Pharmacology and controlled release of hirudin for cardiovascular disorders

ELSEVIER

Pharmacology and Controlled Release of Hirudin for Cardiovascular Disorders Dae-Duk Kim, PhD,*t Thomas A. Horbett, PhD,** Marc M. Takeno, BS,* and Buddy D. Ratner, PhD*t From the *Centerfor Bioengineering and $Department of Chemical Engineering, University of Washington, Seattle, Washington; iCollege of Pharmacy, Pusan National University, Pusan, South Korea

Hirudin is the most potent specific inhibitor of thrombin known. Hirudin was originally isolated from leeches,but it is now also available in synthetic form (recombinant hirudin). The inhibitor is currently undergoing clinical trials as a potential replacement for the extensively used thrombin inhibitor heparin. In this review, the biochemical and pharmacokinetic characteristics of hirudin (native and recombinant) and the efficacy of hirudin in treating and preventing cardiovasculardisorders is discussed.The advantagesof local controlled delivery of hirudin for treating cardiovascular disordersare then presented.Severalimplantable polymers applicable for controlled delivery system also are introduced. Finally, the feasibility of controlled delivery of r-hirudin for local therapy of cardiovascular disorders is addressed. 0 1996 by Elsevier Science Inc. Cardiovasc Path01 1996;5: 337-349

Cardiovascular disease still is the number-one killer disease in the United States. Thrombosis remains a significant problem in many clinical settings. New therapeutic agents that can effectively deal with thrombotic complications are widely sought. Recombinant strategies to produce the peptide hirudin in large quantities are now a reality. The widespread introduction of this powerful and safe antithrombotic pharmaceutical agent has the potential to improve many therapies and to reduce mortality in a number of procedures. Thus, a broad perspective documenting the potential of hirudin is of value to a large community of researchers and clinicians. This review article aims to highlight important aspects of hirudin action, pharmacology, and clinical application, with a special emphasis on controlled delivery.

History of Leeching and Hirudin The art of bloodletting is an ancient one that archeologists have now dated to the Stone Age after discovering ManuscriptreceivedMarch6, 1996;accepted May 7, 1996. Addressfor reprints:ThomasA. Horbett,PhD,Centerfor Bioengineering andDepartmentof ChemicalEngineering,Universityof Washington, Box 351750,Seattle,WA 98195.1750. Cardiovascular PathologyVol.5, No.6, November/December 1996:337-349 0 1996by ElsevierScienceInc. 655Avenueof theAmericas,NewYork,NY 100IO

bloodletting tools in that culture (1). The earliest clearly documented record of leeches being used for remedial purposes appears in a painting of an Egyptian tomb around 1500 B.C. The physicians believed that bloodletting would rid the body of noxious substances produced by disease. The popularity of leeching increased in Europe during the eighteenth and nineteenth centuries and reached its zenith in France in the early 1800s. By 1900, physicians became increasingly disenchanted with leeching, and the medical use of leeches was largely discredited in the West. However, leeches were still being used occasionally in the 1940s (2). Leeches have enjoyed a renaissance in microsurgery since the 1980s. When other methods fail in the reattachment of severed body parts, such as fingers, toes, and ears, the leech can play an important adjunctive role because of its anticoagulant and blood-removing properties (3,4). When microsurgeons undertake to reattach a finger, for example, anastomosis of arteries is a relatively simple task, but the successful establishment of venous return flow is often difficult or impossible because of local blood clotting. Leeches placed on the reattached part close to the line of demarcation or margin of the anastomosis draw out the congested blood while an anticoagulant in their saliva prevents

1054-8807/96/$15.00 PI1S1054-8807(96)00045-2

338

KIM ET AL. HIRUDIN FOR CARDIOVASCULAR

clotting. After 3 to 5 days, the venous return flow from the graft will be reestablished and the potential for rejection of the attached part greatly diminished. The process of leeching is not painful because the saliva of the leeches acts as a local anesthetic. In 1884, Haycraft demonstrated that medicinal leeches contain a substance that can prevent blood from clotting, and, as a result, the wound produced by the leech continues to bleed long after the animal has detached itself. Until heparin was discovered several years later, leeches were the only means to prevent blood from clotting. In 1904 Jocoby isolated the active anticoagulant ingredient from leech heads and suggested the name hirudin based on the leech’s name Hirudo medicinalis. In 1957 Markwardt succeeded in isolating the pure active anticoagulant substance produced by the peripharyngeal glands of the leech. This agent was determined to be a polypeptide that selectively inhibits thrombin (2). Although early pharmacological studies on the naturally occurring thrombin inhibitor showed that hirudin is a pronounced and specific antithrombotic agent, its clinical use remained limited because this substance was not available in adequate amounts for therapeutic purpose. A solution to the problem awaited the application of recently developed methods of peptide isolation and genetic engineering in order to obtain a sufficient amount of purified material. In 1986 Harvey et al. (5) reported that they had isolated clonal DNAs that encode a variant of hirudin.

Hirudin

(‘audiova\i: Pathd Vol. 5. bm R Novemher/Deic~lrhz~ 1996: i 1’7. Wi

DISORDERS

versus Heparin

Thrombin is a pivotal enzyme in thrombogenesis; it converts fibrinogen to fibrin, triggers the conversion of soluble fibrin into cross-linked fibrin, efficiently promotes platelet activation, and upregulates the activities of the coagulation cascade by activating coagulation cofactors V and VIII. Accordingly, thrombin inhibition is considered to be the most effective antithrombotic strategy (6). Both heparin and hirudin effectively inhibit thrombin circulation in plasma, but by different mechanisms; heparin depends mainly on plasma cofactor antithrombin III to exert its effects, whereas hirudin is a direct active-site inhibitor of thrombin. On a weight basis, hirudin is a three to five times stronger anticoagulant than heparin; however, the relative anticoagulant actions of this inhibitor are assay dependent (7). What differences between hirudin and heparin might render hirudin a better antithrombotic drug? First, hirudin does not require any endogenous cofactors, such as heparin cofactor II and antithrombin III to mediate its anticoagulant effects. Hirudin will not consume antithrombin-III. whereas heparin infusion results in the progressive depletion of this natural anticoagulant. Thus, hirudin might be used in patients with antithrombin III deficiency, for effective anticoagulation without the need for antithrombin III replacement therapy. Second, hirudin is not inactivated by antiheparin proteins such as platelet factor IV, histidine-rich glycoprotein,

or vitronectin, whereas elevated plasma concentrations of these substances can cause heparin resistance in patients with inflammatory or malignant disorders (8). Third. hirudin has no direct effects on platelets or endothelial cells, whereas such effects of heparin may contribute to the associated risk of bleeding. Use of hirudin might also avoid the problem 01 heparin-induced immune thrombocytopenia and associated thrombosis (8). Hirudin may therefore be a more effective anticoagulant than heparin in arterial thrombosis, including thrombosis following angioplasty or thrombolytic therapy.

Native Hirudin Most leeches used for therapeutic purposes belong to the species of Hirudo medicinalis. Native hirudin is extracted from the homogenized heads of the medicinal leeches and enriched by precipitation procedures followed by ion zxchange chromatography and gel filtration (9-10). Affinity chromatography on matrix-bound thrombin was also used to obtain a highly purified preparation (1 I )”

Biochemistr?, The primary structure of hirudin is a polypeptide chain (65 amino acids) with a molecular weight of 7,000 daltons and three internal disulfide bridges (13). In studies of Ihe amino acid sequence of highly purified hirudin preparations, several isoforms of hirudin with a very similar primary structure were found ( 14,lS). These variants showed different N-terminal amino acids and certain differences in the amino acid sequence between positions 34 and 38. The unique clustering of acidic amino acid residues in the C-terminal half remained constant with all variants ( 1.5). Native hirudins are tight-binding, thrombin-specific inhibitors that form equimolar complexes with thrombin whereby the active center of the enzyme is blocked. These complexes have a very low equilibrium dissociation constant (/Cd) in the 10.. I2 mole/L range. From this complex, hirudin is liberated only by synthetic thrombin inhibitors such as benzamidine or by heat denaturation of thrombin ( 15I. It was proposed that the binding of hirudin to thrombin involves two steps. The initial interaction is diffusion controlled and involves an ionic interaction at a place separate from the thrombin active site. In a second step. the thrombinhirudin complex rearranges to form a tighter union in which hirudin also is bound at the thrombin active site (16.17). The compact N-terminal portion of the hirudin molecule, stabilized by disulfide bridges. corresponds to the apolal binding site regions of thrombin. Presumably. the C-terminal segment of hirudin binds to the anionic binding site region, and a basic side chain of the inhibitor occupies “‘the arginine side chain pocket” near the catalytic site. This basic group belongs to Lys-47, which is noncleavable because of its being flanked by two prolines. Binding studies with acylated thrombin in which the active site serine is covalently

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FOR CARDIOVASCULAR

Table 1. PharmacokineticData of Hirudin in Different Speciesafter a Single IV Injection Rat (19)

Dog (19)

Assay

1251-hirudin

‘251-hirudin

Dose

1 .O (w&9

0.5 bwW

ha W

0.21

hP

(hr)

I .08

vc UJW V&s WW CL, (ml/min) CL,, (mhmin)

0.39 0.74 2.83

Dog

(20)

Chromogenic

Man (21)

Chromogenic

Radioimmunobioassay

1,000 (ATUikg)

0.5 hiVW

0.21 0.94 0.19 0.30 117.67

Man (21)

0.17 0.95 0.11 0.12 178 128

600-1,000

(ATUikg)

0.15

0.15

0.84

I .08 0.24 187.1 79.9

0.18

230 99.5

Abbreviations: tu20 = distribution (a-phase) half-life, tt& = elimination (P-phase) half-life, V, = central volume of distribution, Vd,, = steady-state volume of distribution, CL, = total body clearance, CL,, = renal clearance.

blocked showed that hirudin would still bind, suggesting that the catalytic site is not necessary for hirudin-thrombin complexing (15). The analysis of the anticoagulant effect of hirudin showed that it is a unique thrombin inhibitor in that it is highly specific for thrombin without affecting other closely related serine proteinases. Because of its high affinity for thrombin, relatively low inhibitor concentrations are necessary to prevent coagulation. In the hirudin-thrombin complex, all proteolytic functions of the enzyme are blocked. Thus, hirudin prevents not only fibrinogen clotting but also further thrombin-catalyzed hemostatic reactions such as the activation of clotting factors V, VIII, and XIII and thrombin-induced platelet reactions. Therefore, by instantaneous inhibition of the small amount of thrombin generated after activation of the coagulation system, the positive feedback on prothrombin activation also is blocked, which can prevent accelerated generation of further thrombin (18).

appearance,first-order elimination kinetics was observed with a half-life of 0.84 hours. The cumulative urinary excretion of hirudin showed that 30 to 40% of the administered hirudin was excreted in the urine within 24 hours in active form. In one study, the pharmacokinetics and the anticoagulant effects of native hirudin were investigated in 12 healthy vol-

c 5 .-2 I

i

lb;“““’

Phannacokinetics The efficacy of hirudin as an anticoagulant agent in vivo depends on the maintenance and control of an adequate blood level of hirudin. Therefore, a knowledge of its pharmacokinetics is an essential prerequisite for using hirudin as an antithrombotic agent. The results of pharmacokinetic studies with hirudin in various species after intravenous (IV) injection are summarized in Table 1. Pharmacokinetic data resulting from blood levels and urinary excretion have shown that hirudin given IV to experimental animals is rapidly eliminated from the body (t&3 = 1 hour) (19,20). Hirudin is distributed into the extracellular space, and more than 70% is eliminated in active form through the kidneys by glomerular filtration (20). Although the studies on experimental thrombosis have shown that hirudin is a potent antithrombotic agent, clinical use of hirudin has remained limited to a few pilot trials because of the limited availability of the substance. Hirudin levels in plasma after an IV injection of 1,000 ATU hirudin/kg were investigated in three volunteers (21). After the initial dis-

IO

2

3

5

6

7

a

I 5

I 6

I 7

I 6

Time (hr)

A

IO].,

I 0

6

4

12

I 3

I 4

Time (hr)

Figure 1. Hirndin plasmaconcentrationlevel after (A) IV injection of 1,000 ATUlkg in healthy human subject and (B) SC injection 600 ATU/kg. Replotted from (22).

of

340

KIM ET AL. HIRUDIN FOR CARDIOVASCULAR

DISORDERS

unteers after single subcutaneous (SC)or IV bolus administrations (22). The elimination half-life after IV injection was 1 hour, and the total plasma clearance was 187 ml/min (Figure 1A). The extracellular volume corresponded to the whole extracellular water compartment of the body. After subcutaneous administration, the hirudin plasma concentration increased steadily and reached its maximum after 2 hours (Figure 1B). Thereafter, it decreased, and after 24 hours it was close to or below the detection limit of the assay (5 rig/ml). The maximum of the hirudin plasma concentration (60-l 20 rig/ml) was dependent on the dose of hirudin administered. All test subjects tolerated the hirudin injection without visible or measurable side effects. Immune reactions to hirudin were not observed after a single parenteral administration.

Recombinant

Hirudin

Native hirudin must be isolated from leeches. Leeches are available in limited numbers, and breeding trials have failed. Some species of these animals have been placed on the Endangered Species List. Under these circumstances, hirudin is an appropriate candidate for genetic engineering production. Several groups studied cloning and expression of a cDNA coding for hirudin and succeeded in producing recombinant hirudin (r-hirudin) (5,23-26). The recombinant DNA technology has greatly increased the availability of hirudin. Thus, it became possible to provide hirudin for therapeutic purposes. That was the starting point for reconsidering hirudin as a therapeutic anticoagulant (27). Its introduction into clinical medication reflects the first substantial change in, the prophylaxis and therapy of thrombosis in a long time. A list of companies interested in the development of r-hirudin and of functional preparations is given in Table 2 (7). Biochemistry r-Hirudin can now be produced in different biologic systems (28). The DNA containing the coding sequence of the

Table 2. Manufacturers

of Natural

and Recombinant

Company Biophram (UK) Ciba-Geigy Pharmaceuticals (Switzerland) Farmitalia Carlo Erba (Italy) Genbiotec (Germany) Hoechst AG (Germany) Knoll Pharmaceuticals (Germany) Merrell Dow (USA) Mitsui Co. (Japan) Pentapharm. Ltd. (Switzerland) Plantorgan Werk (Germany) SRI International (USA) Transgene (France) Source: Based on (7).

inhibitor protein was isolated or chemically synthesized based on the amino acid sequence of hirudin. The hirudin gene was fused to other protein genes and expressed in bacteria (Escherichia coli) or yeast cells (Suc-c~h~11.011/!,(.1’.5 (‘f’)f~visiar). After controlled incubation, hirudic was cxrraeted from the lysed E. ~oli cells or the yeast culture broth and purified via several chromatographic steps until homogeneir~ was achieved. The recombinant form of hirudin expressed was desulfatohirudin, as it was missing a sulfate residue on Tyr-63. IYowever, the recombinant products had the same configuration in other respects as native hirudin, including all three disulfide bonds. Further genetically engineered bariants were pi-clduced by exchanging individual amino acids at the N-&knal and at position 47 ( 15). The various types of desulfato-hirudin\ are selective tight-binding thrombin inhibitors. The anticoagulant activity in clotting assays was as high as that of native hirudin. But the complex of thrombin with desulfato-hirudin showed a 1O-fold higher Kd value than for native hirudin. When Lq s-47 is substituted with Asn, the affinity for thrombin is further decreased (29). Both domains of hirudin. the N-temminai core and the Cterminal tail. contribute to the formation ot’ a tight complex with thrombin ( 17). The N-terminal core hinds near the active site of thrombin, whereas the C-terminal tail recognizes the fibrinogen-binding site of thrombin (30,3 1). One study investigating the effects of covalently bound r-hirudin to albumin on cl-thrombin inhibition (32) found that the covalently bound r-hiruditialbumin complex still maintains potent thrombin affinity while inhibiting the thrombin enzyme activity. In recent studies (33-36), hirudin variants have been isolated from Hirudinuria manillensis, the buffalo leech, a species that is significantly more specialized for mammalian parasitism than H. medicinalis ( I3 ). The primary structures of hirudin isoforms from H. r~za~~if/rn.si.s (35) show significant sequence similarity (60-75%) to those ~)f H. mcdiciw/is, and, in particular. contain six cysteine residues at the same highly conserved positions, thus signifying the hame

Hirudins

and Related Agents -----

Product

.._..

--.--.

Natural hirudin r-Hirudin (E. coli and yeast) Newer r-hirudin r-Hirudin (25.co/i) r-Hirudin (yeast), other expressions r-Hirudin (E. co/i), r-hirudin conjugates r-Hirudin, synthetic fragments [--Himdin (Rtrcillus suhtilis) Natural hirudin Natural and r-hirudin (E. co/i) r-Hirudin, hirudin fragments r-Hirudin (yeast) -_-----__

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KIM ET AL. HIRUDIN FOR CARDIOVASCULAR DISORDERS

CardiovascPath01Vol. 5, No. 6 November/December1996:337-349

Table 3. PharmacokineticData of r-Hirudin (absentTyr-63) in Different Animal Speciesafter a Single IV Bolus Injection Dosehzkg) ti/zQ (hr) tld (W v, VW Vd,, (Lkz) CL, (mllmin) CL,,, (mpmin)

Rat (27)

Rat (27)

Rabbit (37)

Dog (38)

1.0 0.15 1.07 0.31 0.66 3.05

2.0 0.08 1.18 0.17 0.63 2.09

I.0 0.1 I 1.16 0.07 0.23 12.38 -

0.5 0.25 1.22 0.18 0.28 183 152

Abbreviations: tu20 = distribution (o-phase) half-life, tt& = elimination (B-phase) half-life, V, = central volume of distribution; Vd,, = steady-state volume of distribution; CL, = total body clearance, CL,,, = renal clearance.

pattern of disulfide formation. Hirudin from H. rnunillensis was found to be glycosylated and not sulfated (35). A 64residue hirudin variant, HM2, from H. rnunillensis was expressed in E. coli using a synthetic gene (36). The recombinant HM2, lacking posttranslational modifications, possessed inhibitor activity for thrombin comparable to that of other recombinant hirudins.

Pharmacokinetics It is known that pharmacologic properties of genetically engineered desulfato hirudin are similar to those of native hirudin and have the same amino acid sequence, including both pharmacokinetic and pharmacodynamic characteristics. The pharmacokinetic characteristics of r-hirudin were intensively studied in various animal species (27,37,38). Table 3 shows the pharmacokinetic parameters obtained in these animal species after a single IV bolus injection of r-hirudin. The elimination half-lives (t&3) were almost identically short at the respective dosages (0.5 to 2 mg/kg) in the rat, rabbit, and humans (ti,# = 1.0 hr). As with native hirudin, a relatively small amount (15%) of active r-hirudin was found in rat urine (27), whereas in dogs almost the total quantity of administered r-hirudin was eliminated through the kidneys in active form (38). In the dog studies, the renal clearance approximated total clearance, and the urinary recovery of active r-hirudin exceeded 80 to 85% of the applied dose. Thus, only a small amount of r-hirudin might possibly be stored or degraded. From the distribution volume, it was evident that r-hirudin is distributed into the extracellular space. From the constant fractional efflux and reflux, it could be concluded that there was a relatively rapid transfer between the intravascular and extravascular compartments. After subcutaneous administration of r-hirudin in dogs, the pharmacokinetics also corresponded to an open onecompartment model for extravascular application (38). The apparent elimination half-life of r-hirudin was prolonged, so that 8 hours after a single IV dose of 0.5 mg/kg, a hirudin plasma level of 0.15 p,g/ml was measured. About 10% of the administered r-hirudin plasma level of 0.15 kg/ml was

measured. About 10% of the administered r-hirudin dose was excreted per hour. Recently, the pharmacokinetic profiles of r-hirudin variant 2 (rHV2) with a lysine residue in position 47 (rHV2-Lys 74) also was compared in male dogs (39). The pharmacokinetic parameters obtained after IV and SC dosing are listed in Table 4. Clinical pharmacologic studies also were performed in human volunteers after single IV and SC doses of 0.1 and 0.5 mg/kg, as well as after repeated SC injections (40). Thrombin time and partial thromboplastin time were prolonged, depending on the r-hirudin level in plasma. Figure 2 shows the concentration-time profiles of r-hirudin in human plasma after IV and SC injection. The pharmacokinetic data analyzed from the hirudin plasma concentration and urinary excretion are summarized in Table 5. In the human studies, r-hirndin was rapidly distributed into the extracellular space after IV injection and eliminated with a half-life of 1 to 2 hours. After SC administration, the r-hirudin level in blood reached plateau values within 60 to 120 minutes. The high recovery of unchanged r-hirudin in the urine identified renal excretion as the predominant route of r-hirudin clearance. Subcutaneous injection of 0.1 mg/kg r-hirudin at g-hour intervals resulted in a therapeutically relevant long-lasting r-hirudin level of 0.5 to 0.2 pg/ml (Figure 3). Accumulation

Table 4. PharmacokineticParametersObtained after IV and SC Administration of 1-mg/kg rHV2-Lys 47 in Male Dogs Parameter ke ( l/hr) G/2 (W AUC,, kg hW AUMCo, (pg h*/ml) MRT (min) CLt (ml/kg/hr) Vd Wk)

IV Dose 1.06 (% 0.83 (k 3.97 (5 3.37 (2 49.62 (? 258.39 (” 263.67 (2

0.46) 0.01) 0.78) 1.58) 14.01) 44.06) 77.58)

SC Dose 1.96 (2 0.35 (ir 21.7 (i 69.15 (5 199.63 (2 258.36 (5 106.75 (2

0.2) 0.01) 7.24) 19.16) 30.15) 43.89) 42.47)

Abbreviations: ke = elimination constant, t,,* = elimination half-life, AUC= area under the curve, AUMCb = area under the moment curve, MRT = mean residence time, CL, = total body clearance, Vd = volume distribution. Source: Based on (39).

KIM ET AL. HIRlJDiN FOR CARDIOVASCULAR DISORDERS

0

100

200

300

400

0

500

4

8

12

Figure 2. Concentration-time profile for r-hirudin in human plasma after 0.5 mg/kg IV (solid squares), 0.1 mg/kg IV (opm squares), 0.5 mgikg SC (solid circles), and 0.1 mg/kg SC (open circles) injection. Replotted from (40).

of r-hirudin in plasma was not observed under these conditions. Recently, the pharmacokinetics of r-hirudin (Ciba-Geigy, CGP 39 393, MW 6964) in healthy volunteers after IV administration was investigated (41). A total of 77 plasma profiles following a single IV bolus dose of either 0.1, 0.3, 0.5, or 1.O mg/kg of r-hirudin was used for the evaluation. The kinetics of r-hirudin after a bolus IV injection were best described by a three-compartment open model. Plasma concentrations, and especially area under the curve (AUC) values, were proportional to the dose. The mean apparent terminal half-life was 2.8 hours, and the total clearance was 0.138 L/h&g.

Preclinical Studies After genetically engineered recombinant desulfato-hirudin was proven to be as potent a anticoagulant as native hirudin, preclinical studies were initiated. To accurately compare the efficacy of r-hirudin and of native hirudin, identical thrombosis models were used for both types of hirudin. The results showed that r-hirudin was antithrombotically effective in various models of experimental thrombosis (27,42). The doses and plasma concentrations of r-hirudin required for antithrombotic action were within the same range as those of native hirudin isolated from H. medicinalis. The antithrombotically effective plasma concentrations of hirudin was different among the thrombosis models used. However, the outstanding efficacy of r-hirudin became particularly obvious in various thrombosis models because r-hirudin immediately prevented the thrombin effect on fibrinogen as well as on other reactions in the coagulation system. r-Hirudin also was effective in models of stasis-induced venous thrombosis and in microthrombosis.

16

20

24

28

32

Time (hr)

Time (min)

Figure 3. Course of r-hirudin level in human plasma after repeated SC administration of 0.1 mg/kg. Arrows. r-hirudin administration. Replotted from (40).

The antithrombotic effects of r-hirudin also were demonstrated both in arterial and in arteriovenous shunt thrombosis, although higher doses were required than in venous thrombosis (15). This could be due to the important role of platelets in thrombus formation on damaged endothelium or on an artificial surface. In such circumstances, thrombus formation is initiated by platelet adherence to the respective surface followed by activation of the plasmatic coagulation system leading to the formation of fibrin. Studies with the genetically engineered recombinant desulfato-hirudin revealed not only that the pharmacologic properties are similar to those of native hirudin, but also its outstanding potency as an antithrombotic agent that was well tolerated in vivo. The toxicity of r-hirudin was studied in healthy subjects after single IV and SC administration. Generally, administration of r-hirudin was tolerated without undesirable side effects or unexpected effects on the hemostatic system unless administered at considerably higher doses than required for antithrombotic efficiency (40.43,44.).

Table 5. Pharmacokinetics of r-Hirudin in Human Volunteers after a Single IV Dose Parameter

0.1 (mg/kg)

~I/z~ (h) t,/zP (h) AU&, (kg h/ml)

V, (liter) Vd,, (liter) CL, (mllmin) CL,,, (ml/min)

-__

0.5 (mgkg)

0.l.s !.I

ii..+ 2.c

0.64 4.1

?X ._. 45

X.Y IhO ho

I I.4 18h L71 -~___._-_----

Source: Based on (40).

Abbreviations: tli2a = distribution (cx-phase)half-life, t,& y elimination (P-phase) half-life, AUC o-y = area under the curve for 8 hours. Vi = central volume of distribution, Vd,, = steady-state volume of distribution, CL, = total body clearance, CL,,,, ==renal clearance.

Cardiovasc Pathol Vol. 5, No. 6 November/December 1996:337-349

HIRUDIN

Both native and r-hirudin also are known to be weak immunogens (44). Clinicopharmacologic studies revealed that neither IV, SC, or intramuscular (IM) injection of r-hirudin caused sensitization. r-Hirudin-specific antibodies were not found in human serum after r-hirudin treatment two or three times at 3-month intervals (43).

Clinical Aspects of r-Hirudin r-Hirudin characteristics that are particularly therapy are as follows:

useful in

1. Potent anticoagulant; does not require any endogenous cofactors. 2. Pharmacodynamically inert; no effect on blood cells (platelets), plasma proteins, or enzymes; weak immunogen. 3. No endogenous modulation; excreted in active (unchanged) form in urine; no deposition in organs. 4. Reduced bleeding complications at antithrombotically active dosages. 5. Effective in patients with antithrombin III deficiencies; may also be used when platelet defects or thrombocytopenia are present. 6. Quality control and monitoring of therapy easily done by measurement of thrombin clotting time. Dosing studies were performed in patients who were at risk for deep venous thrombosis, restenosis after coronary angioplasty, and acute myocardial ischemia due to coronary artery disease (45). Satisfactory safety profiles in these trials have led to wider scale clinical trials of r-hirudin for a number of applications in humans, as presented in the subsections that follow.

Diffuse Microthrombosis In experimental disseminated intravascular coagulation (DIC), hirudin inhibited the consumption not only of clotting factors (46) but also of antithrombin III, which is its principal advantage over heparin. Consumption of plasma antithrombin III occurs only if heparin is administered, whereas the directly acting thrombin inhibitor hirudin exerts an antithrombin-sparing effect without leading to reduced antithrombin III levels (47). Pilot clinical studies also were conducted in patients suffering from chronic DIC (43). Subcutaneous injection of 0.1 mg r-hirudin/kg at g-hour intervals resulted in normalized fibrinogen and platelet counts and in the disappearance of fibrin monomer complexes and degradation products.

Extracorporeal Circulation r-Hirudin can be used as an anticoagulant in extracorporeal circulation. An arteriovenous shunt model in which the rat carotid artery was connected with the jugular vein by a

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KIM ET AL. DISORDERS

343

polyethylene tubing of defined length was used to elucidate the effects of r-hirudin on clot formation on artificial surfaces (15). In all animals of the control group, a spontaneous occlusion of the shunt occurred within a short time (2.9 ? 1.2 min). In all r-hirudin-treated animals, the time to occlusion was prolonged. No occlusion was observed at doses of 40 pg/kg/min, which corresponds to a plasma level of 2.5 ? 0.6 Pg/ml. After r-hirudin was shown to prevent the thrombotic occlusion of an arteriovenous shunt, the effect of r-hirudin in extracorporeal circulation was studied by using it for anticoagulation in experimental hemodialysis in nephrectomized dogs (48). In contrast to untreated animals, no fibrin deposits were found in the apparatus during a 2-hour dialysis following a single r-hirudin administration of 0.5 mg.

Reduction of Thrombogenicity Prothrombin complex concentrates (PCCs) must be regarded as potentially thrombogenic substances, and patients treated with PCCs have frequently been found to experience thrombotic events. To reduce the risk of thromboembolic complications, the addition of r-hirudin may be advantageous. Pilot studies in rats showed adding r-hirudin to PCCs either reduced or completely abolished their thrombogenicity (49). Thus, r-hirudin seems to be effective for prophylaxis or DIC induced by PCCs or other plasma fractions.

Deep Venous Thrombosis Hirudin was used in 1,120 patients as prophylaxis for deep venous thrombosis after elective total hip replacement (50). It was shown to be twice as effective as heparin in reducing the overall rate of deep venous thrombosis and at least six times more effective than heparin in reducing the development of proximal deep venous thrombosis. Bleeding rates were similar to those for heparin.

Unstable Angina Pectoris Hirudin was compared to heparin in 166 patients with unstable angina pectoris and non-Q-wave myocardial infarction (MI) (51). In this trial, the occurrence of death or MI was approximately three times lower for hirudin (2.6%) than for heparin (8%).

Coronary Thrombosis and Restenosis Thrombosis of a coronary artery is the underlying event in acute MI. Coronary artery obstruction is currently being treated with invasive approaches involving catheter-based angioplasty procedures. Intracoronary lysis (XT) and/or percutaneous transluminal coronary angioplasty (PTCA) are currently the most useful therapeutic means to achieve patency of the occluded area and to limit the extent of myocardial

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Table 6. Therapeutic

Strategies for Restenosis Agent

Mode ot’ Action

Platelet aggregation

Aspirin Echistatin Antibody to IIb/IIIa glycoprotein Platelet-derived growth factor receptor

Prostaglandin synthesis inhibitor Inhibit fibrinogen-mediated platelet adhesion Blocking aggregation Inhibit platelet contribution to prolif’eratioll

Fibrin clot formation

Heparin Hirudinfhirnlog

Bind to antithrombin 111 Binding to thrombin

Cellular proliferation

Angiopeptin Colchicine Antisense c-myb oligonucleotide

Inhibit smooth-muscle cell proliferation Mitotic inhibitor Inhibit mitogen-induced proliferation Antiinflammatory steroidal effect

Pathologic Mechanism

necrosis during the phase of reversible impairment. Recently, balloon angioplasty followed by insertion of an expandable stent has been gaining widespread acceptance as a means for overcoming reobstruction resulting from elastic recoil of the arterial wall (52). However, the arterial stenting procedure itself induces a pronounced pathophysiologic response comparable to balloon dilatation of a diseased artery and leads to reobstruction, a process termed restenosis that is known to affect approximately 50% of stent angioplasty procedures within 6 months of stenting. The process of restenosis involves a complex interaction between endothelial cells, smooth muscle cells, platelets, monocytes, lymphocytes, and lipoproteins (53). Table 6 shows several specific events that predominate in the early development of the restenosis process and potential therapeutic strategies to address these events. Occluding thrombi in coronary vessels develop from a previously existing atherosclerotic stenosis that still continues after ICT or PTCA and thus predisposes the vessels to rethrombosis. Hence, it is a necessity to support successful ICT or PTCA by subsequent anticoagulation. Animal experiments demonstrated that r-hirudin prevented thrombotic coronary artery occlusion (54). 100 1

dextran-r-hirudin --I----t------__-

1 I 0

I 1

I 2

I 3

1 4

Time (hr) Figure 4. Plasma level following an IV injection of 1,000 ATU dextran-r-hirudin or r-hirudin per kg in rats. Replotted from (57).

The preventive effect of r-hirudin on thrombotic occlusion also was shown in experimental angioplasty in the jugular artery of rabbits (37) and in the carotid artery of minipigs (55). Hirudin proved to be particularly useful in preventing reocclusion after intracoronary lysis. This was illustrated by intravascular lysis of a thrombus induced by vessel wall lesions in the jugular artery in rabbits. r-Hirudin administration resulted in continuous patency of the vessel (37). Hirudin and heparin were used in 113 patients with stable angina undergoing elective coronary angioplasty. Restenosis of the dilated site was the primary endpoint of the study. Initial recruitment is completed, and follow-up angiography is now being performed. There was a dramatic decrease in thrombotic complications following the coronary angioplasty. In those treated with heparin. there was a 10.3% incident of death, MI, or need for emergency coronary surgery. This rate was reduced to 1.4% with the use of hirudin (56).

Controlled

Delivery of Hirudin

Pharmacologic studies with native hirudin as well as with r-hirudin preparations have shown that the agent is eliminated from the blood within a relatively short time (about 1 hour). Therefore, a covalent bond between hirudin and a high-molecular-weight carrier (dextran, MW 70,000) was introduced in order to retard its elimination from blood (57). The pharmacokinetic behavior of dextran-r-hirudin was examined following IV administration in rats and compared to r-hirudin (58). Administration of dextran-r-hirudin resulted in a significantly higher plasma level than with r-hirudin and a longer elimination half-life (about 7 hours; Figure 4). The covalent binding of hirudin to polyethylene glycol (PEG) also resulted in a stable and homogenous compound with an elimination half-life between 5 and 9 hours (59). When 1 mg/kg of PEG-hirudin was administered intravenously into DIC-induced rabbits, the thrombin time, as a sign of a direct action of hirudin on thrombin, was considerably longer than in controls, even after 6 hours (60). This is probably due to a longer half-life in circulation of PEG-hirudin than of natural hirudin.

Cardiovasc Pathol Vol. 5, No. 6 November/December 1996:337-349

The use of synthetic polymers to deliver peptide/protein drugs is rapidly becoming an established approach since the development of appropriate delivery vehicles in 1980 by Rhine and his colleagues (61). Drug-polymer composites can be formulated using various polymer-drug dispersions, compression molding techniques, or drug-polymer cosolutions and solvent casting (62). There have been several attempts at using the polymeric drug delivery systems to treat cardiovascular disorders (63,64). Hirulog, a peptide containing a partial amino acid sequence of hirudin, was shown to be released from a silicone rubber matrix at nearly constant release rates for the first 20 days without a burst effect (64). Implantation of hirudin-containing controlled release polymer systems at the site of cardiovascular disease can offer the advantages of both regional high levels of hirudin and lowering systemic hirudin exposure, thereby minimizing the possibility of side effects (63,64). However, no systematic research has been reported on the polymeric controlled delivery of hirudin to date. We have been working on the development of new biomaterials that can release hirudin at a desired therapeutic rate for an extended time (65). The desired release rate of hirudin from the polymer matrix is to be achieved by depositing a rate-controlling barrier using the radio frequency glow discharge plasma deposition technique. The plasma coating is to serve as a diffusional barrier and works to control the release kinetics of hirudin from the matrix by changing the various coating conditions, such as pressure, radio frequency power, and duration of the plasma deposition.

Local Delivery for Cardiovascular Disorders Drug delivered directly to the heart or a blood vessel will more efficiently and effectively treat localized disease processes of interest by increasing the regional levels of drug while minimizing systemic side effects (63). Cardiovascular controlled-release systems using drug-polymer composites implanted in direct contact with the heart have recently come into clinical use and are under active investigation in other areas of possible application. Implantable polymeric systems for drug delivery in the cardiovascular system have been investigated experimentally for a variety of therapeutic purposes besides restenosis. One such system based on the sustained release of dexamethasone from a cardiac pacing catheter is in clinical use at this time (66,67). The slow release of dexamethasone from the ventricular contacting tip of this electrode presumably reduces the extent of regional scar formation and thus provides relief from scar-induced increased electrical impedance at the myocardial interface with the pacing electrode. Thus, these encouraging experimental and clinical results obtained with implantable controlled-release systems for cardiovascular disease processes provide a basis for pursuing similar strategies for pharmaceutic implants for restenosis.

HIRUDIN

FOR CARDIOVASCULAR

KIMETAL. DISORDERS

345

In animal studies, it has been demonstrated that periarterial drug administration using heparin-ethylene vinyl acetate composites significantly inhibited restenosis in a rat arterial injury model (68,69). This initial success of a controlled-release drug-delivery approach to restenosis has stimulated interest in the field. Controlled release may be achieved by formulations of drug polymer composites, either as monolithic matrices or reservoirs with rate limiting membrane configurations. Thus, drug administration can be sustained through the use of polymeric materials. Implanting controlled-release polymer systems at the site of a cardiovascular disease process offers the theoretical advantage of avoiding adverse systemic effects while maintaining sustained release and high local drug concentration. Local therapy for restenosis could be administered to the site of an angioplasty or atherectomy through at least three possible approaches (63; Figure 5): (1) regional infusion of a solution or emulsion or liposome formulation; (2) a coated expandable stent; and (3) periadventitial implantation of a controlled-release matrix around the exterior of a blood vessel. Each of these strategies have some merit, and all have specific disadvantages. Periadventitial administration of a controlled-release matrix would always require open surgical placement. Coated stents may be limited by the relatively small volume of material that can serve as the drug reservoir. Regional infusion requires the temporary isolation of the arterial segment of interest from blood flow, so that the infusion can penetrate the arterial wall.

Drug Carriers for Pharmaceutic Implants A number of currently available polymeric drug-delivery systems are potentially useful for investigations of pharmaceutic implants such as stents. Implants could be formulated with a polymeric matrix system involving either a degradable or nondegradable polymer with a dispersed pharmacologic agent. In addition, a reservoir system consisting of a hollowed chamber with a nondegradable polymeric ratelimiting membrane at its biologic interface offers possibilities for regional administration coupled with the option of altering therapy or even discontinuing treatment by emptying the reservoir system. Obviously, a drug reservoir in the framework of a stent structure would require creative design considerations. Finally, cells as drug-delivery systems, including either genetically modified cells, autotransplanted cells, or other strategies to enhance contiguous arterial wall cell proliferation, constitute another novel drug-delivery strategy that should be considered (70).

Nondegradable Polymer A number of nondegradable polymer matrix materials are currently used in formulating either implants or coatings for implants (70; Table 7). Drug-polymer composites are re-

346

KIM ET AL. HIRUDIN FOR CARDIOVASCULAR DISORDERS

Injected Drug Emulsion

Catheter

Double Balloon Drug Delivery

(Balloon Tip)

Figure 5. Illustration of the various dosage form\ investigated for use to administer local therapy for the prevention and treatment of restenosis. (A) Mi-. croparticle emulsion injection. (B) Polymer drugcoated stent. (C) Periadventitial drug-polymer matrix. Modified from (63). Balloon Expandable

Drug-Polymer

Coated Stent

C

Periadventitial

Drug-Polymer

Maxtrix

ferred to as monolithic matrices. When peptide/proteinloaded nondegradable matrices are used, drug delivery is achieved through sustained release by way of particle dissolution and diffusion through the connecting network of the matrix. Extended drug release is possible through this approach, with formulations reported to release from hours to decades (70). In addition, a number of nondegradable polymers not well explored for drug delivery also could be suitable for use with pharmaceutic devices, including polytetrafluoroethylene, polyvinyl chloride, and various hydrogel formulations (7 1). Obviously the blood compatibility characteristics of any material will be crucial in assessing the likelihood for accelerated arterial obstruction resulting from thrombosis on the pharmaceutic implant (70). Biodegradable

Polymer

Restenosis, while common in the first several months following angioplasty, rarely occurs after 6 months (53). Therefore, perhaps a temporary prosthesis is all that is needed in the first 12 months following angioplasty. Con-

sidering this short-term need and the potential for long-ten-n complications with a metal stem, a stent constructed of material that degrades or is absorbed in a nontoxic manner would be preferable. There have been efforts to develop a biodegradable polymeric endovascular stem, ultimately impregnated with antiproliferative or antithrombotic agents that would be implanted to inhibit locally the restenotic response. Several biodegradable polymers have been screened for other medical device applications, and a few have been employed in humans for local drug-delivery systems 01 wound healing (72). Table 8 shows the biodegradable polymer systems that have been used to formulate drug-delivery matrices and proved to be useful in pharmaceutic stent formulations. One of these polymers, poly(d,l-lactide/glycolide) copolymer, has had extensive clinical use in surgical sutures, clips. and meshes; as bone prostheses for fracture fixation; and as implantable reservoirs for subdermal, oral, and ophthalmologic sustained drug delivery. Poly(hydroxybutyrate/ hydroxyvalerate) copolymer has been used in humans fat surgical sutures and tested in animals for local drug deliv-

Table 7. Nondegradable Polymers for Localized Controlled Drug Delivery Examples of Use

Advantages

Ethylene vinyl acetate

Formulation

Release of heparin, cytokines

Silicone rubber

Anticalcification

Solvent cast, protein compatible Nonsolvent casting

Polyurethane

Antiarrhythmia therapy

Silicone-polyurethane copolymer

Anticalcification

Source: Based on (70).

Hydrophilicity, rapid drug release Thermoplastic

Disadvantages Low melting point Poor blood compatibility, immunogenic Biologic degradation Poor blood compatability --___

KIM ETAL. HIRUDIN FOR CARDIOVASCULAR DISORDERS

CardiovascPath01Vol. 5, No. 6 November/December1996:337-349

347

Table 8. BiodegradablePolymers for Localized Controlled Drug Delivery Examplesof Use

Formulation Collagen High-molecular-weight polyanhydride Polylactic-polyglycolic (PGLA) Alginate Polyalkylcyanoacrylate

acid

Advantages

Disadvantages

Cyclosporin-A BCNU brain tumor

Enzymatically degradable Surface erosion

Potentially immunogenic Inelastic

Antiarrhythmic agents

Suitable for microspheres

Inflammatory response

Encapsulation of islet cell Antibiotics

Low cost Nanoparticle suitability

Potentially immunogenic Potential toxicity

Source: Based on (72).

ery. Other polymers, such as polyorthoester and polycaprolactone, although not yet used clinically, have been successfully screened in animals for biocompatibility and are under evaluation for local or injectable drug-delivery systems.

Perspectives and Conclusions Systemic administration of a number of pharmaceuticals has been tried in various experimental and clinical studies to inhibit restenosis (Table 6). Although many have been successful in animal models, none has been effective clinically. However, the systemic administration of these compounds results in lower arterial wall drug concentrations than would be achieved with regional administration via a pharmaceutic implant. Therefore, any of these compounds can be useful for experimentation with a pharmaceutic implant to enhance regional arterial wall drug concentrations. Considering the relatively small amount of space on the stent structure and implantable device for cardiovascular disorders, a compound with strong activity, such as hirudin, should be incorporated in polymers or coated on stents. r-Hirudin is a recombinant peptide that selectively and irreversibly inhibits thrombin. This new antithrombin offers promise in the treatment of venous and arterial disorders, including deep venous thrombosis, restenosis after coronary angioplasty, and the management of acute ischemic syndromes related to coronary artery disease. Prolonged local drug delivery using drug-containing pharmaceutic stents or related implants seems to be a more effective approach to prevent cardiovascular disorders, while avoiding systemic side effects. The blood compatibility of any material will be crucial in designing implantable polymeric systems. In conclusion, the development of hirudin-impregnated local-controlled delivery system using implantable polymer or coated stents seems to be a promising approach for the treatment of cardiovascular disorders, such as restenosis. The authors would like to gratefully acknowledge the financial support from the National Institutes of Health (Grant No. HL61260) for this work.

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