FLOCOR: a new anti-adhesive, rheologic agent.

FLOCOR (purified poloxamer 188) is a surfactant molecule that represents a new class of pharmacological agent. FLOCOR is different in that its activity does not depend on high affinity receptor binding but rather on altering the way cells and molecules interact with water. Extensive preclinical studies in models of vascular occlusive disorders, including circulatory shock, acute stroke, and myocardial infarction (MI), suggest that iv. administration of poloxamer 188 improves microvascular blood flow in ischaemic tissues by inhibiting adhesive interactions, lowering blood viscosity and reducing friction along the vessel wall. In clinical studies, poloxamer 188 demonstrated statistically significant benefits in patients with acute myocardial infarction (AMI) and acute vaso-occlusive crisis of sickle cell disease. However, these studies were conducted with commercial grades of poloxamer 188 that contained nephrotoxic impurities, and elevated creatinine was observed in a small percentage of patients. A new highly purified version of poloxamer 188 free from impurities has been developed. Highly purified poloxamer 188 (trade name FLOCOR) is better tolerated in models of renal failure and is anticipated to have a significantly improved therapeutic index compared to commercial grade poloxamer 188. Clinical studies are now in progress in order to confirm the therapeutic benefits of FLOCOR (purified poloxamer 188).

Inhibition of cytarabine-induced MDR1 (P-glycoprotein) gene activation in human tumor cells by fatty acid-polyethylene glycol-fatty acid diesters, novel inhibitors of P-glycoprotein function.

Fatty acid ester surfactants Cremophor EL and Solutol HS 15 were described earlier as modulators of multidrug resistance mediated by MDR1 P-glycoprotein (Pgp). We have shown that the most active components of these polydisperse surfactants are fatty acid-polyethylene glycol-fatty acid diesters (FA-PEG-FA). A new generation of Pgp-surfactant inhibitors of defined structure was therefore synthesized. In the present study we show that these compounds are also able to inhibit up-regulation of MDR1 gene expression caused by cytarabine (ARA-C) and doxorubicin in human tumor cell lines H9 and KB 3-1, which express minimal levels of MDR1 mRNA. The surfactant inhibitors, however, had no effect on the induction of MDR1 gene expression by protein kinase C agonists. Using a set of FA-PEG-FA diesters with various fatty acids and different lengths of the PEG domain, we demonstrated that the activity of diester preparations as inhibitors of drug-induced MDR1 activation was in proportion to their activity as inhibitors of Pgp function. Oleic and stearic acid diesters with PEG 900 (20 ethylene oxide units) were the most potent. The poloxamer analogs of these diesters demonstrated similar effects. In contrast, the well-known, structurally unrelated inhibitors of Pgp activity, verapamil, cyclosporin A and PSC 833, had no inhibitory effect on drug-induced MDR1 activation. The ability of FA-PEG-FA diesters to inhibit both Pgp function and drug-induced MDR1 activation suggests that these chemomodulators may be uniquely useful for the prophylaxis of Pgp-mediated multidrug resistance in drug-treated tumors.

Reversal of multi-drug resistance in vitro by fatty acid-PEG-fatty acid diesters.

Fatty acid ester surfactants, e.g., Cremophor EL and Solutol HS 15, that modulate multi-drug resistance (MDR) have been described; however, the drug potential of these preparations is unclear because of the molecular heterogeneity of these and other commercial surfactants. In previous experiments, an active but still polydisperse preparation, designated CRL 1337, was synthesized by reacting purified oleic acid with a 20-fold molar excess of ethylene oxide. We have subjected this preparation to chromatographic separation, and infrared analysis of the active fractions revealed a significant component of diester structures (fatty acid-PEG-fatty acid). A new generation of diester compounds has now been synthesized. Preparations comprised of 99% diesters were significantly more potent than monoester preparations for MDR modification activity in vitro. As previously determined for ethylene oxide-derived preparations similar to CRL 1337, the nature of the fatty acid domains proved to be important for activity, as was the relative length of the polyethylene glycol domain (which determines the hydrophile-lipophile balance). The ester linkage appeared unimportant since homologous diethers and diamides had activity similar to that of diesters. Stearic acid diester was 1.5- to 7-fold more potent than CRL 1337 when tested in cell proliferation inhibition assays. In light of these structural restrictions on drug potentiation, and since these surfactants are active at relatively low concentrations (below 1 microgram/ml), investigations of their mechanism of action were initiated by exploring specific interactions with P-glycoprotein. Both active and inactive diesters inhibited azidopine labeling of P-glycoprotein, suggesting that fatty acid-PEG diesters can interfere with P-glycoprotein substrate binding. Other attributes of these preparations must contribute to their ability to reverse MDR.

Comparison of solutol HS 15, Cremophor EL and novel ethoxylated fatty acid surfactants as multidrug resistance modification agents.

Some well-known fatty acid ester surfactants, e.g., Cremophor EL and Solutol HS 15, are modulators of multidrug resistance in vitro and in vivo. Because they are polydisperse, and their active component(s) have not been identified, the therapeutic potential of such surfactants is unclear. To better define the active components of Solutol HS 15 and to make more potent surfactant multidrug resistance modulators, highly purified C-18 fatty acids were esterified with ethylene oxide at 5-200 molar ratios. Unexpectedly, ethylene oxide esters of pure 12-hydroxy stearic acid, the major components of Solutol HS 15, displayed negligible resistance modification activity compared with Solutol HS 15 itself or to stearic and oleic acid esters synthesized under identical conditions. Since oleic acid esters appeared to have good activity, a series of these compounds was prepared to determine the optimal ethylene oxide/fatty acid ratio. The optimal ratio was found to be 20 mole ethylene oxide: I mole fatty acid, with a steep decline in activity for products made with ratios above and below the optimum. The most active oleic acid ester, designated CRL 1337, was 8.4-fold as potent as Solutol HS 15 and over 19-fold as potent as Cremophor EL in promoting rhodamine 123 accumulation in multidrug-resistant KB 8-5-11 cells in vitro. Our results show that the structure of the hydrophobic domain (fatty acid) of surfactants as well as its hydrophile-lipophile balance are critical in determining the potency of surfactants as reversing agents.

Pharmacokinetics of heparins differing in mean molecular weight using a Xa amidolytic and Heptest clotting method.

The pharmacokinetics of heparins ranging in mean MW from 23,000 to 4,500 were determined using a new clotting test (Heptest) and an anti-Xa, amidolytic method. No significant difference between the two methods was observed in the calculated values for distribution volume (Vd), halflife (t1/2), plasma clearance (Clp) or area under the concentration-time curve (AUC) for heparins of any MW. However, when pharmacokinetic parameters calculated from both methods were compared as a function of MW, significant differences were observed. Values for t1/2 averaged from the two methods increased with decreasing MW. The t1/2 values were 30, 35, and 50 min for the 23,000, 13,300, and 5,100 MW fractions, respectively. Similarly, AUC increased with decreasing MW while Clp decreased. All heparins were observed to have VdS approximating the plasma compartment. A slightly larger Vd was observed with the lowest MW heparin.

In vitro evaluation of heparin fractions: old vs. new methods.

Conventionally, heparin has been evaluated for its anticoagulant effect by various nonspecific, poorly standardized coagulant methods such as the U.S. Pharmacopeial and other global (APTT) tests. However, these assays are relatively insensitive to heparin fractions and fragments, and give varying with the newer modes of heparin administration. It has become necessary, therefore, to develop other methods able to detect a proper therapeutic/prophylactic level and to evaluate a standard potency for these agents. Newer amidolytic assays utilizing synthetic substrates provide for specific anti-factor Xa or anti-factor IIa evaluation. Although these factors are believed to reflect the antithrombotic effect of heparin and its fractions/fragments, conclusive clinical studies are not available at this time. Interactions with non-AT-III-mediated pathways, the contact, kallikrein-kinin, and fibrinolytic system, other coagulant factors, endothelium, and eicosanoid system all can contribute to the overall antithrombotic response of heparin. Some of these actions are not measurable by current in vitro test methods. Amidolytic, immunochemical, and physical assays using various activators to generate endogenous factor Xa, as well as assays for the detection of metabolic or release products of each of these systems, may be more relevant to in vivo conditions. Specific low molecular weight markers such as fibrinopeptide A, which provide the earliest detection of hemostatic activation, can be measured directly, or an in vitro system can be developed to quantitate the relative generation of these markers. These methods can then be modified to assay the in vitro potency of heparin preparations. With proper consideration of reagent specificity and concentration, molecular interactions, reaction time and temperature, ionic type and concentration, reliable accurate methods for a true in vitro representation of in vivo events may be developed. Clinical assays must measure an endogenous response to therapy and not merely an absolute circulating level of drug. However, a standardized pure enzyme system may be ideal to evaluate the potency of these agents. The development of heparin derivatives necessitates a reassessment of currently practiced laboratory technology. Furthermore, well-defined biochemical methods are required for a standardized evaluation of potency and clinical monitoring of these agents.

Laboratory studies on the intravenous and subcutaneous administration of PK 10169 in man.

Intravenous and subcutaneous administration of PK 10169 in healthy human volunteers were studied utilizing numerous old and new laboratory methods for the monitoring of the effects of this agent. In the intravenous studies, individual groups of 10 healthy volunteers were administered with 2,500-12,500 anti-Xa units (25-125 mg) PK 10169 as a single bolus, and blood samples were drawn at 30 min, 1, 4 and 24 h after the administration of the drug. In the subcutaneous studies, 4,000 anti-Xa units (40 mg) b.i.d. of PK 10169 were administered to a group of 10 healthy individuals for 7 consecutive days, and blood samples were drawn 6 h after each dose. Citrated blood samples collected from both studies were centrifuged, plasma was frozen at -70 degrees C in aliquots, and various laboratory parameters were determined in batches. In the intravenous studies, the global clotting assays (activated partial thromboplastin time and thrombin time) were only prolonged at initial stages of treatment, whereas a marked increase in the antiprotease and antifibrinopeptide A generation activity titer was observed for periods of up to 24 h. In the subcutaneous studies, no significant prolongation of the global clotting assays was noted; however, the anti-Xa levels were significantly increased throughout the study period (7 days). Only a trace of anti-IIa activity was observed. In contrast, a strong antifibrinopeptide A generation activity was observed in all samples which persisted after the discontinuation of PK 10169. These studies suggest that PK 10169 may exert its clinical effects by multiple mechanisms which are only partially assessable by routine laboratory methods.

Usefulness of fibrinopeptide A generation tests in experimental and clinical studies with low molecular weight heparin fractions.

FPA generation test provides a sensitive tool to study the events leading to the production of fibrin from fibrinogen. Drug modulation of pathways resulting in thrombin generation can be readily assessed utilizing the FPA generation test. In circumstances in which conventional clotting and amidolytic methods fail to detect any antiprotease effects, the FPA generation test provides the sensitivity to study these actions. The FPA generation test demonstrates the collective action of antithrombotic agents on inhibition of activation of the pathways leading to the generation of thrombin. The generation systems are easily modified by activators to provide identification of sites, components, and modulating interaction of various heparin derivatives on the hemostatic system. The FPA generation test assesses the overall inhibitory effects of heparin and thus provides insight into the pharmacokinetics of the biologically active components of these agents. Currently, the FPA generation test may be utilized to demonstrate pharmacokinetics and pharmacodynamics but has limited quantitative capacity in this regard. The FPA generation test performed on native plasma may provide insight into the great variation in individual hemostatic parameters important to dosage determinations.

Studies on the antithrombotic effects and pharmacokinetics of heparin fractions and fragments.

The pharmacokinetics of heparin differ markedly from those of its fractions both in man and in experimental animal models. The route of administration determines the relative availability of different molecular species that exert the anti-Xa, anti-IIa, fibrinopeptide A generation inhibiting actions and the release of tissue plasminogen activator-like activity from the endothelial cell lining. The bioavailability of heparin fractions has proved to be much greater than heparin after subcutaneous or intraperitoneal administration. Most of the low molecular weight heparin fractions exhibit sustained antiprotease and antithrombotic actions. The pharmacokinetics of the specific anti-IIa and anti-Xa actions of heparin and its fractions is dependent on the molecular composition of these agents. Even if the fractions are standardized for identical potencies by the in vitro assays, the elimination rate of anti-Xa and anti-IIa actions are significantly different for each fraction. The antithrombotic actions of heparin and its fractions also vary widely in the rabbit stasis thrombosis model. Different fractions show variable antithrombotic actions against defined thrombogenic challenges. Moreover, selection and potency of a thrombogenic agent is of crucial importance in these studies. The primate (Macaca mulatta) model offers a useful preclinical model for the pharmacologic evaluation of the low molecular heparin fractions. Since the coagulation system and heparinizability index of this model approximate a human response, the data may be used to reflect therapeutic and prophylactic responses, as well as to assess toxic effects, such as bleeding.

Performance characteristics of a simple radioimmunoassay for fibrinopeptide A.

FPA, although identified 15 years ago, is now becoming an increasingly important diagnostic tool in the evaluation of the hemostatic process. Since this peptide is generated in very small amounts, only very sensitive methods, such as RIA, are useful for its quantitation. Measurement of this peptide allows for a most precise and reliable monitor of any ongoing thrombotic event in which thrombin is generated. Commercial kits have become available for fast and simple clinical evaluations of FPA. The Mallinckrodt RIA Quanti FPA kit has proved its reliability in precision, accuracy, fast turnaround time, and applicability to a routine laboratory setting. This assay kit was evaluated in our laboratory in various aspects. The following points summarize our finding: FPA is a useful diagnostic parameter to evaluate the activation of coagulation pathways in various pathologic states. A study of 170 normal plasma samples resulted in 1.7 +/- 0.5 ng/ml. No significant difference between males and females was noted. FPA levels are evaluated in patients with hypercoagulable states, DIC, and related thrombotic states. Our studies have shown that FPA levels are also elevated in certain cancers, postsurgical states, and certain other conditions in which the coagulation process is activated. During therapeutic heparinization, FPA levels are reduced; thus this form of therapy can be monitored using FPA levels. High-risk population (thrombotic) can be easily screened using FPA measurement. We propose that a multicenter study on FPA levels be conducted to prove its clinical relevance to other diseases.(ABSTRACT TRUNCATED AT 250 WORDS)

Diagnostic efficacy of a simple radioimmunoassay test for fibrinogen/fibrin fragments containing the B beta 15-42 sequence.

A simple RIA method for B beta 15-42 RPs has been evaluated in our laboratory to investigate experimental and clinical fibrinolytic states. The assay utilizes bentonite precipitation to remove cross-reacting fibrinogen. Due to the heterogeneity in molecular weights of the B beta RPs, the results are expressed as nanograms per milliliter. The linear range of the assay is 2 to 40 ng/ml, with a capability of detecting up to 200 ng/ml. A special anticoagulant mixture (heparin or EDTA/aprotinin) is required for sample collection. Certain precautions in the care and handling of specimens are also necessary. Increased levels of B beta RPs were observed in the following conditions: malignancy (associated with increased release of tissue plasminogen activators), pancreatitis, liver diseases, pregnancy, and postexercise testing (associated with increased release of tissue plasminogen activators). Increased levels of B beta RPs were also found during thrombolytic therapy, anabolic steroid treatment, prothrombin complex concentrate therapy, blood component therapy, and low molecular weight heparin subcutaneous therapy (associated with an increase in tissue plasminogen activator release). Our studies suggest that B beta RPs are sensitive molecular markers of the endogenous activation of fibrinolytic system and may provide useful diagnostic information on a pathologic process that often remains undetectable by routine laboratory methods.

Antithrombotic actions and pharmacokinetics of heparin fractions and fragments.

During the last few years many reports on the antithrombotic actions of low molecular weight fractions (LMF) have become available and are being tested in the experimental and clinical settings. Although derived from commercial heparins, the mode of antithrombotic actions, pharmacokinetics and physiologic disposition of these agents is distinct from the parent materials. The antithrombotic potency of these fractions is generally assigned in terms of their ability to inhibit serine proteases such as factor Xa and thrombin in the presence of antithrombin III (AT III). However, these agents are also reported to exert their antithrombotic actions via non AT III mediated inhibition of serine proteases (XIIa, IXa), activation of fibrinolysis via the release of plasminogen activators and charge transition on vascular surfaces. In order to evaluate the relative pharmacologic actions of heparin and its derivatives, we utilized defined animal models to obtain data in terms of pharmacologic parameters. Significant differences between heparin and its derivatives were evident. Human studies on the pharmacokinetics and the endogenous actions of these agents confirmed our preclinical findings. These studies suggest that well-defined preclinical studies are an essential element in the development of newer antithrombotic agents.