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WO2012072799A1 - Procédés inédits de mesure in vitro de l'activité biologique d'un échantillon d'héparine de masse moléculaire ultra-faible - Google Patents

Procédés inédits de mesure in vitro de l'activité biologique d'un échantillon d'héparine de masse moléculaire ultra-faible Download PDF

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Publication number
WO2012072799A1
WO2012072799A1 PCT/EP2011/071633 EP2011071633W WO2012072799A1 WO 2012072799 A1 WO2012072799 A1 WO 2012072799A1 EP 2011071633 W EP2011071633 W EP 2011071633W WO 2012072799 A1 WO2012072799 A1 WO 2012072799A1
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Prior art keywords
ulmwh
standard
sample
arg
activity
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English (en)
Inventor
Hélène RIGAL
Brigitte Bordier
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Aventis Pharma SA
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Aventis Pharma SA
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors

Definitions

  • the present application concerns Ultra Low Molecular Weight Heparins (ULMWH) and the assays for measuring their biological activity.
  • ULMWH Ultra Low Molecular Weight Heparins
  • Low Molecular Weight Heparins are defined in the European Pharmacopoeia (European Pharmacopoeia 6.0 (01/2008:0828, 2041 -2043)) by their molecular mass (less than 8000 Daltons) and their potency (an anti-factor Xa activity not less that 70 IU per milligram and a ratio of anti-factor Xa activity to anti-factor lla activity not less than 1 ,5).
  • semuloparin developed by Sanofi-Aventis (AVE5026), belongs to a new generation of hemisynthetic heparins. It is a new ultra-low molecular weight heparin (ULMWH), with a mean molecular weight of 2400 Daltons and a novel antithrombotic profile resulting from high anti-factor Xa activity (-160 U/mg) and residual anti-factor lla activity (-2 U/mg), as described by C. Viskov et al. in Journal of Thrombosis and Haemostasis, 2009, vol. 7, 1 143-1 151 . Semuloparin, in the form of its sodium salt, is in clinical development for venous thromboembolism prevention.
  • Semuloparin is obtained by selective and controlled depolymerization of heparin, as described in the patent application WO 2004/033503 and in J. Thromb. Hameost. ⁇ ibid). It was shown that its unique structural features result from the highly selective depolymerization of heparin by the phosphazene base that protects the antithrombin (AO- binding site from destruction.
  • LMWH Low Molecular Weight Heparins
  • the anti-factor Xa and anti-factor lla activities are measured by using an International Standard as defined by the European Pharmacopoeia (European Pharmacopoeia 6.0 (01/2008:0828, 2041 -2043)).
  • the patent application WO 2004/033503 teaches to measure the anti-factor Xa activity of semuloparin by the amydolytic method on a chromogenic substrate as described by Teien et al., Thrombosis Research, 10, 399-410 (1977), using as a standard the first International Standard for low molecular weight heparins.
  • it teaches to measure its anti-factor lla activity by the method described by Anderson L. O. et al., Thrombosis Research, 15, 531 -541 (1979), with as a standard the first International Standard for low molecular weight heparins.
  • WO 2005/010051 discloses new heparin-derived oligosaccharide mixtures characterized by a mean molecular weight comprised between 1800 and 2400 Daltons, an anti-factor Xa activity comprised between 190 lU/mg and 450 lU/mg, and virtually no anti-factor lla activity. Said oligosaccharide mixtures are obtainable by a re-depolymerization process of an ULMWH.
  • the anti-factor Xa activity of these mixtures is measured relative to a reference ULMWH having an anti-factor Xa activity of 140 to 180 lU/mg (on dry basis) and an anti-factor lla activity of 2,1 lU/mg, the activity of this reference ULMWH being measured relative to the International Standard for low molecular weight heparins.
  • LMWH Low Molecular Weight Heparins
  • LMMH Low Molecular Mass Heparins
  • the inventors have identified a standard suitable for the in vitro measurement of the biological activity of an ULMWH sample, where said standard comprises said ULMWH sample.
  • the present invention thus concerns a method for the in vitro measurement of the biological activity of an ULMWH sample, wherein said method is carried out relative to a standard comprising the same ULMWH.
  • ULMWH Ultra Low Molecular Weight Heparins, as defined by the European Pharmacopoeia in force, supra. In the framework of the instant invention, it includes heparin derivatives having a molecular weight of 2000 to 4000 Daltons. ULMWH include in particular semuloparin.
  • Semuloparin has a mean molecular weight of 2000-3000 Daltons (about 2.4 kDa on average), a high anti-factor Xa activity (approx. 160 U/mg on dry basis), a low anti- factor lla activity (approx. 2 U/mg on dry basis) and a high anti-factor Xa / anti-factor lla ratio (>30).
  • the term "semuloparin” as used herein is used to refer to semuloparin in the free form or any of its salts, including semuloparin sodium salt.
  • standard refers to the standard, or calibrator used for measurement of in vitro activity to assign the potency of a sample.
  • the standard is generally expressed in units (U).
  • the standard comprising the same ULMWH is herein referred to as "the ULMWH standard”.
  • the ULMWH standard is a preparation of semuloparin.
  • Said preparation is preferably a solution of semuloparin.
  • the ULMWH standard has a pre-determined biological activity; more particularly, the ULMWH standard has a pre-determined anti- factor Xa activity, generally an anti-factor Xa activity of around 160 U/mg on dry basis and an anti-factor lla of around 1 to 5 U/mg on dry basis.
  • the method of the invention is a dilution assay, wherein the ULMWH sample to be measured is supposed to have the same potency as the ULMWH standard.
  • the measurement of the biological activity of an ULMWH sample is carried out by determination of the relative potency(ies) of said sample. This determination requires the parallelism of the curves of the ULMWH sample to be measured and those of the ULMWH standard.
  • the measurement of said biological activity includes the measurement of the anti- factor Xa activity and/or the measurement of the anti-factor I la activity of an ULMWH sample.
  • These assays determine the ability of said sample to accelerate the inhibition of factor Xa (anti-factor Xa activity) and/or of factor I la (or thrombin), by antithrombin III (ATI 11), according to the following reactions: ATIII + ULMWH ⁇ [ATIII-ULMWH]
  • the amount of the paranitroaniline, measured at 405 nm, is inversely proportional to that of the ULMWH sample.
  • curves refers to the graphic representation obtained by plotting the optical density measured in the above reaction as function of the concentration of the sample.
  • the measurement of said biological activity is preferably made by adapting the method described in the monograph on LMWHs of the European Pharmacopoeia in force, such as European Pharmacopoeia 6.0 (01/2008:0828, 2041 -2043).
  • said method comprises an amidolytic assay on a chromogenic substrate, according to the method described in the monograph on LMWHs of the European Pharmacopoeia in force, as above.
  • Said chromogenic substrate depends on the activity to be measured.
  • Suitable chromogenic substrates for measuring the anti-factor Xa activity may be chosen from the group consisting in S-2222TM (Bz-lle-Glu(g-OR)-Gly-Arg-pNA-HCI) and S-2765TM (N-a-Z- D-Arg-Gly-Arg-pNA 2HCI also referred to as N-a-benzyloxycarbonyl-D-arginyl-L-glycyl-L- arginine-4-nitroanilide dihydrochloride) both available from Chromogenix; CBS 31 .39 (CH 3 -S0 2 - D -Leu-Gly-Arg-pNA) available from Stago; BIOPHEN® CS-1 1 (22) (Mixture (50%-50%) of Bz-lle-Glu (YOCH3)-Gly-Arg-pNa (form 1 ) and Bz-lle-Glu (yOH)Gly-Arg
  • Suitable chromogenic substrates for measuring the anti-factor I la activity may be chosen from the group consisting in S-2238TM (H-D-Phe-Pip-Arg-pNA-2HCI also referred to as D-phenylalanyl-L-pipecolyl-L-arginine-4-nitroanilide dihydrochloride) available from Chromogenix, CBS 34.47 (H- D -CHG-But-Arg-pNA) and CBS00.68 both available from Stago, BIOPHEN® CS-01 (81 ) (Tos-Gly-Pro-Arg-pNa.
  • BIOPHEN® CS- 01 (38) H-D-Phe-Pip-Arg-pNa, 2HCI) both available from HYPHEN BioMed. More preferably, said chromogenic substrate for measuring the anti-factor lla activity is S- 2238TM.
  • the method of the invention may be conducted in solutions of biological samples such as plasma, or in buffer solutions, preferably in buffer solutions.
  • Buffers may be chosen from any known buffers, in particular those having a pH close to biological media, such as those buffers having a pH comprised between 6.5 and 9.
  • Buffer solutions include in particular tris(hydroxymethyl)aminomethane sodium chloride pH 7.4, tris(hydroxymethyl)aminomethane-EDTA pH 8.4, and tris(hydroxymethyl)aminomethane-NaCI-PEG pH 7.4. They may be prepared according to routine, well-known procedures.
  • the method of the invention comprises the following steps:
  • Step a determining the biological activity of the ULMWH sample against the calibration curve obtained in step b).
  • the ULMWH standard in a form such as a water for injection solution, may generally be prepared by diluting a given amount of the ULMWH into a given amount of the chosen solution such as buffer solution.
  • the given amounts are advantageously expressed in volumes.
  • Said ULMWH standard and ULMWH sample are generally processed in the same way, in the same medium, e.g. buffer solution.
  • the ULMWH sample may be prepared in solution of water for injection, by weighing an appropriate quantity in order to obtain activities in U/mL equivalent to those of the ULMWH standard. The sample is then diluted in the same medium (e.g. buffer solution) as the ULMWH standard.
  • medium e.g. buffer solution
  • a potency (e.g. anti-factor Xa or anti-factor I la activity) expressed in U/mL is assigned to the given amount of the ULMWH, before dilution. A corresponding potency may thus be calculated for the diluted sample, depending on the dilution rate.
  • From 4 to 14 calibration points may be performed.
  • the method of the invention involves 4 or 14 calibration points depending on whether a routine procedure or experimentation procedure is carried out, respectively.
  • the procedure may be carried out according to the 5-parameters methodology (in particular for the 14 calibration point method) or the parallel lines methodology (in particular for the 4 calibration point method).
  • the assay for carrying out the method of the invention may be performed by known procedures, such as on a micro-plate reader and dispenser.
  • the calibration curve may thus be obtained by correlating the calculated potencies with the obtained Optical Density (OD).
  • the activity in U/mL of the ULMWH sample may be obtained by correlating the activity and the obtained Optical Density (OD), according to the calibration curve obtained with the ULMWH standard.
  • OD Optical Density
  • the anti-factor Xa activity assigned to the ULMWH standard before dilution is 100 U/mL.
  • the points of the calibration curve are comprised between approximately 0.002 and 0.8 U/mL for the anti-factor Xa activity curve in the 14 calibration points method.
  • the range of the calibration points for the 4 point calibration curve is generally comprised within the above range, such as 0.04-0.07 ⁇ 0.002 U/mL.
  • the anti-factor I la activity assigned to the ULMWH standard before dilution is 1 .8 U/mL.
  • the points of the calibration curve are comprised between 0.0007 and 0.3 U/mL for the anti-factor I la activity curve in the 14 calibration points method.
  • the range of the calibration points for the 4 point calibration curve is generally comprised within the above range, such as 0.007-0.012 ⁇ 0.001 U/mL.
  • the biological activity (relative potency) of the ULMWH sample is calculated from the calibration curve obtained with the ULMWH standard.
  • the present invention also concerns a standard suitable for measuring the in vitro biological activity of an ULMWH sample, which comprises the same ULMWH than the sample to be measured.
  • said standard comprises semuloparin.
  • Said ULMWH standard is suitable for conducting the method of the invention.
  • Said ULMWH standard is preferably obtained by diluting a sample of the ULMWH to be measured, which is calibrated by comparison with a recognized standard of LMWH, such as the second international standard of LMWH, currently available.
  • the ULMWH standard is generally assigned an anti-factor Xa activity around 160 U/mg on dry basis and an anti-factor I la of around 2 U/mg on dry basis.
  • Figures 1 and 2 illustrate the anti-factor Xa activity of bemiparin and semuloparin according to the 14 calibration points procedure.
  • FIGS 3 and 4 illustrate the anti-factor I la activity of bemiparin and semuloparin according to the 14 calibration points procedure.
  • Figures 5, 6, 7 and 8 illustrate the anti-factor Xa activity and anti-factor lla activity of semuloparin compared with a standard of Low Molecular Mass Heparin for assay Biological Reference Preparation batch 5 (BRP5) according to the 14 calibration points procedure.
  • Figures 9, 10, 1 1 , 12 illustrate the anti-factor Xa activity and anti-factor lla activity of semuloparin compared with a heparin standard (K5, USP357) according to the 14 calibration points procedure.
  • Figure 13 and 14 illustrate the anti-factor Xa activity and anti-factor lla activity of semuloparin and a standard comprising semuloparin (3000ET) according to the 4 calibration points procedure.
  • the curve profile of biological activity of the semuloparin was compared to that of bemiparin, the LMWH reference standard of the European Pharmacopoeia BRP5, and the heparin standard (K5, USP357).
  • the anti-factor lla and anti-factor Xa activities were measured by assays according to the following protocols: PREPARATION OF REAGENTS
  • Equipment is given indicatively - any other equipment validated for this method can be used.
  • PACKARD MULTIPROBE II STD Automated liquid handling system (dilution robot).
  • MR 7000 or DIAS from LABSYSTEM Microplate reader, incubator and dispenser. PREPARATION OF SOLUTIONS TO BE ASSAYED
  • Semuloparin the LMWH standard of the European Pharmacopoeia (BRP5), the heparin standard (K5 USP 357) and the semuloparin reference standard (3000ET) are diluted in the same way, and the ranges in U/mL or UI/mL are given in Tables 1 and 2.
  • Anti-factor Xa activity the LMWH standard of the European Pharmacopoeia (BRP5), the heparin standard (K5 USP 357) and the semuloparin reference standard (3000ET) are diluted in the same way, and the ranges in U/mL or UI/mL are given in Tables 1 and 2.
  • Anti-factor Xa activity the LMWH standard of the European Pharmacopoeia (BRP5), the heparin standard (K5 USP 357) and the semuloparin reference standard (3000ET) are diluted in the same way, and the ranges in U/mL or UI/mL are given in Tables 1 and 2.
  • the assay was performed using MR7000 or DIAS (dynex) from LABSYSTEM.
  • DIAS dinex
  • Results are illustrated in Figures 1 and 2 for the anti-factor Xa activity and in figures 3 and 4 for the anti-factor lla activity.
  • Results are illustrated in Figures 5 and 7 for the anti-factor Xa activity and in figures 6 and 8 for the anti-factor lla activity
  • Results are illustrated in Figures 9 and 1 1 for the anti-factor Xa activity and in figures 10 and 12 for the anti-factor lla activity.
  • This method may be used as a routine procedure. Anticoagulant activity of semuloparin was determined in vitro by chromogenic assays. The methods were adapted from those recommended by the European Pharmacopoeia for low molecular weight heparin (LMWH).
  • LMWH low molecular weight heparin
  • the biological activity of the sample was compared to that of a well characterised in-house semuloparin reference standard.
  • the anti-factor lla and anti-factor Xa activities were measured by assays according to the following protocols:
  • the assay is performed using MR7000 or DIAS from LABSYSTEM, ANTI-FACTOR Xa ACTIVITY:
  • the relative potency is expressed in U (anti-FXa or anti-Flla)/mg on the dried basis.
  • semuloparin was compared to a standard comprising semuloparin (3000ET).
  • the anti-FXa and anti-Flla activities of semuloparin containing samples were determined according to the 4-points calibration methodology disclosed above, using a standard comprising semuloparin.

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Abstract

La présente demande de brevet concerne des héparines présentant une masse moléculaire ultra-faible et les dosages permettant de mesurer leur activité biologique.
PCT/EP2011/071633 2010-12-02 2011-12-02 Procédés inédits de mesure in vitro de l'activité biologique d'un échantillon d'héparine de masse moléculaire ultra-faible Ceased WO2012072799A1 (fr)

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EP10306342.6 2010-12-02

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1070503A1 (fr) * 1999-07-23 2001-01-24 Laboratorios Farmaceuticos Rovi, S.A. Compositions contenant de l'héparin des très bas poids moleculaire
WO2004033503A1 (fr) 2002-10-10 2004-04-22 Aventis Pharma S.A. Melanges de polysaccharides derives d'heparine, leur preparation et les compositions pharmaceutiques les contenant
WO2005010051A2 (fr) 2003-07-24 2005-02-03 Aventis Pharma S.A. Melanges d'oligosaccharides derives d'heparine, leur preparation et les compositions pharmaceutiques les contenant
EP2233145A1 (fr) * 2009-03-19 2010-09-29 Sanofi-Aventis Dose de AVE5026 pour le traitement de thrombo-embolie veineuse chez des patients atteints d'une insuffisance rénale grave

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1070503A1 (fr) * 1999-07-23 2001-01-24 Laboratorios Farmaceuticos Rovi, S.A. Compositions contenant de l'héparin des très bas poids moleculaire
WO2004033503A1 (fr) 2002-10-10 2004-04-22 Aventis Pharma S.A. Melanges de polysaccharides derives d'heparine, leur preparation et les compositions pharmaceutiques les contenant
WO2005010051A2 (fr) 2003-07-24 2005-02-03 Aventis Pharma S.A. Melanges d'oligosaccharides derives d'heparine, leur preparation et les compositions pharmaceutiques les contenant
EP2233145A1 (fr) * 2009-03-19 2010-09-29 Sanofi-Aventis Dose de AVE5026 pour le traitement de thrombo-embolie veineuse chez des patients atteints d'une insuffisance rénale grave

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"The five-parameter logistic: A characterization and comparison with the four-parameter logistic", ANALYTICAL BIOCHEMISTRY, vol. 343, 2005, pages 54 - 65
ANDERSON L. O. ET AL., THROMBOSIS RESEARCH, vol. 15, 1979, pages 531 - 541
C. VISKOV ET AL., JOURNAL OF THROMBOSIS AND HAEMOSTASIS, vol. 7, 2009, pages 1143 - 1151
DUBRUC CATHERINE ET AL: "Pharmacokinetics of a New, Ultra-Low Molecular Weight Heparin, Semuloparin (AVE5026), in Healthy Subjects. Results From the First Phase I Studies", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 14, no. 22, 1 November 2009 (2009-11-01), pages 443, XP009137841, ISSN: 0006-4971 *
DUNN ET AL.: "Measuring Parallelism, Linearity and Relative Potency in Bioassay and Immunoassay Data", JOURNAL OF BIOPHARMACEUTICAL STATISTICS, vol. 15, 2005, pages 437 - 463
GÓMEZ-OUTES ANTONIO ET AL: "New parenteral anticoagulants in development", THERAPEUTIC ADVANCES IN CARDIOVASCULAR DISEASE, SAGE PUBLICATIONS LTD, GB, vol. 5, no. 1, 2 November 2010 (2010-11-02), pages 33 - 59, XP009146821, ISSN: 1753-9447, [retrieved on 20101102], DOI: DOI:10.1177/1753944710387808 *
JESKE W.P. ET AL.: "A common standard is inappropriate for determining the potency of ultra low molecular weight heparins such as semuloparin and bemiparin", THROMB. RES., vol. 128, no. 4, 2011, pages 361 - 367, XP028391145, DOI: doi:10.1016/j.thromres.2011.03.001
LASSEN M R ET AL: "AVE5026, a new hemisynthetic ultra-low-molecular-weight heparin for the prevention of venous thromboembolism in patients after total knee replacement surgery - TREK: A dose-ranging study", JOURNAL OF THROMBOSIS AND HAEMOSTASIS, BLACKWELL PUBLISHING, OXFORD, GB, vol. 7, no. 4, 24 January 2009 (2009-01-24), pages 566 - 572, XP002533591, ISSN: 1538-7933, DOI: DOI:10.1111/J.1538-7836.2009.03301.X *
TEIEN ET AL., THROMBOSIS RESEARCH, vol. 10, 1977, pages 399 - 410
VISKOV C ET AL: "Description of the chemical and pharmacological characteristics of a new hemisynthetic ultra-low-molecular-weight heparin, AVE5026", JOURNAL OF THROMBOSIS AND HAEMOSTASIS, WILEY INTERSCIENCE, ENGLAND, vol. 7, no. 7, 1 July 2009 (2009-07-01), pages 1143 - 1151, XP002545508, ISSN: 1538-7836, DOI: DOI:10.1111/J.1538-7836.2009.03447.X *

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