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Category Anticoagulant; antithrombotic.

         Antithrombin III Concentrate is a freeze-dried preparation of a glycoprotein fraction obtained from human plasma that inactivates thrombin in the presence of an excess of heparin. It is obtained from human plasma that complies with the requirements stated under Plasma for Fractionation, p. 193.

         The antithrombin III is purified and concentrated, and a suitable stabilizer may be added. No antimicrobial preservative is added at any stage of production. The specific activity is not less than 3 IU of antithrombin III per mg of total protein, excluding albumin.

         When reconstituted in the volume of solvent stated of the label, the potency is not less than 25 IU1 of antithrombin III per ml.

Description White, or almost white, friable solid or a powder. Hygroscopic.

Strengths available 500 IU and 1000 IU.

Dose Intravenous, administered at a rate of 50 to 100 IU (not to exceed 100 IU) per minute:

Initial----A sufficient quantity to increase the antithrombin III activity, determined 30 minutes after administration, to 120 per cent of the normal activity.

          Maintenance----A sufficient quantity to increase the antithrombin III activity to 80 per cent or more of the normal activity. Maintenance doses are generallyadministered at 24-hour intervals.

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¹ The potency of Antithrombin III concentrate is expressed in International Unit (IU), defined as the amount of antithrombin III in 1 ml of pooled normal human plasma and is measured against a standard calibrated with a reference preparation from the World Health Organization. 

Warning It may cause nausea, flushing, and headache. Allergic reaction and fever can occur, but rarely.

Additional information

          1. Concomitant use of very large doses of antithrombin III Concentrate and heparin may increase the risk of bleeding. Therefore, the dose of heparin should be monitored carefully when the two drugs are given concurrently.

          2. In pregnant women receiving antithrombin III concentrate and heparin or oral anticoagulants near term, heparin should be discontinued at least 12 hours before delivery and the oral anticoagulant should be terminated several days before delivery or before a therapeutic or elective abortion.

Expiration date The expiration date is not later than 2 years from the date of manufacturing, as indicated on the label.

Packaging and storage Antithrombin III Concentrate shall be kept in tightly closed containers and stored between 2º to 8º, protected from light.

Labelling Complies with the “General Information for Biological Products” p. 177. In addition, the label on the container states (1) the content of antithrombin III expressed in International Units per container; (2) the name and volume of solvent to be used to reconstitute the preparation; (3) the amount of albumin present as a stabilizer; (4) that the transmission of infectious agents cannot be totally excluded when medicinal products prepared from human blood or plasma are administered.

Before carrying out the identification and the tests (except those for constituted solution and water) and the assay, immediately reconstitute the preparation to be examined as stated on the label.

 

Identification

          A. Using a suitable range of species-specific antisera, carry out precipitation tests on the preparation to be examined and stain the gels with acid blue 92. It is recommended that the tests be carried out using antisera specific to the plasma proteins of each species of domestic animal commonly used in the preparation of materials of biological origin in the country concerned. The preparation is shown to contain proteins of human origin and gives negative results with antisera specific to plasma proteins of other species. 

          B. The assay for antithrombin III activity contributes to the identification of the preparation.

pH 6.0 to 7.5 (Appendix 4.11).

Osmolality Not less than 240 mOsmol/kg (Appendix 4.35).

Heparin Not more than 0.1 IU of heparin activity per IU of antithrombin III activity. Carry out the “Biological Assay of Heparin in Coagulation Factors” (Appendix 14.2.1). It is necessary to validate the method for assay of heparin for each specific preparation to be examined to allow for interference by antithrombin III.

Solubility test Reconstitute the preparation as stated on the label. It dissolves completely under gentle swirling within 10 minutes, giving a clear or slightly turbid, colourless solution.

Water Not more than 3.0 per cent w/w (Karl Fischer Method, Appendix 4.12).

Pyrogens Complies with the “Pyrogen Test” (Appendix 8.2), using a volume of the solution containing 50 IU of antithrombin III per kg of the rabbit’s weight.

Sterility Complies with the “Sterility Test” (Appendix 10.1).

Total protein If necessary, dilute an accurately measured volume of the preparation to be examined with water to obtain a solution containing about 15 mg of protein in 2 ml. To 2.0 ml of the solution in a roundbottomed centrifuge tube add 2 ml of a 7.5 per cent w/v solution of sodium molybdate and 2 ml of a mixture of 1 volume of nitrogen-free sulfuric acid and 30 volumes of water. Shake, centrifuge for 5 minutes, decant the supernatant liquid and allow the inverted tube to drain on filter paper. Using residue thus obtained, carry out the “Determination of Nitrogen” (Method II, Appendix 6.7), and calculate the content of protein by multiplying the result by 6.25.

Assay

          The antithrombin III content of the preparation to be examined is determined by comparing its ability to inactivate thrombin in the presence of an excess of heparin with the same ability of a reference preparation of human antithrombin III concentrate calibrated in International Units. Varying quantities of the preparation to be examined are mixed with a given quantity of thrombin and the remaining thrombin activity is determined using a suitable chromogenic substrate.

          Prepare two independent series of three or four dilutions in the range of 1/75 to 1/200 from 1 IU per ml,for both the preparation to be examined and the reference preparation, using tris-EDTA BSA buffer solution pH 8.4 containing 15 IU of heparin per ml.

          Warm 200 μl of each dilution at 37º for 1 to 2 minutes. Add to each dilution 200 μl of a solution of bovine thrombin containing 2 IU per ml in tris-EDTA BSA buffer solution pH 8.4. Mix and maintain at 37º for exactly 1 minute. Add 500 μl of a suitable chromogenic substrate (for example, D-phenylalanyl-L-pipecolyl-Larginine-4-nitroanilide, reconstituted in water to give a solution containing 4 mmol per litre and further diluted for the assay using tris-EDTA BSA buffer solution pH 8.4 without albumin). Immediately start measurement of the change in absorbance at 405 nm, continuing the measurement for at least 30 seconds. Calculate the rate of change of absorbance (ΔA/minute). (Alternatively, an end-point assay may be used by stopping the reaction with acetic acid and measuring the absorbance at 405 nm.)

          The rate of change of absorbance is (ΔA/minute) is inversely proportional to antithrombin III activity. Plot the regression of absorbance of (ΔA/minute) against concentration on a linear scale and determine the potency by comparing the slopes for the reference preparation and the preparation to be examined.

          Check the validity of the assay and calculate the potency of the test preparation by the usual statistical methods for a slope-ratio assay in the “Statistical Analysis of Results of Biological Assays and Tests” (Appendix 9).

          The estimated potency is not less than 80 per cent and not more than 120 per cent of the potency stated on the label. The confidence limits (P = 0.95) are not less than 90 per cent and not more than 110 per cent of the estimated potency.