First-line Herceptin monotherapy in metastatic breast cancer.

The pivotal phase II and III Herceptin trials proved the efficacy and safety of second- or third-line single-agent Herceptin and first-line Herceptin in combination with chemotherapy, respectively. In the current trial, 114 patients were randomized to one of two dose groups of first-line Herceptin monotherapy: standard dose of 4 mg/ kg initial dose followed by 2 mg/kg intravenous (i.v.) weekly; or high dose of 8 mg/kg initial dose followed by 4 mg/kg i.v. weekly. The regimen was generally well tolerated. A similar incidence of adverse events was demonstrated in the two dose groups with the possible exception of acute infusion-related events such as fever and chills as well as rash and dyspnea, which appear to be more prevalent in the higher dose group. The overall response rate was 26% and response rates were similar between the two dose groups (24% for the standard Herceptin dose group and 28% for the high Herceptin dose group). Subgroup analysis determined a higher response rate in IHC 3+ patients (35%) and FISH-positive patients (41%). When women with stable disease for > or =6 months were included with responders, the clinical benefit rate in IHC 3+ patients was 47%. Median survival was 24.4 months, which is comparable with the survival rate seen in the pivotal phase III combination trial (25 months). Therefore, single-agent Herceptin is an important new option for the first-line treatment of HER2-positive metastatic breast cancer patients.

First-line, single-agent Herceptin(R) (trastuzumab) in metastatic breast cancer. a preliminary report.

Following confirmation of the appropriate dosage, safety and potential efficacy of Herceptin(R) (trastuzumab) in small-scale phase I and II trials involving patients with refractory disease, a large trial was conducted in 222 patients with breast cancer who had relapsed after one or two chemotherapy regimens for their metastatic disease. The results showed a positive and durable overall response rate (15% according to a response evaluation committee (REC) assessment) using trastuzumab monotherapy (initial dose 4 mg/kg intravenously (i.v.) followed by 2 mg/kg i.v. weekly). In another recently completed phase II trial, 113 patients were randomised to two dose levels (initial dose of 4 mg/kg i.v. dose followed by 2 mg/kg i.v. weekly, or initial dose of 8 mg/kg followed by 4 mg/kg i.v. weekly) of single-agent trastuzumab as first-line therapy for metastatic disease. The preliminary overall response rate was 23% based on investigator assessment, and tolerability was excellent as in previous trials; efficacy was similar in both dose groups, but the side-effects tended to be more frequent in the higher dose group. The preferred dosage is therefore the same as that currently recommended, i.e. an initial dose of 4 mg/kg i.v. followed by 2 mg/kg weekly i.v. until disease progression.

First-line, single-agent Herceptin(trastuzumab) in metastatic breast cancer: a preliminary report.

Following confirmation of the appropriate dosage, safety and potential efficacy of Herceptin(trastuzumab) in small-scale phase I and II trials involving patients with refractory disease, a large trial was conducted in 222 patients with breast cancer who had relapsed after one or two chemotherapy regimens for their metastatic disease. The results showed a positive and durable overall response rate (15% according to a response evaluation committee (REC) assessment) using trastuzumab monotherapy (initial dose 4 mg/kg intravenously (i.v.) followed by 2 mg/kg i.v. weekly). In another recently completed phase II trial, 113 patients were randomised to two dose levels (initial dose of 4 mg/kg i.v. dose followed by 2 mg/kg i.v. weekly, or initial dose of 8 mg/kg followed by 4 mg/kg i.v. weekly) of single-agent trastuzumab as first-line therapy for metastatic disease. The preliminary overall response rate was 23% based on investigator assessment, and tolerability was excellent as in previous trials; efficacy was similar in both dose groups, but the side-effects tended to be more frequent in the higher dose group. The preferred dosage is therefore the same as that currently recommended, i.e. an initial dose of 4 mg/kg i.v. followed by 2 mg/kg weekly i.v. until disease progression.

Platelet activation in patients after an acute coronary syndrome: results from the TIMI-12 trial. Thrombolysis in Myocardial Infarction.

UNLABELLED: This study was designed to determine the magnitude and time course of platelet activation during therapy of acute coronary syndromes with an oral platelet antagonist. BACKGROUND: Platelet activation and aggregation are central to the pathogenesis of the acute coronary syndromes (ACS). However, few data are available on levels of platelet activation over time in patients with ACS, especially in the setting of chronic glycoprotein (GP) IIb/IIIa inhibition. METHODS: The Thrombolysis in Myocardial Infarction (TIMI) 12 trial was a phase II, double-blind trial evaluating the effects of sibrafiban, an oral, selective antagonist of the platelet glycoprotein IIb/IIIa receptor in patients stabilized after an ACS. A subset of 90 of the 329 patients in the study had measurement of platelet activation as assessed by the expression of platelet associated P-Selectin on days 0, 7 and 28. Platelet activation was measured in blood samples that were fixed either immediately (spontaneous activation) or after 5 minute incubation with 0, 1 microM or 5 microM ADP in order to assess platelet responsiveness to very low or moderate stimulation. RESULTS: At baseline there was a significant elevation of spontaneous platelet activation as compared to samples obtained from normal donors or from patients who did not have acute coronary syndromes (ACS patients 27.6+/-18.7%, Normal controls 8.5+/-4.4%, Patient controls 10.9+/-7.1%, p < 0.005 for both). In addition, there was a significant decrease in the levels of platelet activation with time during the 28 days of treatment with sibrafiban. Nevertheless, even on day 28, the TIMI-12 patients continued to show elevated platelet activation in comparison to the control groups (p < 0.05 for both). CONCLUSIONS: These results suggest that platelets remain activated long after clinical stabilization post ACS. Although platelet activation decreased after one month of oral GPIIb/IIIa inhibition, levels remained higher than normal, suggesting the need for long-term antiplatelet therapy following ACS.

Randomized trial of an oral platelet glycoprotein IIb/IIIa antagonist, sibrafiban, in patients after an acute coronary syndrome: results of the TIMI 12 trial. Thrombolysis in Myocardial Infarction.

BACKGROUND: Inhibitors of the platelet glycoprotein IIb/IIIa receptor given intravenously have been shown to be effective in reducing ischemic complications after coronary angioplasty and in unstable angina, making this a promising new class of agents for the treatment and prevention of ischemic events in patients with acute coronary syndromes. Sibrafiban (Ro 48-3657) is an oral, peptidomimetic, selective antagonist of the glycoprotein IIb/IIIa receptor. METHODS AND RESULTS: The Thrombolysis in Myocardial Infarction (TIMI) 12 trial was a phase II, double-blind, dose-ranging trial designed to evaluate the pharmacokinetics (PK), pharmacodynamics (PD), safety, and tolerability of sibrafiban in 329 patients after acute coronary syndromes. In the PK/PD cohort of TIMI 12, 106 patients were randomized to receive one of seven dosing regimens of sibrafiban, ranging from 5 mg daily to 10 mg twice daily for 28 days. In the safety cohort, 223 patients were randomized to one of four dose regimens of sibrafiban (ranging from 5 mg twice daily to 15 mg once daily) or aspirin for 28 days. High levels of platelet inhibition were achieved: mean peak values ranged from 47% to 97% inhibition of 20 micromol/L ADP-induced platelet aggregation on day 28 across the seven doses. Twice-daily dosing provided more sustained platelet inhibition (mean inhibition, 36% to 86% on day 28), whereas platelet inhibition returned to baseline levels by 24 hours with once-daily dosing. Major hemorrhage occurred in 1.5% of patients treated with sibrafiban and in 1.9% of patients treated with aspirin. Protocol-defined "minor" bleeding, usually mucocutaneous, occurred in 0% to 32% of patients in the various sibrafiban groups and in none of the patients treated with aspirin. Minor bleeding was related to total daily dose (P=.002), once- versus twice-daily dosing (P<.0001), renal function (P<.0001), and presentation with unstable angina (P<.01). CONCLUSIONS: The oral glycoprotein IIb/IIIa antagonist sibrafiban achieved effective, long-term platelet inhibition with a clear dose-response but at the expense of a relatively high incidence of minor bleeding. Oral IIb/IIIa inhibition deserves further study as a new treatment strategy in patients after acute coronary syndromes.

Pregnancies in a patient with congenital absence of prothrombin activity: case report.

Congenital hypoprothrombinemias are very rare, inherited disorders in which factor II (prothrombin) levels and/or activity are extremely low or absent. We report eight pregnancies in a patient with this disorder. Obstetric complications attributed to the coagulation disturbance included first-trimester bleeding in each pregnancy, miscarriage in four of the pregnancies, spontaneous maternal subarachnoid hemorrhage in one, and postpartum hemorrhage in one of four term pregnancies despite administration of clotting factor concentrate. The management of pregnancy in congenital hypoprothrombinemia, and issues of coagulation factor replacement, are discussed.

Agmatine, a bioactive metabolite of arginine. Production, degradation, and functional effects in the kidney of the rat.

Until recently, conversion of arginine to agmatine by arginine decarboxylase (ADC) was considered important only in plants and bacteria. In the following, we demonstrate ADC activity in the membrane-enriched fraction of brain, liver, and kidney cortex and medulla by radiochemical assay. Diamine oxidase, an enzyme shown here to metabolize agmatine, was localized by immunohistochemistry in kidney glomeruli and other nonrenal cells. Production of labeled agmatine, citrulline, and ornithine from [3H]arginine was demonstrated and endogenous agmatine levels (10(-6)M) in plasma ultrafiltrate and kidney were measured by HPLC. Microperfusion of agmatine into renal interstitium and into the urinary space of surface glomeruli of Wistar-Frömter rats produced reversible increases in nephron filtration rate (SNGFR) and absolute proximal reabsorption (APR). Renal denervation did not alter SNGFR effects but prevented APR changes. Yohimbine (an alpha 2 antagonist) microperfusion into the urinary space produced opposite effects to that of agmatine. Microperfusion of urinary space with BU-224 (microM), a synthetic imidazoline2 (I2) agonist, duplicated agmatine effects on SNGFR but not APR whereas an I1 agonist had no effect. Agmatine effects on SNGFR and APR are not only dissociable but appear to be mediated by different mechanisms. The production and degradation of this biologically active substance derived from arginine constitutes a novel endogenous regulatory system in the kidney.

Diamine oxidase is the amiloride-binding protein and is inhibited by amiloride analogues.

Diamine oxidase (histaminase), an enzyme that oxidatively deaminates putrescine and histamine, was purified from human placenta and from pig kidney. Both NH2-terminal sequences are highly homologous to the human kidney amiloride-binding protein, previously thought to be a component of the amiloride-sensitive Na+ channel. Monoclonal antibodies raised against the pig kidney amiloride-binding protein immunoprecipitate a polypeptide with the same M(r) (105,000) as that of pig kidney diamine oxidase. That polypeptide has both diamine oxidase activity and the capacity to bind [3H]phenamil, a tritiated amiloride derivative. Cells stably transfected with human kidney amiloride-binding protein cDNA express a high diamine oxidase activity. In transfected cells as well as with the purified enzyme, this activity was inhibited by amiloride and by some of its derivatives, such as phenamil and ethylpropylamiloride. Amiloride inhibition seems to be due to drug binding at the active site of the enzyme. These data indicate that human placental diamine oxidase is identical to the human kidney amiloride-binding protein and that amiloride analogues may have wider physiological effects besides those on epithelial ion transport.

Identification of novel heparin-releasable proteins, as well as the cytokines midkine and pleiotrophin, in human postheparin plasma.

The heparin-releasable proteins are a group of proteins that are targeted to the endothelial surface by attachment to glycosaminoglycans and may have functions specific to the endothelium-blood interface. In this study, heparin-affinity chromatography of human postheparin plasma was used as a method to identify and study novel heparin-releasable proteins. Six proteins seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels have increased levels in plasma after intravenous heparin. The six proteins are platelet factor 4, midkine, pleiotrophin, and several novel proteins. Midkine and pleiotrophin are related cytokines that are developmentally regulated, neurotrophic, and mitogenic. Additional studies show that levels of midkine and pleiotrophin peak at 10 to 30 minutes after injection of heparin. Heparin-releasable midkine and pleiotrophin do not originate from blood cells or the kidney. Heparin-releasable midkine may originate from endothelial cells. Soft agar culture of an adenocarcinoma cell line (SW-13) demonstrates growth-stimulating activity similar to that described for pleiotrophin in the heparin-agarose eluate of postheparin plasma but not in the heparin-agarose eluate of preheparin plasma. It is concluded there are more heparin-releasable proteins than previously identified, including midkine and pleiotrophin, and that heparin-affinity chromatography of postheparin plasma is a useful technique for identifying novel heparin-releasable proteins.

Lipoprotein associated coagulation inhibitor, factor VII, antithrombin III, and monocyte tissue factor following surgery.

Fifteen patients undergoing major surgical procedures were evaluated for lipoprotein associated coagulation inhibitor (LACI) antigen, factor VII (F VII), antithrombin III (AT III), and peripheral blood monocyte tissue factor (TF) activity immediately before surgery and on following days. A peak in monocyte TF activity occurred between the first and fifth days after surgery in 10 of the patients, while LACI, F VII, and AT III levels dropped in a qualitatively parallel manner in 8 of these patients. LACI, F VII, and AT III levels decreased after surgery in two additional patients even though TF activity also decreased after surgery in these patients. In the remaining 3 patients who developed infections during the study, TF activity rose within 2 days of the diagnosis of infection in addition to the postoperative peak. In two of these patients, LACI levels increased dramatically near the end of the study period without concomitant changes in F VII and AT III. Overall, the presurgical TF levels in disrupted monocytes varied 52-fold and the maximal TF activity varied 24-fold among patients. The TF response following surgery is therefore heterogenous in both temporal occurrence and magnitude of the postsurgical peak. The patients also varied considerably in the presurgical levels of monocyte TF activity. A possible association between the level of presurgical TF activity and the magnitude of the postsurgical peak was noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of lipoprotein-associated coagulation inhibitor (LACI) on thromboplastin-induced coagulation of normal and hemophiliac plasmas.

Lipoprotein-associated coagulation inhibitor (LACI) is a plasma-derived protein that inhibits the tissue factor/factor VIIa/calcium/phospholipid complex in a factor Xa-dependent manner. Recombinant LACI (rLACI) added exogenously to plasma prolonged the activated partial thromboplastin time (APTT) and prothrombin time (PT) as a function of rLACI concentration in linear and curvilinear manners, respectively. Under these standard assay conditions, the amounts of rLACI required to double the APTT and PT were approximately 350- and 90-fold the plasma concentration of LACI, respectively. Likewise, addition of antibodies against LACI to pooled normal, factor VIII-deficient, or factor IX-deficient plasma had no effect on their respective APTTs and PTs, demonstrating the insensitivity of these assays to endogenous LACI. The prothrombin time assay was modified by using dilute thromboplastin. Unlike the standard prothrombin time assay, the clotting times were prolonged for factors VIII- or IX-deficient plasma relative to pooled normal plasma in this modified PT assay. Additionally, the degree of factor deficiency, as determined by the APTT assay, was correlated with that determined by the modified PT assay using dilute thromboplastin. When antibodies against LACI were added to pooled normal, factor VIII-deficient, or factor IX-deficient plasma and the prothrombin time assay initiated using dilute thromboplastin, the clotting times for antibody-treated plasma were shorter than for the corresponding plasma in the absence of antibodies. Moreover, the clotting times for factors VIII- and IX-deficient plasmas treated with antibodies raised against LACI were at least as fast as pooled normal plasma in the absence of LACI antibodies when dilute thromboplastin was used to initiate clotting. These results suggest that the prothrombin time assay using dilute thromboplastin may more accurately reflect what occurs in vivo and that LACI may play an important role in the prolonged bleeding of those with hemophilia A or B.

Purification and properties of heparin-releasable lipoprotein-associated coagulation inhibitor.

The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin-releasable LACI (HRL), as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

Plasma antigen levels of the lipoprotein-associated coagulation inhibitor in patient samples.

Human plasma contains an inhibitor of tissue factor-initiated coagulation known as the lipoprotein-associated coagulation inhibitor (LACI) or also known as the extrinsic pathway inhibitor (EPI). A competitive fluorescent immunoassay was developed to measure the plasma concentration of LACI in samples from normal individuals and patients with a variety of diseases. The LACI concentration in an adult control population varied from 60% to 160% of the mean with a mean value corresponding to 89 ng/mL or 2.25 nmol/L. Plasma LACI levels were not decreased in patients with severe chronic hepatic failure, warfarin therapy, primary pulmonary hypertension, thrombosis, or the lupus anticoagulant. Plasma LACI antigen was decreased in some, but not all patients with gram-negative bacteremia and evidence for disseminated intravascular coagulation. Plasma LACI levels were elevated in women undergoing the early stages of labor (29%), in patients receiving intravenous tissue-type plasminogen activator (45%), and in patients receiving intravenous heparin (375%). A radioligand blot of the pre- and post-heparin plasma samples shows the increase to be in a 40-Kd form of LACI. Very low levels of plasma LACI antigen were found in patients with homozygous abetalipoproteinemia and hypobetalipoproteinemia, diseases associated with low plasma levels of apolipoprotein B containing lipoproteins. Following the injection of heparin into one patient with homozygous abetalipoproteinemia, the plasma LACI antigen level increased to a level comparable with that in normal individuals after heparin treatment.

The lipoprotein-associated coagulation inhibitor.

TF mediated initiation of coagulation appears to play a critical role in normal hemostasis and probably pathologic thrombosis as well. Although teleological considerations would seem to suggest that a specific regulator of this process should exist, and although the presence in plasma of such an inhibitor was documented many years ago, it was not until the past five years that the inhibitor was characterized and its mechanism of action defined. LACI produces factor Xa-dependent feedback initiation of the VIIa/TF catalytic complex. The mechanism of this feedback inhibition is novel. First, LACI, a multi-headed protease inhibitor, binds factor Xa, a product of VIIa/TF catalysis, at one of its inhibitory domains. The Xa-LACI complex, possibly acting as a pseudosubstrate, then is able to bind to VIIa/TF in an appropriate conformation such that a second inhibitory domain of LACI is positioned to interact with factor VIIa in the VIIa/TF complex. Whether such a unique means of eliciting feedback inhibition in a protease cascade is repeated in nature is unknown. The existence of LACI appears to help explain the clinical need for both "extrinsic" and "intrinsic" coagulation pathways. In addition, data to the present are consistent with the notion that, in normal hemostasis at least, TF is responsible for an initial burst of factor Xa generation which provides sufficient thrombin to induce the aggregation of platelets and the activation of the critical coagulation cofactors factor V and factor VIII. Ultimate and persistent hemostasis, however, appears to require the continued production of additional factor Xa through the action of factor IXa and factor VIII. The fact that patients with factor XI deficiency suffers a variable but usually mild bleeding diathesis suggests that under certain conditions the initial burst of factor IXa formed through the action of VIIa/TF is insufficient and supplemental factor IXa generated by factor XIa is needed for normal hemostasis. The mechanism by which this factor XIa is generated in vivo, however, has not been determined. We stress that the predicted in vivo role of LACI is simply that--a prediction based on its known in vitro properties. Documentation of its physiologic importance remains to be provided and is an area of active research. Further, although significant progress has been made over the past few years in the characterization of LACI, many questions remain unanswered. For example: What is the mechanism for LACI's association with lipoproteins in plasma? What function, if any, does the third Kunitz-type protease inhibitor domain in LACI serve? (ABSTRACT TRUNCATED AT 400 WORDS)

Endogenous phosphorylation of the lipoprotein-associated coagulation inhibitor at serine-2.

Lipoprotein-associated coagulation inhibitor (LACI) inhibits activated Factor X (Xa) directly and, in an Xa-dependent fashion, inhibits Factor VIIa-tissue factor (TF), presumably by forming a quaternary Xa-LACI-VIIa-TF complex. LACI isolated from the conditioned media of HepG2 cells grown in the presence of [32P]orthophosphate was observed to be covalently phosphorylated. Dephosphorylation of 32P-LACI with phosphatase resulted in an almost complete removal of the radiolabel. Phosphoamino acid analysis of the purified 32P-LACI established that the phosphorylation occurred on (a) serine residue(s). At its N-terminus, LACI contains a cluster of acidic residues C-terminal to the serine-2 residue. Such a site is characteristic of the sites phosphorylated by casein kinase II (CKII) in protein substrates. Edman degradation of endogenously labelled 32P-LACI revealed that the serine-2 residue was a major site of phosphorylation. Phosphorylation of purified LACI by bovine CKII was observed to occur in vitro; amino acid sequence analysis demonstrated that CKII phosphorylated LACI at the serine-2 residue. Recombinant LACI expressed from mouse C127 fibroblasts transfected using a bovine-papilloma-virus expression vector was found to be endogenously phosphorylated. By using site-directed mutagenesis, an altered form of LACI was produced in which the serine-2 residue had been changed to alanine. This altered LACI, although expressed in similar quantity to the wild-type LACI, was not detectably phosphorylated. Using the altered LACI in functional studies demonstrated that a serine residue at position 2, and thus the phosphorylation of this site, was not essential for LACI's inhibition of Xa and VIIa-TF activities.

Inhibition of factor VIIa-tissue factor coagulation activity by a hybrid protein.

Lipoprotein-associated coagulation inhibitor (LACI) appears to inhibit tissue factor (TF)-induced blood coagulation by forming a quaternary inhibitory complex containing factor Xa, LACI, factor VIIa, and TF. A genetically engineered hybrid protein consisting of the light chain of factor Xa and the first Kunitz-type inhibitor domain of LACI is shown to directly inhibit the activity of the factor VIIa-TF catalytic complex. Unlike inhibition of factor VIIa-TF activity by native LACI, inhibition by the hybrid protein is not dependent on factor Xa. In an assay of TF-induced coagulation, 50% TF inhibition occurs with hybrid protein at 35 nanograms per milliliter, whereas LACI at 2.5 micrograms per milliliter is required for an equivalent effect. gamma-Carboxylation of glutamic acid residues in the factor Xa light chain portion of the hybrid protein is required for inhibitory activity, indicating that the first Kunitz-type domain of LACI alone is not sufficient for inhibition of factor VIIa-TF.