Synthesis and pharmacology of modified amidine isoxazoline glycoprotein IIb/IIIa receptor antagonists.

Selective antagonism of the platelet GPIIb/IIIa receptor represents an attractive mechanism for the prevention and treatment of a number of thrombotic disease states. The antiplatelet activity of the oral GPIIb/IIIa receptor antagonists DMP 754 and DMP 802 have been disclosed. In this paper, the synthesis and biological evaluation of a series of potent N-substituted benzamidine isoxazolines are explored. The effect of benzamidine substitution on the duration of antiplatelet efficacy in dog is presented.

Inhibition of neointima formation by a nonpeptide alpha(v)beta(3) integrin receptor antagonist in a rabbit cuff model.

This study was performed to determine whether a highly selective nonpeptide alpha(v)beta(3) antagonist (SH306) would prove effective in inhibiting neointima formation in a rabbit cuff model. The animals were dosed with SH306, 5 mg/kg i.v., followed by 10 mg/kg s. c., 3 times daily for 3 days, or with vehicle (10% DMAC). Rabbits were sacrificed and perfused on days 1, 3, and 21; the vessels were paraffin embedded. A reduction in the intima/media (I/M) of the SH306-treated rabbits, as compared with the vehicle-treated control group, was noted (0.20 vs 0.36 [n = 4]). A significant increase in the area of the media was observed in the SH306-treated group versus the control group (0.20 vs 0.13). No difference was observed in cell proliferation between SH306 and vehicle after 1-day and 3-day dosing. Thrombi were found in 43% of the control vessels and in only 14% of the drug-treated vessels. No anticoagulant was used during the surgical procedure. No increase in inhibition of GPIIb/IIIa was observed in SH306-treated animals, as compared with the vehicle control group. We conclude that selective inhibition of alpha(v)beta(3) reduced neointima formation in a rabbit model at 3 weeks.

alphavbeta3, alphavbeta5, and osteopontin are coordinately upregulated at early time points in a rabbit model of neointima formation.

Both smooth muscle cell migration and replication are known to be responsible for neointima formation. Recent reports based on in vitro studies and animal models of neointima formation highlight the possible importance of alphavbeta3 and alphavbeta5 integrins in mediating neointima formation. Clinical data suggest that specific alphavbeta3 blockade may limit restenosis. The aim of this study was to identify the expression of alphavbeta3 and alphavbeta5 and their ligand osteopontin in the very early phases of neointima formation in a rabbit model. A non-occlusive cuff placed around the rabbit femoral artery resulted in a complete, concentric neointima that formed by 14 days. Antibodies specific for the integrin heterodimers and for osteopontin, along with a probe specific for osteopontin mRNA, were used to identify expression at early time points (6 h, 1 day, 3 days, 5 days) post-cuffing. Immunohistochemistry and in situ hybridization expression results were quantitated by image analysis and tested for statistical significance by a two-tailed t-test. The data demonstrated the rapid (within 6 h) and abundant upregulation of alphavbeta3 and alphavbeta5 integrins and their ligand during very early time points of neointima formation. The very early (6 h) upregulation of alphavbeta3 underscores a potentially important clinical intervention point in limiting restenosis following clinical angioplasty procedures.

Orally active isoxazoline glycoprotein IIb/IIIa antagonists with extended duration of action.

Modification of the alpha-carbamate substituent of isoxazoline GPIIb/IIIa (alphaIIb beta3) antagonist DMP 754 (7) led to a series of alpha-sulfonamide and alpha-sulfamide diaminopropionate isoxazolinylacetamides which were found to be potent inhibitors of in vitro platelet aggregation. Aryl- and heteroaryl-alpha-sulfonamide groups, in conjunction with (5R)-isoxazoline (2S)-diaminopropionate stereochemistry, were found to impart a pronounced duration of antiplatelet effect in dogs, potentially due to high affinity for unactivated platelets. Isoxazolylsulfonamide 34b (DMP 802), a highly selective GPIIb/IIIa antagonist, demonstrated a prolonged duration of action after iv and po dosing and high affinity for resting and activated platelets. The prolonged antiplatelet profile of DMP 802 in dogs and the high affinity of DMP 802 for human platelets may be predictive of clinical utility as a once-daily antiplatelet agent.

Oral antiplatelet efficacy and specificity of a novel nonpeptide platelet GPIIb/IIIa receptor antagonist, DMP 802.

This study was undertaken to define the platelet glycoprotein alphaIIb beta3 integrin (GPII/IIIa) affinity, specificity, and oral antiplatelet efficacy of DMP 802, a small-molecule nonpeptide antiplatelet agent. Platelet GPIIb/IIIa integrin binding affinity and specificity for DMP 802 were determined by using binding and adhesion assays with cells from various species, including human. DMP 802 demonstrated a potent antiplatelet efficacy [median inhibitory concentration (IC50), 0.029 +/- 0.0042 microM] in inhibiting human platelet aggregation induced by 10 microM adenosine diphosphate (ADP), as assessed by light-transmittance aggregometry. DMP 802 inhibited 125I-fibrinogen binding to activated (ADP, epinephrine, and arachidonic acid at 100 microM each) gel purified human platelets with an IC50 of 0.012 +/- 0.003 microM. DMP 802 demonstrated tight association with unactivated human, baboon, or canine platelets (t(1/2) of dissociation, 32 +/- 2, 32 +/- 13, and 11 +/- 1 min, respectively). DMP 802 binds with high affinity to both unactivated and activated human platelets (Kd = 0.61 +/- 0.17, 0.57 +/- 0.21 nM, respectively). DMP 802 demonstrated species specificity in inhibiting platelet aggregation with IC50 values ranging from 0.025 to 0.092 microM (human, guinea pig, dog, swine, hamster) and 0.88-1.0 microM (rabbit and rat) in platelets obtained from these various species. DMP 802 demonstrated a high degree of specificity for platelet GPIIb/IIIa (alphaIIb/beta3) as compared with other integrins including alpha(v)beta3 (IC50, >10 microM), alpha(v)beta5 (IC50, >100 microM), alpha4beta1 (IC50, >100 microM), and alpha5beta1 (IC50, >10 microM). Oral antiplatelet efficacy of DMP 802 was examined after single oral (0.05-0.20 mg/kg) and after repeated oral dosing at 0.05 mg/kg daily for 5 days in mongrel dogs. Dose-dependent antiplatelet efficacy with an extended duration of antiplatelet efficacy was demonstrated based on ex vivo inhibition of platelet aggregation induced by 100 microM ADP. DMP 802 has an oral bioavailability of 14.9% in dogs. In conclusion, the alpha sulfonamide isoxazoline analog, DMP 802, is a novel oral antiplatelet agent with high affinity, relatively slow dissociation rate and specificity for human platelet GPIIb/IIIa receptors.

Neoadjuvant chemotherapy with accelerated CNF plus G-CSF in patients with breast cancer tumors larger than three centimeters: a pilot study.

From February 1992 to November 1993, forty patients with operable breast cancer tumors larger than three centimeters were enrolled in this study of accelerated neo-adjuvant chemotherapy. Thirty-seven patients are evaluable: one patient was excluded from the protocol and two refused to continue treatment after the first cycle. Chemotherapy consisted of three presurgical cycles of CNF [cyclophosphamide at 600 mg/m2, mitoxantrone (Novantrone) at 10 mg/m2 and 5-fluorouracil at 600 mg/m2] administered every 2 weeks, plus G-CSF (5 microg/kg s.c./day on days 7-12). Twenty-six of 37 patients (70%) achieved objective tumor response and were submitted to quadrantectomy. Toxicity was easily manageable. After a median 55-month follow-up (range 48-70), no locoregional recurrences were observed. Distant metastases occurred in 12/37 (32%) patients. The five-year disease-free (DFS) and overall (OS) survival were 58% and 80%, respectively. Accelerated CNF plus G-CSF proved to be a safe and tolerable regimen yielding a good clinical response thereby increasing the possibility of breast conservation surgery for patients otherwise candidates for mastectomy.

Discovery of an orally active series of isoxazoline glycoprotein IIb/IIIa antagonists.

Using isoxazoline XR299 (1a) as a starting point for the design of highly potent, long-duration GPIIb/IIIa antagonists, the effect of placing lipophilic substituents at positions alpha and beta to the carboxylate moiety was evaluated. Of the compounds studied, it was found that the n-butyl carbamate 24u exhibited superior potency and duration of ex vivo antiplatelet effects in dogs. Replacement of the benzamidin-4-yl moiety with alternative basic groups, elimination of the isoxazoline stereocenter, and reversal of the orientation of the isoxazoline ring resulted in lowered potency and/or duration of action.

Antithrombotic actions of selective inhibitors of blood coagulation factor Xa in rat models of thrombosis.

The antithrombotic actions of selective factor Xa (FXa) inhibitors, recombinant tick anticoagulant peptide (rTAP) and DX-9065a, were evaluated in experimental thrombosis models in anesthetized rats. In the first model, thrombosis was induced by exposing flowing blood to a silk thread anchored in an arteriovenous (AV) shunt. rTAP, DX-9065a and heparin, given as an iv infusion 1 hr before blood was circulated in the AV shunt, had ID50s of 0.007, 0.6 mumol/kg/hr and 16 U/kg/hr, respectively. In the model of venous thrombosis which was induced by hypotonic saline (0.225%) followed by 15-min stasis of abdominal vena cava, rTAP and heparin had ID50s of 0.007 mumol/kg/hr and 3.5 U/kg/hr, respectively. In both models, full inhibition of thrombus formation was achieved with FXa inhibition at doses which only modestly increased ex vivo plasma clotting time APTT (1.26 to 1.82 over the baseline). By contrast, the maximum antithrombotic effect of heparin was associated with high and significant APTT prolongation (> 5 fold over the baseline). Therefore, our study suggests that FXa inhibitors are effective agents in preventing thrombosis in both rat thrombosis models and may have therapeutic antithrombotic potential.

A monoclonal antibody that recognizes the GPIIb/IIIa antagonist DMP 728. Reversal of the effects of DMP 728 on platelet aggregation and bleeding time in the dog.

Since hemorrhagic events represent a major safety concern associated with the use of new antithrombotic therapies such as glycoprotein (GP) IIb/IIIa receptor blockade, we evaluated the ability of a monoclonal antibody recognizing DMP 728 (cyclic [D-2-aminobutyryl-N2-methyl-L-argininyl-glycyl-L-aspartyl-3- aminomethyl-benzoic acid] methanesulfonic acid salt), a potent GPIIb/IIIa receptor antagonist, to reverse the pharmacological actions of DMP 728 in the dog. DC11 was chosen for in vivo evaluation based on its ability to inhibit the binding of [3H]DMP 728 to activated platelets and to attenuate the inhibition of ADP-induced aggregation on platelet-rich plasma ex vivo by DMP 728. After anesthesia mongrel dogs were given DMP 728 (20 micrograms/kg body wt IV) infused into the femoral vein, bleeding times were determined using a Simplate device from incisions on the backside of the tongue, and platelet aggregation was determined ex vivo. Nearly complete inhibition of platelet aggregation was observed for the dogs treated with DMP 728 (20 ug/kg IV) for up to 210 minutes, and bleeding times were prolonged > 15 minutes for 2 hours and remained elevated for more than 4 hours. DC11 (0.2 or 1.0 mg/kg body wt IV) given to dogs 10 minutes after DMP 728 resulted in 50% attenuation of the effect of DMP 728 on aggregation at 3 hours. Approximately 34% inhibition of the DMP 728-mediated bleeding time was achieved at 1 hour with the 0.2 mg/kg dose, whereas approximately 50% inhibition of the bleeding time was observed for the 1 mg/kg dose at 1 hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Mathematical model of serine protease inhibition in the tissue factor pathway to thrombin.

A mathematical model has been developed to simulate the generation of thrombin by the tissue factor pathway. The model gives reasonable predictions of published experimental results without the adjustment of any parameter values. The model also accounts explicitly for the effects of serine protease inhibitors on thrombin generation. Simulations to define the optimum affinity profile of an inhibitor in this system indicate that for an inhibitor simultaneously potent against VIIa, IXa, and Xa, inhibition of thrombin generation decreases dramatically as the affinity for thrombin increases. Additional simulations show that the reason for this behavior is the sequestration of the inhibitor by small amounts of thrombin generated early in the reaction. This model is also useful for predicting the potency of compounds that inhibit thrombosis in rats. We believe that this is the first mathematical model of blood coagulation that considers the effects of exogenous inhibitors. Such a model, or extensions thereof, should be useful for evaluating targets for therapeutic intervention in the processes of blood coagulation.

Appropriateness of the use of clinical and radiologic examinations and laboratory tests in the follow-up of surgically-treated breast cancer patients. Results of the Working Group on the Clinical Aspects of Follow-up.

BACKGROUND: The aim of this working group was to assess the appropriateness of use of radiologic examinations, laboratory tests and periodic check-ups for surgically-treated, disease-free breast cancer patients. MATERIALS AND METHODS: A total of 252 clinical scenarios (36 for each of the 8 exams considered: clinical examination, mammography, chest roentgenography, hematochemical tests, markers, bone scan, liver echography/abdominal CT scan), each representing a specific surgically treated and disease-free breast cancer patient, were rated by the members of the panel. A 3 point scale was utilized as to whether the test in question was judged inappropriate (1), questionable (2), or appropriate (3) (in the latter case the panel member was also asked for the advised frequency of the exam expressed in months). RESULTS: After two assessment sessions, consensus among members of the panel was reached on 216 of the 252 scenarios; disagreement remained on only 36 clinical scenarios. CONCLUSIONS: The panel agreed that only clinical examinations and mammographies should be recommended for routine clinical follow-up of surgically-treated breast cancer patients. Given the current available therapeutic options, these would assure adequate medical care and psychological aid.

Recombinant plasminogen activator inhibitor type 1: a review of structural, functional, and biological aspects.

Plasminogen activator inhibitor type 1 (PAI-1), a member of the serpin family of serine protease inhibitors, inhibits both tissue-type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA). High PAI-1 levels are associated with an increased risk of thromboembolic disease while PAI-1 deficiency may represent an inherited autosomal recessive bleeding disorder. This review describes the biochemistry of PAI-1 including its purification, conversion between active and latent forms, and interaction with its target serine proteases and its protein cofactor, vitronectin. In addition, an overview of animal studies with PAI-1 is presented to examine its role in regulating fibrinolysis in vivo. For this review, particular emphasis is placed on studies with a recombinant form of bacterially expressed PAI-1 (rPAI-1), which shares many features in common with the active form of native PAI-1.

Mammography and morphobiologic characteristics of human breast cancer.

AIMS: A comparative analysis was performed to verify a possible correlation between mammographic features and morphobiologic characteristics of the tumor in a series of 176 invasive primary breast cancer patients. METHODS: Breast cancers were grouped according to mammographic features as follows: tumor mass with spiculated borders; tumor mass with well-circumscribed borders; tumor with density alteration of parenchyma with no clear borders; a cluster of microcalcifications as the only sign of tumor presence; tumor without mammographic abnormality. The tumor tissue biologic characteristics investigated were: hormone receptor content, tumor proliferative activity, DNA content and cytohistologic tumor-grade differentiation. RESULTS: Spiculated tumors showed a significantly higher percentage of estrogen-receptor-positive cases with respect to circumscribed tumors, independently of the patient's menopausal status. Tumors with only microcalcifications were all from premenopausal patients and showed a significantly higher percentage of progesterone-receptor-positive cases (83%). Tumor proliferative activity did not significantly differ in the 5 mammographic breast cancer groups; aneuploidy was less frequent in tumors with spiculated borders than in mammographic types (39% vs 57%; p = 0.05); percentages of G1-G2-G3 tumors did not differ significantly among the mammographic groups considered. CONCLUSIONS: Certain relationships between mammographic features and biologic characteristics could be of potential clinical interest and stimulate more detailed studies on this issue.

Recombinant plasminogen activator inhibitor-1 protects platelets against the inhibitory effects of plasmin.

Plasmin-induced degradation of platelet glycoprotein Ib (GPIb), the von Willebrand factor (vWF) receptor, has been implicated as a mechanism contributing to the development of platelet dysfunction following cardiopulmonary bypass (CPB). The goal of this study was to assess whether biologically active recombinant plasminogen activator inhibitor-1 (rPAI-1), could antagonize the inhibitory effects of plasmin on GPIb. GPIb function, as evaluated by measuring vWF-dependent, ristocetin-induced platelet agglutination in human platelet rich plasma (PRP) was significantly impaired following incubation with plasmin (60 +/- 14% inhibition, p < 0.01). Inclusion of rPAI-1 (10 micrograms/ml) in the PRP antagonized this plasmin effect, restoring agglutination to 92 +/- 8% of the control value (p < 0.01). The effect of rPAI-1 on the enzymatic activity of plasmin was further evaluated in an amidolytic assay with the plasmin substrate S2251 where an apparent second order rate constant of plasmin inhibition by rPAI-1 of 9.4 x 10(4) M-1 S-1 was determined. Our results suggest that rPAI-1, by inhibiting both tissue plasminogen activator-induced plasmin generation and plasmin activity directly, may have clinical value for improving platelet function during and after CPB.

Mammographic aspect, cell kinetics and hormone receptor status of operable breast cancer.

To investigate the relationships between the mammographic aspects (according to Broberg), proliferative activity and hormone receptor status, we studied 165 patients subjected to radical mastectomy for operable breast cancer. The highest percentage of estrogen receptor-positive cases was observed in mammograph class I (p < 0.02) while classes III and V were highest in progesterone receptor-positive cases. Proliferative activity (111 cases) was significantly lower in class I and IV with respect to all other classes (p < 0.03 and p < 0.01, respectively). This study suggests that certain mammographic signs can be related to the biological characteristics of breast cancer.

Neutralization of the antithrombotic effects of heparin and Fraxiparin by protamine sulfate.

In general, the in vitro anti Xa activity of low molecular weight heparins is neutralized to a lesser degree than the anti Xa activity of unfractionated heparin. To determine whether these differences occur in vivo, a rabbit stasis thrombosis model and a rat laser-induced thrombosis model were utilized. In the laser model, a similar degree of neutralization of the antithrombotic activity of heparin and Fraxiparin was obtained. However, in the stasis thrombosis model, significant antithrombotic activity of Fraxiparin remained after equigravimetric protamine administration. Ex vivo APTT, thrombin time, Heptest, amidolytic anti Xa and anti IIa assays were performed. A coefficient (r = .806) was obtained for the correlation of Heptest activity to antithrombotic effect in the stasis thrombosis model, while the coefficients obtained for the other tests ranged from .152-.570. However, after neutralization by protamine, the thrombin time exhibited the highest correlation coefficient (r = .685) between ex vivo activity and residual antithrombotic effect. Since Fraxiparin retains antithrombotic activity after protamine administration, clinical benefit may be observed for this low molecular weight heparin as compared to unfractionated heparin after neutralization.

Effect of thrombin inhibitors on platelet functions: comparative analysis of DuP 714 and hirudin.

Since thrombin plays an important role in platelet-mediated arterial thrombosis, we have examined the antiplatelet activity of a synthetic thrombin inhibitor, DuP 714 (Ac-(D)Phe-Pro-boroArg), in comparison with that of the naturally occurring inhibitor hirudin. Hirudin was slightly more potent than DuP 714 in inhibiting thrombin-induced aggregation in washed human platelets (IC50s of 72 nM and 150 nM, respectively) and in inhibiting the secretion of plasminogen activator inhibitor-I from human platelets (IC50s of 300 nM and 900 nM, respectively). In contrast, DuP 714 was more potent than hirudin in inhibiting thrombin-induced [125I]fibrinogen binding to gel purified platelets, and in inhibiting thrombin-induced intracellular calcium mobilization in washed platelets. These results indicate that the tripeptide DuP 714 has comparable antiplatelet activity to the 65 amino acid hirudin. We conclude that DuP 714 may have clinical utility in the prevention of platelet-dependent, arterial thrombotic processes.

Ex vivo activity of heparin is not predictive of blood loss after neutralization by protamine.

To study whether the ex vivo activity of heparin and Fraxiparin correlates to and predicts the extent of blood loss induced by the heparins (pre- and post neutralization by protamine), a rat tail transection model and a rabbit ear bleeding model were used. In the rat model heparin (2 mg/kg i.v.) significantly prolonged the bleeding time, while this dose of Fraxiparin had no effect. In the rabbit ear blood loss model, heparin (2 mg/kg i.v.) produced significant increases in blood loss while Fraxiparin (2 mg/kg i.v.) produced approximately 30% of the blood loss induced by heparin. Equigravimetric protamine reduced the heparin-induced blood loss by approximately 50%, however, significant blood loss, thrombin time and Heptest activity remained. Heparin and Fraxiparin (3 mg/kg s.c.) did not cause any increased bleeding. While, all activities of heparin were completely neutralized by protamine, the Heptest activity of Fraxiparin was resistant to neutralization. The ex vivo activity of heparins after neutralization by protamine does not correlate to the extent of blood loss which suggests it may not be necessary to neutralize all ex vivo activities of the heparins to baseline values to be assured that blood loss is reversed.

Comparison of recombinant plasminogen activator inhibitor-1 and epsilon amino caproic acid in a hemorrhagic rabbit model.

A rabbit ear model of blood loss was developed to compare the effects of an active form of recombinant plasminogen activator inhibitor-1 (rPAI-1) with epsilon amino caproic acid (EACA) in antagonizing tissue-type plasminogen activator (r-tPA)-induced blood loss. The antagonism of both rebleeding, which occurs as a result of hemostatic plug degradation, and r-tPA-induced hemorrhage, where rabbits lose approximately 30% of their blood volume, was studied. rPAI-1 (1 mg/kg i.v.) or EACA (70 mg/kg i.v.) antagonized the rebleeding induced by r-tPA (10 micrograms kg-1 min-1) to a similar extent. In the hemorrhagic studies, rPAI-1 effectively antagonized the r-tPA-induced hemorrhage with an ED50 of 3 mg/kg i.v., while the ED50 obtained for EACA was 230 mg/kg i.v. rPAI-1 may be of value in reversing r-tPA-induced blood loss during thrombolytic therapy or in clinical situations where excessive fibrinolysis contributes to bleeding.

[Lymph nodes of the neck. Diagnosis using magnetic resonance with gradient-echo technique]. / I linfonodi del collo. Diagnosi mediante risonanza magnetica con tecniche a eco di gradiente.

As for the pathologic conditions of neck lymph nodes, the clinician needs to know if the involved node is reactive, phlogistic, or neoplastic in nature. If accurate tumor staging is required, imaging techniques play a fundamental role. Our study was aimed at assessing the actual role of MR imaging in the evaluation of neck lymph node involvement. The study was performed using an MR Max Plus by General Electrics operating with an 0.5 T superconductive magnet. We employed gradient-echo (GE) pulse sequences with TR 500, TE 15 ms and 90 degrees flip angle for T1-weighted images, and with TR 500, TE 30 ms and 25-30 degrees flip angles for T2-weighted images; for Pd-T2-weighted images, TR was 520, TE 30 ms, and flip angles were 40-45 degrees. The results were correlated with histopathologic findings obtained at biopsy. The advantages of GE sequences were: 1) whole neck imaging--thus saving time, and reducing radiation dose and contrast media; 2) optimal anatomical and topographic evaluation of the lesion; 3) imaging of the longitudinal diameter of the node; 4) higher sensitivity for lymph node tissue modifications; 5) imaging of necrosis, hemorrhage, and/or fibrosis. GE sequences were especially useful for accurate tumor staging, in the follow-up, and to verify response to therapy. However, even though MR imaging has proven to have high sensitivity, its specificity was similar to that of contrast-enhanced CT. Further studies with the use of paramagnetic contrast media are needed to solve these problems.