In Vitro Bioactivity of a Synthesized Prostaglandin E,- Heparin Conjugate

HARVEY JACOBS AND SUNG WAN KIM' Received Jul 1 1, 1985, from the Depamnent of Phamaceu~cs, Unkersity of Utah, Salt Lake CHy, UT 84 1 12. November 1 i 1985.

Accepted for publication

AbstraclO A covalently bound conjugate of commercial grade heparin and prostaglandin El (PGEl) was synthesized to provide the dual pharmacological role of decreasing the extent of platelet aggregation and Inhibiting fibrin formation during thrombogenesis. The compound was synthesized using a modified mixed carbonic anhydride method of amide bond formation between the carboxylic acid moiety of PGE, and a primary amine group on heparin. Ouantitation of coupling was measured spectrophotometrically by monitoring a degradation product of the prostaglandin E14eparin amjugate (prostaglandin Bl-heparin amjugate). Bioactivity tests on the conjugates (activated partial throm- boplastin time and platelet aggregation) confirmed that both the antico- agulant activity of heparin and the inhibitory effect of PGEl on platelet aggregation were maintained.

Thrombus formation and platelet aggregation are the two main mechanisms governing hemostasis in animals. The two mechanisms are separate, yet synergistic, in controlling bleeding and providing a defense mechanism against vascu- lar injury and foreign surfaces.

Fibrin formation involves activation of the intrinsic coagu- lation m a d e . The cascade is initiated by plasma proteins adsorbing onto an activating surface (e.g., polymers, nega- tively charged collagen, or activated platelet membranes). Various proteolytic enzymes are involved in the caecade, leading to the conversion of prothrombin to thrombin. Thrombin then converta fibrinogen to fibrin monomers which are subsequently crosslinked to form a fibrin network (clot).'

Platelet aggregation and adhesion are initiated by expo- sure of blood to activating agents, such as polymer surfaces, sublayer structures of injured vaeculature (microfibrils, basement membranes, or collagen), established fibrin clots, or various chemicals (ADP, serotonin). Platelets attach to eurfaces, then activate and contract to release the contenta of their intracellular granules (ADP and AW). These agents stimulate further platelet aggregation to ultimately form a platelet plug and release platelet factora.2

Fibrin formation and platelet aggregation are controlled by heparin and prostaglandins, respectively. Specifically, heparin is an effective antithrombotic agent for certain conditions. Its interactions with antithrombin III and pre- vention of fibrin clot formation are well documented.'ss Prostaglandins (PG12, PGE1, and PGD2) interact with mem- brane bound platelet receptors to inhibit platelet adhesion and aggregation.a.4

The intrinsic coagulation pathway and platelet aggrega- tion mechanisms are synergistic and the stimulation of one pathway leads to the activation of the other mechanism. Considering this fact, it may be desirable to control both thrombus formation and platelet aggregation by one macro- molecule.

An approach studied by Hennink et al .6 was to covalently bind heparin to human albumin and preadmrb this conjugate onto polymer surfaee. This method utilized the fact that albumin preadsorbed onto surfaces with reduced platelet adhesion: while heparin was able to interact with anti-

thrombin 111, preventing thrombus formation. The investiga- tore found that the heparin-albumin conjugate significantly reduced both the amount of fibrin formation and the extent of platelet aggregation. This indicates that it is possible to prevent both mechanism of thrombogeneeis by a single marromolecule.

In this study, it is proposed to covalently link heparin and PGE1, thereby utilizing a dual pharmacological approach to reduce platelet aggregation with the potent antiplatelet agent, WE1, and to prevent fibrin formation with heparin. This paper describes the synthesis, characterization, and in vitro testing of the PGEl-heparin conjugate (PGEI-HEP).

Experimental Section MaterialeHeparin from porcine m u m a (165 IUlmg) was ob-

tained from Diosynth, Chicago, IL. FGE, was purchased from Cayman Chemicals, Ann Arbor, MI. FGF= was purchased from the Upjohn Company, Kalamamo, MI. Radiolabeled ["ClPGF2, was purchased from New England Nuclear, Boston, MA. Bio-Gel P-6 was obtained from BioRad Company, Richmond, CA. Activated Thrombo- fax Reagent-Optimized and calcium chloride were purchased from Ortho Diagnostic Systems, Raritan, NJ. All other chemicals were reagent grade (Sigma, St. Louie, MO) and used without further purification.

Quantitation of Free Amino Groups on Heparin-The first step in coupling FGEl to heparin via an amide bond was to quantitate the number of free amino groups on the heparin molecule. This was accomplished using the 2,4,6-trinitrobenzeneeulfonic acid (TNBS) spectmphotometric method of derivatization of free amines.7 A glucosamine-TNBS derivative was used to generate a standard curve for the W detection of the TNBS derivative of heparin. Stock solutions of gluwaamine (0.004-0.1 mg/mL, 2 x lO-'A x lo-' M) were prepared and used. One milliliter of 0.5 M phosphate buffered d i n e (pH 8) and 1 mL of 0.01% TNBS solutions were added to 0.5 mL of each gluwaamine solution. The mixtures were stirred at 40°C for 90 min, after which time 1 mL of 6 M HCl was added. The absorbance was read at 348 nm on a W spectrophotometer (Perkin- Elmer Lambda 7 UV-VIS Spectrophotometer). Stock solutions of heparin, (1-10 mg/mL), were prepared, and TNBS derivatives were made as described above.

Synthesis of the Prostaglandin-Heparin cod jugate-The mixed anhydride method of amide bond formation was utilized for the coupling of WEl to heparin. Other methods had been attempted, specifically the active ester method and the NJV'dicyclohexylwbo- diimide method, yet the mixed anhydride method proved to be the faatest, moat efficient, and contained the least amount of contaminat- ing by-product.a.8

In a typical experiment, 5 mg (1.4 x lo-' moll of WEl was diasolved in 0.5 mL of dimethylformamide. This was stirred in an ice bath at 0°C. Chilled (0°C) isobutyl chloroformate (2 pL, 1.6 x lo-' moll and triethylamine (4 pL, 1.6 x lo-' moll were added to the above solution. ARer 15 min, 8 pL (3.2 x mol) of triethylamine was added, followed by the addition of 350 mg of heparin dissolved in 2 mL of 1:l H,O:dimethylformamide (pH = 8.0) solution, and chilled. The reaction was maintained at 0°C for 1 h, and then at Mom temperature for 18 h.

The WE1-HEP coqjugate waB then lyophilized, redissolved in a minimal amount of water, and isolated by precipitation with a large excB88 of acetone. The precipitate was removed by filtration. washed with acetone, and then dried.

172 / Journal of Phamaceutjcal Sdences Vol. 75, No. 2, Februaty 1986

0022-3549/86/0200-0172$01.00/0 Q 1986, Americen Pharmaceutical A m a t i o n

Fractionation of the Prostaglandin-Heparin Conjugate-PGF, and ["CIPGF,, were used to determine if a covalent amide bond was formed during the reaction. PGFh has similar chemical reactivity and structure to PGE, and was available from previous stocks.

A stock solution of [14ClPGFz&nlabeled PGF, was made by mixing 250 pL of ['4C]PGFzo [3.8 x lo6 disintegrations per minute (dpm)/mLl with 100 mg of unlabeled PGF, and diluting to 5 mL with dimethylformamide. This created a solution of 1.9 x lo6 dpm/mL (24 x lo3 SD), or 9.3 x lo3 d p d m g of PGF,,. The chemistry for coupling PGFh to heparin was identical to that used for the synthesis of PGEl-HEP.

ARer the reaction, the solution was applied to a gel-permeation chromatography column (30 x 1.5 cm) packed with Bio-Gel P-6. Distilled water waa eluted through the column at a flow rate of 20 mL/h. Aliquots (0.5 mL) of collected fractions were mixed with scintil- lation fluid (Formula-95OA, New England Nuclear) and the radioactiv- ity (dpm) waa determined on a Beckman LS 7500 liquid scintillation counter. A volume (25 p L ) of each fraction was also qualitatively tested for heparin using the toluidine blue chromogenic assay.9

Quantitation of Prostaglandin El Covalently Bound to Hepa- rin-Roataglandin El undergoes a base-catalyzed dehydration (15- OH) to form a conjugated ketone diene (PGB1) with a Am= = 282 nm, at pH > 11.10 Several samples of varying concentrations of PGE, (1 x 10-6-1 x M) were dissolved in 1 M NaOH and heated to 60°C for several hours. The absorbance at 282 nm was measured on a UV spectrophotometer, and a standard curve was constructed. PGE1- HEP conjugates were analyzed for the PGE, to PGBl degradation by a similar procedure.

Quantitation of Free Prostaglandin Contamination-An experi- ment was performed to determine the amount of free prostaglandins contaminating the PGE1-HEP conjugate (unbound). Again, radiola- beled PGFh was used to model the free PGEl concentrations that may remain after the PGEI-HEP acetone precipitation. ["CIPGF, (0.025 mL1.1 x lo7 dpdmL, 58 pCi/pmol) and PGE1-HEP (50 mg) were dissolved in a minimal amount of water. The mixture was added to a large excess (20 times the volume) of acetone and the PGE,-HEP conjugate was precipitated. The precipitate was removed by filtration, washed with acetone, and the concentration of [14ClPGF,, contaminating the conjugate was determined on a liquid scintillation counter.

Bioactivity Tests of the Prostaglandin El-Heparin Conjugates- New Zealand white rabbits (male, 2-3 kg) were anesthetized with ketamindpromethazine, and 100 mL of blood was drawn from the femoral artery into citrated (3.8%, 1:9 dilution) syringes. The blood was centrifuged at 3OOxg for 15 min to obtain platelet-rich plasma and finally a t 1500xg for 20 min to obtain platelebpoor plasma.

Platelet aggregation measurements were performed on a Chrono- Log aggregometer (model 335). For platelet aggregation experi- ments, solution concentrations are reported as the final cuvette concentration after dilution with platelet-rich plasma and ADP. Platelet-rich plasma (450 pL) and PGEl (25 &, 1 x 10-"--1 x lo-' mol/mL) were incubated at 37°C for 2 min. Adenosine diphosphate (25 pL, 20 pM) was added and the extent of platelet aggregation afbr 5 min was recorded. Varying concentrations (0.05-2.5 mg/mL) of PGE,-HEP conjugate were evaluated for inhibiting effects on platelet aggregation by an identical procedure.

Heparin and PGE, were mixed in amounts equal to the concentra- tion of each drug as determined to be present in the PGE1-HEP conjugate (based on 3.2 x lo-' mol of PGEl/mg of HEP). A stock solution of 50 mg/mL HEP (in phosphate buffered saline) was mixed with 1.6 x mol(0.566 mg) of PGE1. Varying concentrations of this stock solution (5 X mg/mL HEP plus 1.6 x lo-'' rnol of PGEl to 2.5 mg/mL of HEP plus 8 x lo-' mol of PGEJ were evaluated for inhibition of platelet aggregation as described. Also, pure HEP (5 x 10-3-2.5 mg/mL) was evaluated for platelet responsiveness as described.

Activated partial thromboplastin time was determined on a Fibro- system fibrometer. Heparinized platelet-poor plasma (0.1-0.5 U, 100 &) was incubated with 100 pL of activated Thrombofax Reagent for 2 min, after which time 100 pL of 0.02 M CaC1, was added and the time for a fibrin clot to form (mechanical end point) was recorded. This procedure established a heparin standard curve to evaluate subsequent PGEl-HEP conjugates. The PGEI-HEP conjugate was diluted in platelet-poor plasma (0.1 to 0.5 U/mL, based on weight of heparin). Evaluations for prolongation of clotting times were per- formed as previously described.

Results Quantitation of the Free Amino G r o u p s on Heparin-

The heparin-TNBS derivative absorbance was extrapolated from the glucosamine-TNBS UV standard curve to estimate the number of free amino groups on commercial grade heparin. The concentration was determined t o be 4.0 x mol of NHz/mg of HEP (50.5 x

Separation of [14C]Prostaglandin Fk-Heparin-Figure 1 shows the elution profi le for the products of the [ 14C]PFzR-heparin coupling reaction. As c a n be seen, there are two distinct radioactive peaks. The first radioactive peak contained the [14ClPGF2R-HEP conjugate, as shown by the dpm counts and the chromogenic detection of heparin. The s e c o n d r a d i o a c t i v e p e a k c o n t a i n e d o n l y u n r e a c t e d [l4C]PGFk, since n o heparin was detected. The specific activity of [l4C]PGFZa used in the reaction was known, therefore, by summing the dpm under each curve. It w a s estimated that approximately 3.5 x lo-' mol of PGFza were coupled per milligram of PGF2,HEP conjugate, assuming near complete recovery of the conjugate.

Quantitation of Prostaglandin El Bound to Heparin- The base hydrolysis of PGEl to PGBl is a known quantitative tool for measuring PGEl concentration.10 A standard curve for pure PGEl was constructed. Known amounts of PGE1- HEP were degraded to PGBl-HEP and the absorbance re- corded (282 nm). The results indicate approximately 3.2 x lo-' mol of PGEl were coupled per milligram of PGE1-HEP conjugate (50.2 x SD).

SD).

140r

FRACTION NUMBER

FI ure 1-Gel-permeation chromatographic elution profile of

with toluidine blue. I' B C]PGF,,-H€P. Key: (1-1) represents qualitative detection of heparin

i I z

I 2ot i _ / m -

O -I: 'I, -:o -b " -; -; -'6

LOG ESTIMATED PGE, (rnollrnL)

Flgure 2-Dose response curves for prostaglandin E, (0) and prosta- glandin €,-heparin (0) inhibition of ADP induced platelet aggregation.

Journal of Pharmaceutical Sciences / 173 Vol. 75, No. 2, February 1986

Quantitation of Free Prostaglandins-The results of the contamination experiment determined the unbound PGFzo concentration to be -2.5 x mol [14ClPGF2, unbound per milligram of PGE1-HEP.

Bioactivity Tests-Platelet Aggregation Results-The amount of pure PGEl needed to cause 50% inhibition of platelet aggregation (IDs0) in the rabbit model was deter- mined to be 1.0 x lo-'' mol of PGEl/mL.ll It was found that 0.34 mg/mL of PGE1-HEP (corresponding to 1.1 x lo-' mol of PGEl attached to HEP) was required for 50% inhibition of platelet aggregation. The dose response curves for pure PGEl and PGE1-HEP are shown in Fig. 2. The dose response data for the mixture of PGEl and HEP and for HEP alone are summarized in Table I.

Activated Partial Thromboplastin Results-The results of the tests (Fig. 3) show that the heparin standard curve is linear (r = 0.97) and that the clotting times for the equiva-

Table I-Dose Response Data for Platelet Aggregatlon Experlments

Prostaglandin Heparin, Inhibition of El, mol/mL mg/mL Aggregation, %

100 Prostaglandin El 1 x 10-8 - 1 x 10-9 - 100

50 1 x 10-10 - 6 1 x 10-11 - 0 1 x 10-12 -

Heparin' - 0.5 0 - 5 x 10-2 0 Prostaglandin El- 8 x lo-' 2.5 100

Heparin 4.2 x lo-' 1.3 100 1.6 X lo-' 0.5 70 9.6 x 10-9 0.3 38 3.3 x 10-9 0.1 1 0

Prostaglandin El + 1.6 x lo-' 5 x lo-' 100 Heparinaed 1.6 x 10-10 5 x 10-3 75

1.6 x 1 0 - l ~ 5 x 1 0 - ~ 20 1.6 x 10-12 5 x 10-5 0

a Represents the final cuvelte concentration after diluting with 0.45 mL platelet-rich plasma and 0.025 mL ADP. Prostaglandin El-heparin is the covalently bound conjugate. 'Calculated prostaglandin El con- centration. dProstaglandin El + heparin is the mixture of pure prosta- glandin El and heparin.

l o t 1 I I

0.2 0.3 0.4 0.5 HEPARIN CONCENTRATION [units h L )

Flgure 3-Activated partial thromboplastin time measurements of pros- taglandin €,-heparin conjugate (0) against the heparin standard cuwe (-1 *

lent PGE1-HEP conjugate concentrations were within the error measurements obtained for the heparin standards. This indicates that the ability of the conjugate to increase clotting time has been maintained, with respect to native heparin.

Discussion Reports on both the type and amount of several functional

groups on heparin have been cited,12 however, the number of free amino groups available for derivatization has not been reported.

Derivatization with TNBS proved to be a satisfactory method to quantitate the number of free amino groups on heparin. The results presented here show that the amount of PGEl that coupled to heparin correlated well with the number of free amino groups on heparin, as determined by this method. Also, the results of the radioactive gel-permeation chromatography experiment indicate that a stable covalent bond has been formed.

An important criteria established by the contamination experiment was that the amount of free PGFzU, which can be correlated to free PGEl concentrations, was -40 times less than the platelet aggregation IDso for pure PGE1. Therefore, these results imply that any biological activity of PGEl is elicited by the conjugate, and not from any free PGEl in the solution.

Studies13.14 have described a direct antagonistic effect of high doses of heparin on PGEl in evaluating platelet respon- siveness. Therefore, heparin alone and a mixture of PGEl and heparin were used as a control to evaluate the response of the conjugate to inhibition of platelet aggregation. The results indicate that in rabbit plasma, there was no effect of heparin towards platelets, and the mixture of PGEl and heparin showed a profile similar to pure PGE1. This implies that the extremely large doses of heparin found on the conjugate, compared to the PGEl concentration, did not influence the efficacy of PGE1, nor the responsiveness of the platelets.

However, the results of the platelet aggregation experi- ments indicate that the bioactivity of coupled PGEl is less than pure PGE1. This decrease in bioactivity may therefore be attributed to several other factors. The reaction was performed in an alkaline environment (pH 8) to insure that a percentage of amino groups would not be protonated in order to optimize the mixed anhydride coupling. However, a t a pH of 8, there is some base-catalyzed degradation of the PGEl molecule, which may account for the decreased activity. Another possibility is a restriction of motion of the PGEl molecule attached to a macromolecule. Bamford et a1.l6 reported a 50- to 100-fold decrease in activity when prosta- glandin analogues were coupled to grafted copolymers. This may prevent the molecule from interacting efficiently with the platelet receptors. This was compared to PGEl in solu- tion, where there is no restriction of interaction between PGEl and the receptor site. The bioactivity of PGEl may be improved by attaching the molecule to heparin via a spacer group. This would extend PGEl away from the immediate environment of the heparin molecule, which would permit it to interact with the receptors.

The results of the activated partial thromboplastin time tests revealed that the biological activity of heparin is retained when coupled with PGE1. This was also verified by Ebert et a1.,1* who reported no decrease in activity when amino groups were N-acetylated to prevent cross-linking during carboxylic acid group derivatization. However, deri- vatization of carboxylic acid, hydroxyl, and sulfate groups led to significant losses in bioactivity.

174 / Journal of Pharmaceutical Sciences Vol. 75, No. 2, February 1986

Heparin and PGEl have been investigated for use in controlled-release devices (blood contacting).le Both agents were stable and active in the polymer matrix and when released into solution (buffer or plasma). However, the diffu- sional properties of heparin and PGEl were vastly different; thus, it proved difficult to control the optimum release rate of each agent when mixed together.17 It is expected that the diffusional properties of the conjugate will be more like heparih, and the release kinetics will be easier to control.

Heparin and PGEl have also been immobilized on separate polymer surfaces, and the bioactivity of each drug was maintained.1s20 However, in the heparin-immobilized sys- tem,19 fibrin formation decreased while the extent of platelet aggregation was still significant. In these systems it was noted that the immobilization yields of heparin and PGEl were low, and there were no attempts to immobilize both heparin and PGEl on the surface together. The conjugate has the advantage of immobilizing only one macromolecule on the surface, thereby increasing the efficiency of the reaction.

References and Notes 1. Bi gs, R.; Denson, K. “Human Blood Coagulation, Haemostasis

an5 Thrombosis”, 2nd ed.; Biggs, R., Ed.; Blackwell Scientific Publication: London, 1976; pp 143-167.

2. Aiken, J . J. Cardwvasc. Phurmacol. 1984, 6, 5413. 3. Damus, P. S.; Hicks, M.; Rosenberg, R. D.Nature (London) 1977,

246, 355. 4. Whittle, B.; Moncada, S.; Vane, J . R. Prostaglandins 1978, 16,

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5. Hennink, W.; Feijen, J . ; Ebert, C.; Kim, S. W. Thromb. Res.

6. Hennink, W.; Dost, L.; Feijen, J . ; Kim, S. W. Trans. Am. Soc.

7. Yosizawa, K.; Kotoku, T.; Yamauchi, F. Biochem. Biophys. Actu

8. Meienhofer, J . “The Pe tides”; Gross, E.; Meisenbhofer, J., Eds.;

9. Smith. P.: Mallin. A.: Hermanson. dl Anal. Biochem. 1980,109.

1983,29, 1.

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466. 10. Stehle, P. G.; Gesterling, T. 0. J. Pharm. Sci. 1977, 66, 1590. 11. Dost, L., unpublished results. 12. Ebert, C.; Lee, E.; Deneris, J . ; Kim, S. W. “Biomaterials: Interfa-

cial Phenomena and Applications”, Coo er, S., Ed.; Advances in Chemistrv Series. no. 199: American 8hemical Societv: Wash- indon. DQ. 19821 DD 161-176. * r . ~

13. EPdor,A.; Leuksler, B. Thromb. Res. 1979,16,617. 14. Salzman, E. W.; Rosenberg, R. D. J. Clin. Invest. 1980, 65, 64. 15. Bamford, C.; Middleton, I.; Sataka, Y. Polym. Prepr. Am. Chem.

SOC. Div. Polym. Chem. 1984,25, 27. 16. Ebert, C.; McRea, J . ; Kim, S. W. “Controlled Release of Bioactive

Materials”; Baker, J . , Ed.; Academic Press: New York, 1980; pp . A ” .... l U ’ I - l Z l .

17. Kim, S: Ebert, C.; Lin, J . ; McRea, J . Tmns. Am. Soc. Artif

18. Kim, S. d, Ebert, C.; McRea, J.Ann.N.Y. Acad. Sci. 1983,416,

19. Heyman, P.; Cho, C.; McRea, J . ; Kim, S. W. J. Biomed. Mat. Res.

20. Goosen, M.; &Ron, M. J. Bwmed. Muter. Res. 1983,17, 359.

Intern. 6 r ans 1983,6, 76.

513423.

1985,19,419-436.

Acknowledgments The authors wish to acknowledge and thank Dr. T. Okano for his

surgical skills, Mr. L. Dost for the PGEl measurements, and Ms. L. Seminoff for her editorial comments. This research was supported by NIH Grant HL20251 and HL07520 (training grant).

Journal of Pharmaceutical Sciences / 175 Vol. 75, No. 2, February 1986