGD)-containing synthetic peptide glycoprotein (gp)IIbIIIa antagonist as a bolus of 0.31 mg/kg, followed by a 4-h infusion of 0.5 mg/kg/h. rt-PA was administered as a modification of the weight-adjusted standard regimen. Heparin was administered as a bolus followed by an infusion producing a 1.5- to 2-fold increase in the activated prothromboplastin time (aPTT) above baseline values. Aspirin was administered orally, approximately 24 and 2 h before TP-9201. TP-9201 had a plasma clearance of 9.9 +/- 2 ml/min/kg and a volume of distribution that was larger than plasma volume. Administration of heparin and aspirin with TP-9201 did not affect the clearance of TP-9201, whereas rt-PA resulted in a faster clearance (p = 0.05). Whether the faster clearance is physiologic or a result of rt-PA interference in the TP-9201 assay is unclear. TP-9201 completely inhibited ADP-mediated platelet aggregation. After discontinuation of TP-9201, recovery of platelet aggregation had a half life (t1/2) of 2-3 h and was complete < or = 24 h. Coadministration of heparin did not interfere with TP-9201 pharmacodynamics, whereas aspirin and rt-PA slowed the recovery of platelet aggregation. The template bleeding time profile for the TP-9201-treated animals was similar to that of the aspirin-treated animals.

The endogenous oestrogen metabolite 2-methoxyoestradiol inhibits angiogenesis and suppresses tumour growth.

The formation of new blood vessels (angiogenesis) is critical for the growth of tumours and is a dominant feature in various angiogenic diseases such as diabetic retinopathy, arthritis, haemangiomas and psoriasis. Recognition of the potential therapeutic benefits of controlling pathological angiogenesis has led to a search for angiogenesis inhibitors. Here we report that 2-methoxyoestradiol, an endogenous oestrogen metabolite of previously unknown function, is a potent inhibitor of endothelial cell proliferation and migration as well as angiogenesis in vitro. Moreover, when administered orally in mice, it strongly inhibits the neovascularization of solid tumors and suppresses their growth. Unlike the angiostatic steroids of corticoid structure, it does not require the co-administration of heparin or sulphated cyclodextrins for activity. Thus, 2-methoxyoestradiol is the first steroid to have high antiangiogenic activity by itself. Our results suggest that this compound may have therapeutic potential in cancer and other angiogenic diseases.