Unit dose formulation of antidotes for factor Xa inhibitors and methods of using the same

A unit dose, inhibitor technology, applied in the direction of anti-toxic agents, biochemical equipment and methods, medical preparations containing active ingredients, etc., can solve problems such as low procoagulant activity

Active Publication Date: 2012-08-01
PORTOLA PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unlike rfVIIa which has very low procoagulant activity in the absence of its cofactor tissue factor, native fXa is a potent enzyme and ha

Method used

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  • Unit dose formulation of antidotes for factor Xa inhibitors and methods of using the same
  • Unit dose formulation of antidotes for factor Xa inhibitors and methods of using the same
  • Unit dose formulation of antidotes for factor Xa inhibitors and methods of using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0368] Example 1. Preparation of Gla-depleted anhydro-fXa by chymotrypsin digestion

[0369] According to Morita (Morita, T.) et al., Journal of Biochemistry (J.Bio.Chem.), 1986,261 (9): 4015-4023 program by anhydrous-fXa ​​(wherein dehydroalanine De-Gla anhydrous-fXa ​​was prepared by incubating with chymotrypsin in 0.05M Tris-HCl, 0.1M NaCl at pH 7.5 and 22°C for 60 minutes. In a typical experimental setup, 0.5 milligram per milliliter (mg / mL) anhydrous-fXa ​​was incubated with 5 units / milliliter (U / mL) α-chymotrypsin-agarose beads under gentle agitation. At the end of the reaction, remove the α-chymotrypsin-agarose beads by centrifugation or filtration. This was subsequently combined with the excess inhibitors 4-amidino-phenyl-methane-sulfonyl fluoride (APMSF), tosyl-L-lysine chloromethyl ketone (TLCK), and tosyl-L- Phenylalanine chloromethyl ketone (TPCK) was incubated with to quench residual fXa activity or any chymotrypsin activity that might be leached from the beads....

Embodiment 2

[0373] Example 2. Analysis of Thrombin Generation in Platelet Poor Plasma (PPP) or Platelet Rich Plasma (PRP)

[0374] In this example, human platelet-poor or platelet-rich plasma samples were prepared from blood of healthy donors drawn into 0.32% citrate. PRP and PPP were prepared by spinning anticoagulated blood at about 100X gravity or 1000X gravity, respectively, for 20 minutes at room temperature. Mix 75-100 microliters (uL) of plasma with CaCl 2 Mix with Z-Gly-Gly-Arg-aminomethylcoumarin (Z-GGR-AMC, fluorogenic substrate for thrombin). Tissue factor (Innovin, Dade Behring) was added to initiate thrombin generation. For a typical experiment, the reaction mixture contained 15 millimolar (mM) Ca 2+ , 100 micromolar (μM) Z-GGR-AMC, and 0.1 nanomolar (nM) tissue factor (TF) (Innovin). Thrombin formation was monitored continuously at 37°C by a fluorescent plate reader (Molecular Devices) measuring relative fluorescence units (RFU). When present, inhibitors and antidotes w...

Embodiment 3

[0376] Example 3. Coagulation Prolongation Analysis

[0377] Two coagulation assay formats were used to test the effect of factor Xa inhibitors and antidotes on prolonged coagulation. In the first format, a 96-well plate is used to measure multiple samples simultaneously. In a second assay format, aPTT was measured using a conventional coagulation instrument (MLA Electra 800 Automatic Coagulation Timer).

[0378] In the 96-well plate format method, human platelet-poor plasma or platelet-rich plasma was prepared in a procedure similar to that in Example 2. Utilize 75-100uL of plasma with CaCl 2 Recalcify, incubate at 37°C for 3 minutes and remove calcification by adding tissue factor (Innovin, Dade Behring) or aPTT reagent (Actin FS, Dade Behring) ) to initiate blood clot formation. Changes in OD405 were continuously monitored by a plate reader (Molecular Devices). Coagulation time was defined as the time (in seconds) when the half-maximum change in absorbance (OD405nm) wa...

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Abstract

The present invention relates unit dose formulations of antidotes to anticoagulants targeting factor Xa. Disclosed herein are methods of stopping or preventing bleeding in a patient that is currently undergoing anticoagulant therapy with a factor Xa inhibitor.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 61 / 225,887, filed July 15, 2009, under 35 U.S.C. § 119(e), which is hereby incorporated by reference in its entirety. technical field [0003] The present invention relates to unit dose formulations of antidotes that reverse and / or neutralize factor Xa inhibitors. In particular, the antidote may be a Factor Xa (fXa) derivative having reduced or no intrinsic procoagulant activity, but which is also capable of binding and / or neutralizing the fXa inhibitor , thus acting as an antidote to anticoagulants targeting fXa. The invention also relates to methods of using specific doses of antidotes. Background technique [0004] There is a need in the market for anticoagulants in the treatment or prevention of unwanted thrombus formation in patients prone to blood clot formation while inactive or undergoing medical procedures (eg, those with coagulation disor...

Claims

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Application Information

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IPC IPC(8): A61K38/48C12N9/64A61P7/00A61P7/04
CPCC12N9/6432A61K38/4846C12Y304/21006A61P7/00A61P7/04A61P39/00A61P43/00A61K9/0019
Inventor U.辛哈卢根民A.哈特查理拉哈S.J.霍伦巴克
Owner PORTOLA PHARMA INC
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