The effect of molecular weight on the bioavailability of heparin

THROMBOSIS RESEARCH 48; 591-596, 1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.

BRIEF

COMMUNICATION_ __.._ -.

THE EFFECT OF MOLECULAR WEIGHT ON TEE BIOAVAILABILITY OF HEPARIN

R. M. Emanuele

and

J. Fareed

Department of Pharmacology and Pathology, Maywood, Medical Center, 2160 S. First Ave.,

Loyola University Illinois

(Received 1.6.1987; Accepted in revised form 15.9.1987 by Editor D.F. Mosher)

INTRODUCTION Currently there is interest in the development of low molecular weight heparins for use in the prophylaxis of thromThese agents have been shown to be effective botic disorders. antithrombotic agents in both experimental and clinical studies Although the predicted reduction in hemorrhagic potent(1,293). ial has not been convincingly demonstrated, low molecular weight heparins have shown other beneficial properties (3,4,5). Among a circulating half-life and these advantages are their greater bioavailability characteristics (3,4,5). Subcutaneous injection appears to be an effective route of administration for both heparin and its low molecular weight fractions (2,6). When administered by this route, low molecular weight heparins have been shown to display greater bioavailabilIn order to investigate the ity compared to native heparin (5). relationship between molecular weight and bioavailability, we have determined the absolute bioavailability for different molecular weight fractions obtained from one source of heparin in a primate model. For comparative purposes, a commercial low molecular weight heparin (CY 216) and unfractionated heparin were also studied.

MATERIALS 8 METHODS Unfractionated

Key

words:

heparin,

porcine

molecular

sodium

weight, 591

heparin

(M.W.

12,575),

bioavailability

BIOAVAILABILITY OF HEPARIN

592

Vol. 48, No. 5

molecular weight fractions derived from this source by geland CY 216 (M.W. filtration (M.W.'s 23,000 ; 13,300 ; 5,100) 5,400) were obtained from Institute Choay (Paris, France). The molecular weight characteristics of all heparins were determined by HPLC-GPC chromatography (7) using Waters 840 software. The absolute bioavailability of the different molecular weight heparins was studied in the primate Macaca mulatta (n = Intravenous injections (250 ug/Kg) of each fraction were 5). administered through a superficial leg vein. Blood samples were obtained by venipuncture at 0, 5, 10, 15, 30, 60, 180 and 360 minutes post injection and collected into glass tubes containing 3.8 % citrate. The same fractions were administered to the same five primates via subcutaneous injection (1.0 mg/kg) at a site in the lower abdomen . Blood samples were obtained at 0, 2, 4, 6, Plasma was prepared from all 8, 10 and 12 hours post injection. blood samples by centrifugation. Heparin concentrations in the sample plasmas were determined using Heptest clotting assay (Haemachem Inc. St. Louis, MO). The assay was performed by incubating 100 ul of undiluted titrated plasma sample for exactly 120 seconds at 37O C. The plasma was recalcified by the addition of 100 ul of Recalmix@ and Calibration clotting time was measured using a fibrometer (8). curves were used to convert the assay parameter (clotting time) into heparin concentrations. These curves were constructed using the time zero plasma for each primate. The area under the concentration time curve (AUC) values were calculated for each test heparin in each primate using the trapezoidal rule. Average AUC values were determined from the five primates for both intravenous and subcutaneous routes of administration. The average AUC values were used to determine the absolute bioavailability of each test heparin by using the following formula. Absolute

Bioavailability

= subcutaneous intravenous

AUC / dose AUC / dose

RESULTS

The determined

molecular weight characteristics of the test by HPLC-GPC chromatography are shown in table TABLE

Molecular

Test

Heparin

Unfractionated Fraction I Fraction II Fraction III CY 216

Weight

Mean

12,575 23,000 13,300 5,100 5,400

1

Characteristics

M.W.

Peak

heparins 1. The

M.W.

13,550 22,000 13,000 3,800 4,400

of the Test

M.W.

Heparins

Distribution 44,000 44,000 22,000 14,000 14,000

-

Range

1,500 12,500 7,000 1,500 1,000

593

BIOAVAILABILITY OF HEPARIN

Vol. 48, No. 5

three gel-filtered fractions significantly differed in mean peak molecular weight and molecular weight molecular weight, The unfractionated heparin had a similar mean distribution. molecular weight as gel-filtered fraction II, however it conweight components. CY tained a much broader range of molecular 216 and gel-filtered fraction III displayed similar values for all characteristics. Table 2 shows the AUC values obtained after subcutaneous and intravenous administration of the test heparins.

TABLE AUC

Test

Values

for

Different

AUC (up

agent

Unfractionated Fraction I Fraction II Fraction III CY 216

Molecular

4.26 3.99 4.36 6.38 6.25

Test

Agent

Heparins

AUC S.C. (ug hr/ml)

lr.3 k.19 k.5 2.6 t.57

TABLE Bioavailability

Weight

I.V. hr/ml)

For both routes of administration molecular weight. The calculation of absolute data is shown in table 3.

Absolute

2

AUC

increased

6.81 0.47 1.54 23.7 22.0

& ? + + *

with

decreasing

bioavailability

from

1.4 .38 .2 .9 2.2

the

AUC

3

of Different

Molecular

Weight

Bioavailability

Calculation

Heparins

Unfractionated Heparin

AUC AUC

SC = IV =

6.81/1.0 4.261.25

= 6.81 _ 17.0

Bioavailability

= 4o %

Gel-filtered fraction I

AUC AUC

SC = Iv =

0.47/1.0 3.g9/.25

= 0.47 = 15.9

Bioavailability

= 3 %

Gel-filtered fraction II

AUC AUC

SC = IV =

1.54/1.0 4.36/.25

= 1.54 = 17_4

Bioavailability

= 9 %

Gel-filtered fraction III

AUC AUC

SC = IV =

23.1/1.0 6_38,.25

= 23.1 = 25.5

Bioavailability

= 93 %

CY

AUC AUC

SC = IV =

21.9/1.0 6.25/.25

= 21.9 = 25.0

Bioavailability

= 88 %

216

The absolute bioavailability of increased with decreasing molecular M.W.) displayed a bioavailability of increased to 9 % for fraction II

the gel-filtered fractions weight. Fraction I (23,000 3 %. Bioavailability (13,300 M.W.) and 93 % for

BIOAVAILABILITY OF HEPARIN

594

Vol. 48, No. 5

The bioavailability of CY 216 showed fraction III (5,100 M.W.). a similar molecular weight dependence. It was interesting to note that although the unfractionated heparin and gel-filtered fraction II were of similar mean molecular weight, the unfractionated heparin displayed much better bioavailabiiity. To determine the relationship between bioavailability and molecular weight, correlations were calculated between bioavailability and the percent composition of molecular weight compoThe results are shown in table 4. nents for each test heparin. A high correlation (r = .99) was observed between bioavailability and the percent of components with molecular weights of 10,000 or less.

Correlation

Test

Agent

Unfractionated fraction I fraction II fraction III CY 216

The correlation weight components

Between

TABLE 4 Molecular Weight

% of Components M.W.'s less than

With 10,000

and

Bioavailability

Absolute Bioavailability

38 2 7 96 95

between bioavailability and the % of less than 10,000 molecular weight was

40 % 3% 9% 93 % 88 %

molecular .99.

DISCUSSION

In this study the absolute bioavailability of different molecular weight heparins was determined by comparing AUC values obtained after intravenous and subcutaneous administration in Bioavailability calculations determined in this manner primates. are valid provided the rate constant of elimination after both routes of administration is similar. Heparin is known to display both dose dependent elimination and incomplete absorption after subcutaneous administration (3). For these reasons a higher dose of the test heparins was injected subcutaneously to minimize absorption and subsequent elimination differences. This technique favored the lower molecular weight fractions and may have yielded a slightly better bioavailability profile for the lower molecular weight fractions relative to those of higher molecular weight. It is important to mention that in this study bioavailability was calculated from drug concentrations determined using a clot based assay (Heptest). For this reason, bioavailability values were relative to the circulating anticoagulant actions of the molecular weight fractions. The Heptest assay uses a factor Thus bioavailXa activation system with a clot based endpoint. ability profiles determined using this assay should have reflected both circulating anti Xa and anti IIa actions.

Vol. 48, No. 5

595

BIOAVAILABILITY OF HEPARIN

Within the previously defined limits, the results of these studies demonstrated that the bioavailability of heparin is Lower molecular weight heparins affected by molecular weight. displayed better bioavailability characteristics compared to those of higher molecular weight. It was interesting that although the 13,300 M.W. fraction and native heparin had similar mean molecular weights, significantly better bioavailability was displayed by the native This difference was probably due to the molecular heparin. The unfractionated heparin weight distributions of these agents. was composed of molecular weight components ranging from 44,000contained components 1,500 M.W., while the 13,300 M.W. fraction The presence of a greater ranging from 22,000 - 7,000 (table 1). percentage of low molecular weight components in the unfractionated heparin contributed to its enhanced bioavailability. The differences in absolute bioavailability between the test heparins suggested a molecular weight dependent threshold This for the absorption of heparin after subcutaneous injection. threshold appeared to be around 10,000 M.W. based on correlations content of components between bioavailability and the percent less than this M.W. for each fraction. that the bioavailability of In conclusion it appears Furthermore, heparin increases with decreasing molecular weight. heparins bioavailability and the the high correlation between percent of components less than or equal to 10,000 M.W. suggests that a size exclusion limit exists for the absorption of heparin after subcutaneous injection. This absorption threshold appears to influence the absolute bioavailability of heparin preparations.

REFERENCES KAKKAR V. Prevention of post-operative venous thrombo1. embolism by a new low molecular weight heparin. Nouv. Rev. Fr. Hematol. 26, 277-282, 1984 D, HEDNER U, SJORN E, HOLMER E. 2. BERGQVIST Anticoagulant effects of two types of low molecular weight heparin administered subcutaneously. Thromb. Res. 32, 381-391, 1983 J, WALENGA J, WILLIAMSON K, EMANUELE 3. FAREED HOPPENSTEADT D. Studies on the antithrombotic pharmacokinetics of heparin fractions and fragments. Hemost. 11, 56-74, 1985. 4. SALZMAN E. Low molecular weight N. Ennl. J. Med. 315, 957-959, 1986

heparin

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Is small

5. HARENBERG J, WURZNER B, ZIMMERMANN R, SCHETTLER G. ability and antagonization of the low molecular weight 216 in man. Thromb. Res. 44, 549-554, 1986

beautiful?

Bioavailheparin CY

6. BENTLEY P, KAKKAR V, SCULLY M, MAC GREGOR F, WEBB F, CHAN P, JONES H. An objective study of alternative methods of heparin administration. Thromb. Res. 18, 177-187, 1980

BIOAVAILABILITY OF HEPARIN

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7.

HARENBERG

J,

DE VRIES

J.

performance size exclusion 261, 287- 292, 1983 8. Heptest

Package

Insert.

Vol.

48,

No.

Characterization of heparins by high liquid chromatography. J. Chromatog.

Haemachen,

Inc.

St. Louis,

MO

1985.

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