Indium-111 DTPA-Heparin: Radiolabeling, Pharmacokinetics, and Biodistribution Following Intravenous Administration in Rat and Rabbit

THROMBOSIS RESEARCH

Pergamon

ThrombosisResearch89 (1998)23-30

REGULAR ARTICLE

Indium-111DTPA-Heparin:Radiolabeling, Pharmacokinetics,and BiodistributionFollowing IntravenousAdministrationin Rat and Rabbit RobertW. Buntingl,RobertA. Wilkinsonz, RonaldJ. Callahanz, H.W.Strauss3and AlanJ. Fischman2 ~Department of Hematology,SpauldingRehabilitationHospital,Boston,MA,USA ‘Departmentof NuclearMedicine,Massachusetts GeneralHospital,Boston,MA,USA 3Departmentof NuclearMedicine,StanfordUniversity,PaloAlto,CA,USA. (Received2 July1997by EditorS.Niewiarowski; revised/accepted 8 October1997)

Abstract Heparin was coupled to DTPA using the bicyclic anhydride and labeled with Indium-111. This resulted in a radiochemically pure preparation (greater than 95% activity in one peak) as determined by high pressure liquid radiochromatography and did not affect the anticoagulant properties of heparin. Biodistribution in the rat at 1,20, and 60 minutes after intravenous injection showed rapid blood clearance with uptake in the liver followed by bone and kidney when expressed as percent injected total dose per organ and liver followed by kidney and spleen when expressed as percent injected total dose per gram. Blood elimination in the rabbit was 18.5minutes which decreased to 7.5 minutes when followed by the injection of protamine.Radioactivitycleared from the liverand lungs as a single exponential with a half-time of 30 minutes, but there was very rapid increase of radioactivityin the lungs,peaking at 1–2minutes,followingthe injection of protamine. Indium-111DTPA-heparin may be used to study in vivo pharmacokinetics and biodistribution of heparin. @1998 Elsevier Science Ltd. Abbreviations: HPCL, high pressure liquid radiochromatography;

P’IT, partial thromboplastine times. Corresponding author: Robert Bunting, Spaulding Rehabilitation Hospital, 125Nashua Street, Boston, MA 02114;Tel: 617-854-6300; Fax: 617-720-6575.

Key Words: Intravenous; Heparin;Biodistribution; Pharma-

cokinetics

A

lthough heparin has been widely used since the 1930sfor the treatment of thrombotic diseases, its pharmacokinetics and biodistribution have not been studied extensively[1].Because there is no chemical assay for heparin, its kinetic behavior has most often been determined by measurement of its anticoagulant properties in plasma, and biodistribution studies have been limited becausethese measurements could not be made non-invasivelyin tissue [1]. Heparin labeled with a gamma emitting radionuclide which would permit external detection with scintillationcameras would be useful in the study of heparin pharmacokinetics, especiallyin humans. To date, heparin labeled with technetium-99m[2-4] is the most widelyused radiolabeled heparin. Although technetium-99mheparin has excellentimagingproperties, it is of limited use in extended pharmacokinetic studies due to its 6-hour half-life.In addition, in our hands, using previously published methods [2,3],a radiochemically pure preparation could not alwaysbe achievedwhen analyzedby high pressure liquid radiochromatography (HPLC). In this study, we have coupled DTPA to heparin using the cyclicanhydride, a bifunctional chelating agent to which several radionuclides including Indium-111 with its 2.8-day half-life, can readily be attached. This procedure preserves in vitro pharma-

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R.W.Buntinget al./Thrombosis Research89 (1998)23-30

cologicalactivity. We report the pharmacokinetics and biodistributionof intravenousheparin usingthis new radiopharmaceutical. 1. Materials and Methods 1.1. CouplingHeparinwithDTPA Prior to coupling, 5 to 10 ml of preservative-free heparin (Apothecon, Bristol-Myers Squibb, Plainsboro, NJ) at 1000 units/ml (11 mgs/ml of sodium heparin from porcine intestine) was dialyzed overnight against 0.1 M NaHC03 in membrane tubing with a 12,000–14,000Dalton exclusion limit. To each 1 ml of heparin was added 1 mg of DTPAcyclic anhydride (diethylene triamine-pentacetic dianhydride from Aldrich, Milwaukee, WI) in .02 ml DMSO. This solution was stirred at room temperature for 1 hour and then exhaustively dialyzed against 0.15 M NaC1.As a control for subsequent clotting tests, heparin was similarly prepared except that DTPA-cyclic anhydride was omitted from the DMSO. Aliquots containing 500 units of DTPA-heparin were stored in unit dose vials at –70”C. 1.2. RadiolabelingwithIndium-111 Unit dose vials were allowed to come to room temperature. Indium-111chloride (Amersham, Princeton, NJ) was then added dropwise to a final concentration of 1 mCi/1000 units DTPA-heparin and incubated for 15minutes at room temperature. The amounts of Indium-111DTPA-heparin used thereafter were expressed in microcuries (uCi.) Radiolabeling of control uncoupled heparin was performed under the same conditions. 1.3. RadiochemicalPurityDetermination A 20-microliter sample of Indium-111 DTPA-heparin was applied to an HPLC column, TSK-250, and eluted isochratically with a mobile phase of 0.075 M NaC1. Both radioactivity and ultraviolet (205 nm) tracings were recorded [5]. Retention time, column recovery, and percent radioactivity associated with heparin was determined. Radiochemical purity determination of labeled uncoupled heparin was determined under the same conditions.

1.4. Effect of HeparinandDTPA-Heparin on PartialThromboplastinTime (PTT) Blood from normal human volunteers was drawn into 4.5-mlvacuum tubes containing 0.5 ml of 3.8% sodium citrate solution and plasma prepared. To 1 ml plasma was added 1, 2, 2.5, or 3 microliters of control heparin or DTPA-heparin solution, each diluted with 0.15 M NaCl to a concentration of 100u/ml. Partial thromboplastin times (PTT) were determined immediately with a General Diagnostics Coag-A-Mate XC (n=6 for plasma, n=5 for heparin plasma, and n=7 for DTPA-heparin plasma). 1.5. ProtaminePrecipitation One hundred microliters of control heparin, DTPA-heparin or 0.15M NaCl was added to duplicate glass tubes. Indium-111 Cl was added to each tube. The tubes were incubated at room temperature for 30 minutes. Protamine (200 microliters at 10 mg/ml, Elkins-Sinn, Inc., Cherry Hill, NJ) was then added to each tube followed by 1 ml of .15 M NaC1.The tubes were centrifuged at 1500gfor 15 minutes and the supernatant decanted. The percent radioactivity in the precipitate and supernatant was determined. This experiment was repeated on three separate occasions. 1.6. Biodistributionof Intravenously InjectedIndium-111DTPA-Heparinin Rats The biodistribution of Indium-111 DTPA-heparin was determined in 18 male Sprague-Dawley rats weighing 80–100grams following intravenous tail vein injection of 6 uCi of Indium-111 DTPA-heparin. Groups of six animals each were sacrificed by ether overdose at 1, 20, and 60 minutes following injection. These and all other animals in the study were cared for in compliance with the American Convention on Animal Care; these animals were approved for study by the animal studies committee. Samples of blood, muscle, and bone and the entire heart, lungs, liver, spleen, kidney, stomach, GI tract, and testes were collected, weighed, and counted using a 30Y0window around both photopeaks of Indium-111(LKB 1282Compu-Gamma counter). Using appropriate standards of the injected dose, the percent of the administered dose per organ and per gram of tissue were calculated.

25

R.W.Buntinget al./Thrombosis Research89 (1998)23-30

1.7. Blood Clearanceof Intravenously InjectedIndium-111DTPA-Heparin and Indium-111DTPA-Heparin Followedby Protaminein Rabbits New Zealand White rabbits (n=3) were anesthetized with ketamine/xylazine and a carotid artery was cannulated with PE-50 polyethylene tubing (Clay Adams, Parsippany, NJ) which was connected to a fraction collector (Gilson Medical Electronics Co., Middletown, WI) using a P-1 Peristaltic Pump (Pharmacia, Gaithersburg, MD). Following the injection of 200 microcuries of Indium-111 DTPA-heparin into a marginal ear vein, 0.1 ml blood samples were collected into weighted tubes at l-minute intervals for a total of 60 minutes. Indium-111radioactivity per gram of blood was measured with a LKB 1282 Compu-Gamma Counter and expressed as percent maximum counts. The intravenous injection of Indium-111DTPAheparin in three rabbits was followed by an intravenous injection of 400 units of protamine 14 minutes later. Blood samples were similarly collected and counted. 1.8. Imagingof IntravenouslyInjectedIndium111 DTPA-HeparinandIndium-111DTPAHeparinFollowedby Protaminein Rabbits Following the intravenous injection of 200 microcuries of Indium-111 DTPA-heparin into male New Zealand White rabbits (n=3), serial anterior gamma camera images were acquired for 60 minutes using an Ohio Nuclear large fieId of view 410 gamma camera equipped with a medium energy collimator and 20Y0 windows around both photopeaks. Regions of interest were drawn over the liver, lungs, and kidneys in all images using a Technicare 560 computer. Data were expressed as percent maximum counts and plotted versus time for lung and liver. An additional three rabbits were injected as above with Indium-111 DTPA-heparin followed 1-4 minutes later by the intravenous injection of 400 units of protamine and similar analyses were performed. 2. Results 2.1. RadiochemicalPurityDetermination A typical HPLC chromatogram of Indium-111 DTPA-heparin is shown in Figure 1. Tracings in-

clude UV at 205 nm and radioactivity (kcps). Indium-111 DTPA-heparin had a retention time of 7.06~.08 minutes (n=15) UV followed 35 seconds later by radioactivity consistent with the delay between detectors. Similar results were obtained with unlabeled heparin. The corresponding retention times for Indium-111 DTPA (n=3) and for Indium-111 chloride (n=3) were 10.75~.01 minutes and 11.12~.02 minutes respectively. 97.4f3.0% of Indium-111 activity applied to the column was recovered within the first 15 minutes and of this 99.2* 1.OYO (n=18) was associated with the heparin peak at 7 minutes. In all products used in subsequent experiments greater than 98Y0of the radioactivity was determined to be associated with this peak prior to use. When Indium-111and uncoupled heparin were applied to the HPLC column, only 4.75~2.470 of Indium-111radioactivitywas recovered in the first 15 minutes; 77.26f6.34°/0 was associated with the peak at 7 minutes. 2.2. Effect of HeparinandDTPAHeparinon PartialThromboplastinTimes Figure 2 showsthe results of the P’IT plotted against the concentration of heparin or DTPA-heparin in plasma. The anticoagulant effect of heparin was largely preserved after coupling with DTPA. Increasing doses of control and DTPA-heparin over the range of added 0-0.3 units/ml plasma resulted — RADIOACTIVITY

Iridium- ffl DTPA - Heparin Retention Time 7.2 min

1

&

Fig.1. HPLCradiochromatogram of Indium-111DTPAheparinshowsaretentiontimeof7.2minutesbyradioactivity and UV.

R.W.Buntinget al./Thrombosis Research89 (1998)23-30

120-

followinginjection,organ distribution remained relatively stable with liver and bone accounting for approximately535%0 of the administered dose (dose/ organ). Kidney activity decreased throughout the observation period.

100.

40

---+---

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:.DO

0.05

0.10

0.15

0.20

Hep.min-DTPA

0.25

0.30

Units/ml Plasma

Fig. 2. Effectofincreasing concentrations ofcontrolheparin(squares)andDTPA-heparin(dots)addedtoplasmaon thepartialthromboplastin time(n=6plasma,n=5 heparin, n=7 DTPA-heparin).

in linear increases in partial thromboplastin times; however the slope of the line for DTPA-heparin (y=26.7+342x; RZ=0.998)was approximately 60% of uncoupled heparin (y=32.2+204x; R2=0.976). Paired t-test of differences in PTTs after addition of 1, 2, and 2.5 microliters of heparin or DTPAheparin solutions,however, failed to show a significant difference in these values with significanceof 0.9, .051,and .096,respectively. 2.3. ProtaminePrecipitation The addition of an excess of protarnine to solutions of Indium-111DTPA-heparin, Indium-111added to control heparin, or Indium-111 without heparin in NaCl resulted in the precipitation of 96.5tl.5?40, 52.5f5.9Yo, and 10.2f4.7940of the Indium-111activity, respectively. 2.4. Biodistribution of Intravenously InjectedIndium-111DTPA-Heparinin Rats The biodistribution of intravenously injected bolus Indium-111DTPA-heparin is shown in Figure 3 as percent mean injected dose per gram and Figure 4 expressed as mean percent injected dose per organ. This compound is cleared from the blood with a half-lifeof approximately 8.9 minutes with accumulation in the liver, spleen and kidney, and bone expressed as dose/gram and liver, bone, kidney, and GI tract expressed as dose/organ. By 20 minutes

2.5. Blood Clearanceof Intravenously InjectedIndium-111DTPA-Heparinin Rabbits Figure 5 shows that the blood elimination of Indium-111 DTPA-heparin was monoexponential (y= 108.33x1O(-.O165); R2=0.998)with a half-life of 18.2minutes. Blood elimination of heparin-protamine complex was considerablymore rapid with a half-life of 7.5 minutes. 2.6.Imagingof Intravenously InjectedIndium-111DTPA-Heparin andIndium-111DTPA-Heparin Followedby Protaminein Rabbits Region of interest analysis showed (Figure 6) that radioactivity clears from the lungs as a single exponential with a halftime of 30 minutes after intravenous injection of Indium-111 DTPA-heparin. In contrast, when protamine was injected after Indium-111 DTPA-heparin, there was very rapid increase of radioactivity in the lungs peaking at 1–2 minutes following injection of protamine. Figure 6 shows the results from one animal after injection of Indium-111 DTPA heparin was followed by protamine. Lung radioactivity returned to the same levels as control animals within 3–15minutes after the injection of protamine. 3. Discussion The first attempts to study heparin kinetics using radiochemicals were made with 35S sodium sulfate labeled heparin which had been prepared from the liver of dogs previously injected with 35S sodium sulfate [6]. It was subsequently shown, however, that reabsorption of the labeled sulfur from the urine and incorporation into other mucopolysaccharides made these measurements unreliable [7]. A tritium labeled heparin was developed which was more stable than the 35S preparation [8], but the use of this compound is limited because h is a beta emitter and cannot be imaged externally or

R.W.Buntinget al./Thrombosis Research89 (1998)23-30

‘i

1,2 J

E m h k a al

Fig.3. Biodistribution ofIndium-lllDTPAheparin after bolus intravenousinjection was givento rats expressedas mean percent~SD (n=6) of the total doseinjected per gram of tissue1, 20, and 60 minutes afterinjection.

27

I“1’

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[:)

1 MIN

,

20 MIN

M

60 MIN

~ ~ U ~ ~ c (u ;

used inhuman studies. The first heparins that could be externally imaged with a gamma camera were labeled with iodine 125 [9] and later Technetium99m [2, 3], but use of these reagents is limited by a difficult labeling procedure for the former and the short half-life of the latter. Bifunctional chelates coupled to proteins and carbohydrates [10]can be easilylabeled with radio-

active metals such as Indium-111. These chelates do not interfere with function [11]and high avidity of the metal for the chelate (equilibrium constant of 10 to the 28th for DTPA) results in a minimal rate of transchelation in vivo [12].In order to overcome the limitations of previously reported methods and provide a radiolabeled heparin product which allows for possible extended monitoring of pharmacokinetics, we evaluated the use of these bifunctional chelating agents coupled with the 2.8

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4

a

12

16

20

24

20

32

36

40

44

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52

Time (rein)

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Fig.4. Biodistribution ofIndium-111 DTPA-heparinafter bolusintravenousinjectionwasgivento rats expressedas mean percent*SD(n=6) of the total dose injectedper organ1,20,and 60minutesafterinjection.

Fig.5. Bloodclearancecurvesafterintravenousinjection ofIndium-111 DTPA-heparinin rabbitsandafterintravenousinjectionof Indium-111DTPA-heparinis followed by protamine.Eachpointis mean*SDof three determinations.

R.W.Buntinget al./Thrombosis Research89 (1998)23-30

28

160-

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100-

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Heparin-Protamine

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Fig. 6. Pulmonary clearance curve after intravenous injection of Indium-111 DTPA-heparin in rabbits (squares) (each point is meantSD of three determinations) and after intravenous injection of Indium-111 DTPA-heparin was followed by protamine (dots) in one rabbit.

day gamma emitting metal ion Indium-111 to label heparin. This method resulted in a high labeling efficiency with minimal effect of the labeling procedure on the anticoagulant activity of heparin. There was a slight but insignificant decrease in the slope of the curve when increasing amounts of DTPA-heparin were added to plasma and plotted against the resultant partial thromboplastin time when compared to control heparin. These curves are similar to previouslyreported results [13].The number of DTPA molecules attached per heparin molecule could not be determined, however, and it is possible that uncoupled heparin present in this preparation accounts for the residual activity in these clotting studies. The labeling procedure, however, had no effect on the ability of DTPA-heparin to bind to protamine both in vitro and in vivo, experiments in which heparin molecules which had been coupled and labeled were certainly studied. The labeling efficiency was over 98°L and the radioactivity was found in only the heparin peak on HPLC in comparison with several peaks which were frequently found when technetium-99m labeled heparins were studied with HPLC. Initial labeling studies were performed using both citrate and oxalate as a carrier for the Indium-111, but it was found that the labeling could be as successfully accomplished in the .15M saline which initially was used

as a control and without the formation of colloids. It is possible that the non-specific binding of Indium-111 which occurs to- heparin enables DTPA-heparin to act as its own carrier. It seems likely that any Indium-111 which is initially bound to heparin non-specifically is subsequently chelated to the DTPA molecule. There was minimal recovery of Indium-111 activity when uncoupled labeled heparin was subjected to HPLC, suggesting that this activity is stripped from the heparin by the HPLC procedure. There was, however, nearly complete recovery when Indium-111 DTPA-heparin was similarly studied. These results suggest that there is little unchelated Indium-111 remaining in the DTPA-heparin labeling mixture because it would have been stripped off by the HPLC procedure and the recovery would not have been nearly complete. This direct addition of Indium-111 contributes to the simplicity of the method, and it can be performed in laboratories where a technetium generator is not readily available. The biodistribution of intravenously injected Indium-111DTPA-heparin is consistent with those previously reported for other radiolabeled heparins in sacrificed animals including tritium [14],Cr51 [15], iodine 125 [9], and technetium-99m [2-4] although in all of these previous studies heparin biodistribution was determined at only one time point. Maximum uptake by the liver was demonstrated in all of these except for technetium heparin in which counts/gram of tissue were highest in the kidneys three hours after injection. These were the only experiments, however, in which dogs were used, suggesting a possible species difference. In none of these previous studies were as many tissues examined as in this report. The only studies in which uptake by bone was examined were those using technetium-99m heparin [2,3] in which the ratio of liver to bone activity was approximately the same as that reported here. Although it is possible that transchelation of Indium-111 accounts for some of the bone activity in this study, this would not be the case for technetium heparin, These results are consistent with the observation that osteoporosis is a complication of long-term administration of heparin [16]. The blood clearance of intravenously injected heparin has been shown to be dose dependent [17], the half-life increasing as larger doses are administered, and it is therefore difficult to compare the

R.W.Buntinget al./Thrombosis Research89 (1998)23-30

half-life of Indium-111 DTPA-heparin obtained in this study with many other reported values. In one study [18], however, the doses of standard iodinated heparin administered versus the half-life were reported. The half-life of iodinated heparin for the approximate doses used in this study was 14.7minutes, similar to the 18.6minutes found for DTPA-heparin. The blood clearance of Indium111DPTA-heparin was accelerated by the injection of protamine. This result has not previously been reported since most clearance studies have been performed usinganticoagulantactivitywhichcannot be measured after protamine administration. Imaging of the lungs after injection of Indium111 DTPA-heparin in rabbits showed a gradual decline of activity as a single exponential. In contrast, when protamine was administered there was an increase in the activity in the lungs; in two of the animals studied the peaks were approximately the same and there was a rapid return to baseline; in a third animal the peak was much higher and persisted longer. Further study of these findings is necessary to determine whether larger aggregates of heparin and protamine were formed in this latter animal. The relationship of these precipitates to possible adult respiratory distress syndrome has been previously considered [19].These results are also compatible with previous studies in sheep using labeled protamine [20]in which it was shown that prolonged pulmonary clearance of Indiumlabeled protamine occurs in animals that have been previously anticoagulated with heparin. DTPA can easily be coupled to heparin using bifunctional chelates and subsequently radiolabeled with radioactive metals. Although Indium111was chosen in this study, it is possible that other radiochemicals includingtechnetium-99m could be used to label the DTPA-coupled heparin. Although it is theoretically possible that Indium-111 DTPA-heparin could be used in long-term studies this has not been demonstrated here, and it is possible that some transchelation with ferritin would occur. This method does not substantially affect heparin function. Since commercial grade heparins were used for labeling in this method, these compounds could be readily adapted for use in human studies of the pharmacokinetics of heparin. It is also likely that this approach could be used to label low molecular weight heparins in order to further

29

study the comparative pharmacokinetics of this important class of drugs. References 1. Hirsh J. Heparin. NEJM 1991;324:1565-74. 2. Kulkarni PV, Parkey RW, Buja LM, Wilson JE III, Bonte FJ, Willerson JT. Technetiumlabeled heparin: Preliminary report of a new radiopharmaceutical with potential for imaging damaged coronary arteries and myocardium. J Nucl Med 1978;19:810-5. 3. Kulkarni PV, Parkey RW, Wilson JE HI, Lewis SE, Buja LM, Bonte FJ, Willerson JT. Modified technetium-99m heparin for the imaging of acute experimental myocardial infarcts. J Nucl Med 1980;21:117-21. 4. Laforest MD, Colas-Linhart N, GuiraudVitaux F, Bok B, Bara L, Samama M, Marin J, Imbault F, Uzan A. Pharmacokinetics and biodistribution of technetium 99m Iabelled standard heparin and a low molecular weight heparin (enoxaparin) after intravenous injection in normal volunteers. Br J Haem 1991; 77:201-8. 5. SugisakaN, Petracek FJ. Rapid molecular size characterization of heparins by high pressure liquidchromatography.Fed Proc 1997;36:89-92. 6. Eiber HB, Danishefsky I, Borrelli FJ. Physiological disposition of heparin. Proc Soc Exp Biol Med 1958;98:672-4. 7. Day M, Green JP, Robinson JD Jr. Disposition of (35S)-heparin in the rat. Br J Pharmacol 1962;18:625-9. 8. Barlow GH, Cardinal EV. Preparation and characterization of tritiated heparin. Proc Soc Exp Biol Med 1966;123:831-2. 9. Dawes J, Pepper DS. Catabolism of low-dose heparin in man. Thromb Res 1979;14:845-60. 10. Krejcarek GE, Tucker KL. Covalent attachment of chelating groups to macromolecules, Biochem Biophys Res Commun 1977;77:581–5. 11. Schwarz SW, Mathias CJ, Sun JY, Dilley WG, Wells SA Jr, Martell AE, Welch MJ. Evaluation of two new bifunctional chelates for radiolabeling a parathyroid-specific monoclinal antibody with In-ill. Nuc Med Biol 1991; 18(5):477-81. 12. Welch MJ, Welch TJ. Solution chemistry of

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carrier-free Iridium. In: Subramanian G, Rhodes B, Cooper JF, Sodd VJ, editors. Radiopharmaceuticals. New York: Society of Nuclear Medicine Publications; 1975. 13. De Swart CAM, Nijmeyer B, Roelofs JMM, Sixma JJ. Kinetics of intravenously administered heparin in normal humans. Blood 1982; 60:1251-8. 14. Shanberge JN, Gruhl M, Kitani T, Ambegaonkar S, Kambayashi J, Nakagawa M, Lenter D. Fractionated tritium-labelled heparin studied in vitro and in vivo. Thromb Res 1978;13:767-83. 15. Alant O, Varga L, Antoni F, Karacsonyi S, Failer J. Metabolism and anticoagulant effect of 51Cr-labelled heparin. Acta Physiologic Academiae Scientiarum Hungaricae 1973;43: 261-7. 16. Ginsberg JS, Kowalchuk G, Hirsh J, BrillEdwards P, Burrows R, Coates G, Webber C.

Heparin effect on bone density. Thromb Haemost 1990;64(2):286-9. 17. Boneu B, Caranobe C, Sie P. Pharmacokinetics of heparin and low molecular weight heparin. Balliere’sClinicalHematology 1990;3:53144. 18. Boneu B, Dol F, Caranobe C, Sie P, Houin G. Pharmacokinetics of heparin and related polysaccharides. Ann NY Acad Sci 1989;556: 282-91. 19. Shanberge JN, Murato M, QuattrociocchiLonge T, Van Neste L. Heparin-protamine complexes in the production of heparin rebound and other complications of extracorporeal bypass procedures. Am J Clin Pathol 1987;87:210-7. 20. Montalescot G, Fischman AJ, Strauss HW, Wilkinson RA, Ahmad M, Fitzgibbon C, Robinson DW, Zapol WM. Imaging the ovine heparin-protamine interaction with lllln-protamine. J Appl Physiol 1993;75(2):963–71.