Tải bản đầy đủ - 0 (trang)
1 The GP IIb/IIIa Receptor as a Target for Antithrombotic Therapy

1 The GP IIb/IIIa Receptor as a Target for Antithrombotic Therapy

Tải bản đầy đủ - 0trang

Abciximab: The First Platelet Glycoprotein IIb/IIIa Receptor

Antagonis t



37



surface ( S ) , in addition to an internal pool of receptors that becomes

externalized when the platelet is activated (9).

Independent of the nature of the precipitating injury to the vessel wall

and regardless of the platelet-activating stimuli generated, the GP IIb/lIIa

receptor is the final common pathway for platelet aggregation (10). Thus, it

seemed reasonable to hypothesize that an agent that blocked t h l s receptor

would probably be a more potent irhbitor of platelet aggregation than, for

example, aspirin, whch i h b i t s only thromboxane-mediated aggregation.

The role of the GP IIb/lIIa receptor in platelet aggregation came to light

during investigations of Glanzmann's thrombasthenia, a rare congenital

disorder in which a deficiency of GP IIb/lIIa receptors gves rise to recurrent

mucocutaneous bleeding (1 1, 12). However, the infrequency of visceral

bleeding in patients with this condition suggested that pharmacologc

antagonism of the GP Ilb/IIIa receptor would probably not result in a

dangerous excess of bleeding. Thus, the GP Ilb/lIIa receptor emerged as a

logcal target in the development of therapeutic agents that could efficiently

and safely counteract platelet aggregation (5).



1.2



Development of abciximab



The first step in the development of a therapeutic antagonist of the GP

llb/lIIa receptor was the isolation of antiplatelet antibodies from cell lines

obtained from mice that had been immunized with human platelets (13).

Coller and associates identified one such IgGl antibody with a kappa light

chain (designated 7E3), whch was directed against the GP Ilb/lIIa receptor,

and would interfere with platelet-fibrinogen binding and inhibit platelet

aggregation in response to a variety of stimuli (13, 14).

Since platelets coated with intact antibody could potentially be cleared

or destroyed by the human immune system, murine 7E3 IgG was obviously

inappropriate for in vivo application. To circumvent these problems, the

antibody was subjected to enzymatic digestion by pepsin or papain (15).

Proteolytic digestion produced two antigen-binding fragments, a bivalent 7E3

F(ab')2 fragment derived from pepsin and a univalent 7E3 Fab fragment

derived from papain (Figure 2). Both of these antibody fragments proved to

have platelet-binding affinities comparable to those of the parent 7E3 IgG

molecule. The univalent 7E3 Fab fragment was selected for further

development for several reasons including the expectation that its smaller size

(approximately M i 50,000) would minimize immunogenicity.



38



Robert E. Jordan, Marian T. Nakada, Harlan F. Weisman

Murine 7E3



Replacement of Murine

Constant Regions

with Human

Constant Regions

I



2 Murine 7E3 Fabs



+



Chimeric 7E3



lgGl



Papain



-



2 Chimeric 7E3 Fabs



Figure 2. Schematic drawing of the development of the chimeric 7E3 antibody (c7E3 Fab,

or abciximab). Murine 7E3 IgGl antibody directed against the GP lIbAIa receptor (top left)

was proteolyzed with pepsin to yield a bivalent 7E3 F(ab')z antigen-binding fragment and

with papain to yield two univalent 7E3 Fab fragments (top right). To minimize

immunogenicity, a genetic engineering approach was used to substitute human constant

regions for the murine constant regions linked to the murine variable regions containing the

antigen-binding sites (bottom left). Enzymatic digestion of the genetically engineered

humadchimeric version of 7E3 IgGl yielded chimeric c7E3 Fab, known as abciximab (17).



To further reduce the possibility of a human antimurine antibody

(HAMA) response, the molecular biologtsts at Centocor went on to produce a

humadmurine chmeric version of 7E3 Fab (16). This was acheved by reengtneering the genes that encode the murine 7E3 heavy and light antibody

chains such that human constant domains would be substituted for the

origmal mouse sequences. These human constant domains were linked to the

murine variable regons that contained the antigen-combining sites (see Figure

2). The chmeric 7E3 (c7E3) Fab molecule that resulted from papain digestion

of the chimeric IgG is a 47,600-dalton protein comprising roughly equal parts

of murine variable r w o n and human constant domain sequences and proved



Abciximab: The First Platelet Glycoprotein IlWIIIa Receptor

Antagonist



39



to have a KD for human platelet binding that was equivalent to that of the

parent murine antibody. Importantly, the incidence of human immune

responses to c7E3 Fab, known now as abciximab (marketed name ReoPro),

was shown to be markedly reduced compared with murine 7E3 Fab. In large

clinical trials, the rate of development of serologcally-detectable immune

responses was less than 6% of treated patients (17). No correspondence was

found between the development of immune responses to abciximab and

adverse clinical or safety outcomes. Thus, the dual objectives of equivalent

functionality with reduced immunogenicity had been met.



2.



PRECLINICAL PHARMACOLOGY



2.1



Binding Studies



Quantitative studies using radiolabeled antibodies revealed that

approximately 80,000 abciximab molecules are bound to each human platelet

at saturation (8). The equilibrium dissociation constant (Kd) for the bindmg

of abciximab to human platelets is approximately 5 nM. Abciximab binds

with similar affinity to primate platelets, binds less avidly to dog and rat

platelets than to human platelets, and does not bind appreciably to platelets

from mice, pigs, guinea pigs, or rabbits. A weak binding of 7E3 F(ab')2 to rat

platelets has recently been demonstrated (M. Nakada, unpublished

observation).

The binding of abciximab to platelets is reversible. Under competitive

conditions, about half of platelet-as sociated, radioactively labeled abciximab

was shown to dissociate withm about 4 to 5 hours (8). The dynamic binding

of abciximab to platelets is also evident from flow cytometric studies. In a

mixture of abciximab-coated platelets and noncoated platelets, the

redistribution of abciximab from coated platelets to previously uncoated

platelets is apparent at 30 minutes and is complete at 3 hours as evidenced by

the single unimodal peak of intermediate abciximab binding (Figure 3) (17).



Robert E. Jordan, Marian T. Nakada, Harlan F. Weisman



40



7



t=O



?



30 min.



$7



90 min.



211, hr.



3 hr.



$1



4 hr.



Fzgure 3. Platelet-bound abciximab redistributes to unlabeled platelets under in vitro

mixing conditions. Equal volumes of washed abciximab-treated platelets and control

(saline-treated) platelets were combined and continuously mixed at 37OC. Samples were

periodically removed ftom the incubation and treated with a fluorescein-conjugated rabbit

anti-abciximab antibody preparation and then fixed with 2%formalin. The smaples were

evaluated for the presence of platelet-bound anti-abciximab on a Becton-Dickinson

FACScan flow cytometer. From each sample, 5,000 events were analyzed in the forwardversus-side scatter gate that defined the platelet population. Individual platelet histograms

are shown. The convergene of the two separte peaks into a unimodal pattern indicates that

all platelets eventually acquired comparable amounts of abciximab. From Jakubowski J,

Jordan RE, Weisman HF.Current antiplatelet therapy. In: Uprichard A, ed. Handbook of

Experimental Pharmacology. In press.



Abciximab: The First Platelet Glycoprotein IIb/IIIa Receptor

Antagonist



41



In addltion to binding to the GP IIbAIa receptor, abciximab also binds

with comparable affinity to the related integnn receptor avb3, whch is found

on platelets albeit less abundantly than is GP I I b A a (18). The avb3 receptor

is also expressed by vascular endothelial cells (19), where it may play a role

in wound healing and angogenesis (20), and by vascular endothelial smooth

muscle cells, where it may be involved in cellular migration and proliferation

(21, 22). A recent study has demonstrated that abciximab freely redistributes

between GP IIbAIa and avb3 receptors in vitro (23). T h ~ sfinding implies

that abciximab may not only produce sustained blockade of platelet GP

IIb/IUa receptors but also may exert prolonged actions on vascular cell avp3

receptors.



2.2



Dose-Response Studies



Both in vitro and in vivo studies have consistently demonstrated that

platelet inhibition by abciximab is dose-dependent and correlates directly with

GP llbAIa receptor blockade. In vitro dose-response correlation studies have

been performed by parallel determinations of receptor blockade, estimated by

radiometric binding assay, and of platelet aggregation (24). Platelet

aggregation was measured by the increase in visible light transmission

through a stirred suspension of human platelets (25). At increasing

concentrations of abciximab doses rangmg from 0.75 to 2.0 mg/mL,

increasing levels of receptor blockade rangmg from 35% to 91% and

corresponded to inhibition of ADP-induced platelet aggregation rangmg from

approximately 30% to 100% (Figure 4). Blockade of at least 80% of GP

I I b A a receptors was necessary to achieve complete or nearly complete

inhibition of ADP-induced aggregation.

The results of these in vitro experiments were confirmed by in vivo studies

in whch sequential intravenous doses of abciximab, 0.05 mg/kg, were

administered at intervals to a cynomolgus monkey (17). A cumulative total

dose of 0.25 m a g produced 82% GP IIbAIa receptor blockade and nearly

complete inhibition of ADP-induced platelet aggregation.

When abciximab was administered in a single bolus dose of 0.25 mg/kg to

monkeys, it had a relatively short duration of platelet-inhibiting action. More

than 80% blockade of GP IIbAIa receptors was achleved withm 2 minutes of

injection, but receptor occupancy by abciximab decreased to 75% at 1 hour,

69% at 2 hours, and 50% by 24 hours.



Robert E. Jordan, Marian T. Nakada, Harlan F. Weisman



42



% Receptor c7E3 Fab

Blockade (@rnL)

91

2.0

77

1.75



6



i



1



4



70



1.50



59



1.25



43



0.90



35



0.75



0



0



i



Minutes



Figure 4 Concentration dependence of platelet inhibition by c7E3 Fab (abciximab) and

correlation with GP IIb/IUa receptor blockade. Platelet-rich plasma (250,00O/mL) fiom a

normal donor was incubated with different concentrations of c7E3 and then treated with

ADP to induce platelet aggregation. Each tracing indicates the degree of platelet aggregation

at a given c7E3 dose; greater inhibition of platelet aggregation corresponds with decreasing

visible light transmission througfi the platelet suspension. Corresponding levels of GP

IIbma receptor blockade for each c7E3 dose were determined by radiometric binding

assay. From Jordan RE, Wagner CL, Mascelli MA, et al. Preclinical development of c7E3

Fab; a mousehuman chimeric monoclonal antibody fragment that inhibits platelet fimction

by blockade of GPIIbma receptors with observations on the immunogenicity of c7E3 Fab

in humans. In: Horton MA, ed. Adhesion Receptors as Therapeutic Targets. Boca Raton,

Fla: CRC Press; 1996:281-305.



2.3



Antithrombotic Efficacy Studies In Animals



2.3.1



Abciximab Alone



The efficacy of abciximab has been explored in an array of animal models

of coronary and carotid arterial thrombosis. Repeated experimental

demonstrations that abciximab protected against thrombotic occlusion

following vascular injury laid the foundation for subsequent clinical trials of

GP Ilb/ma receptor blockade in patients undergoing percutaneous

intervention. It should be noted that because of the low affinity binding of

abciximab to dog platelets, the divalent murine 7E3 F(ab‘)2 fragment was

used in canine studies instead of the chimeric antibody.

In dogs whose coronary arteries had been mechanically injured and

constricted by an adjustable cylinder surrounding the artery, 7E3 F(ab’)2

prevented platelet deposition and vascular occlusion (26). 7E3 F(ab’)2

produced similarly encouraging results in a variation of t h l s experiment



Abciximab: The First Platelet Glycoprotein IIb/IIIa Receptor

An tagonis t



43



performed in the carotid arteries of monkeys (27). Importantly, the bleeding

time was only modestly prolonged at doses that blocked 80% of the GP

IIb/IIta receptors, almost completely inhibited platelet aggregation, and

prevented thrombosis.

Other experiments in dogs showed that pretreatment with 7E3 F(ab‘)2, but

not with aspirin or heparin or both, was able to prevent coronary artery

thrombosis and occlusion after electrical injury to a mechanically stenosed

artery (28). Protection against platelet deposition and acute thrombosis at the

site of deep arterial injury lasted for as long as 5 hours. Similar protective

effects were documented with abciximab after electrolytic injury to the carotid

artery in cynomolgus monkeys (29). Another study, with obvious clinical

ramifications, showed that 7E3 F(ab’)2 was more effective than aspirin in

preventing thrombosis and reducing mortality for up to 6 days after balloon

angoplasty in dogs (30).

2.3.2



Abciximab Combined with Fibrinolytic Agents



Since thrombosis represents the culmination of processes involving both

platelet aggregation and coagulation, ample theoretical rationale supports the

combination of abciximab and fibrinolytic agents. In experiments in dogs, the

combination of 7E3 F(ab’)2 with tissue plasminogen activator (tPA)

accelerated the dissolution of thrombin-induced thrombus and prevented

reocclusion in stenotic coronary arteries (3 1). In addition, since platelet-rich

clots are implicated as factors underlyng the resistance of coronary occlusive

thrombus to fibrinolytic therapy, overcoming this resistance might be possible

by the adjunctive use of a GP I I b m a receptor blocker. Indeed, pretreatment

with an intravenous bolus dose of 7E3 F(ab‘)2 prior to tPA administration

was more effective than either aspirin or dipyridamole in preventing

reocclusion in dogs (32). In t h ~ sstudy, seven of eight control animals who did

not receive the GP IIb/IIIa antagonist experienced reocclusion.

In another htghly thrombogenic model in whtch a canine coronary artery

segment was surgcally removed, everted, and re-anastomosed, the

administration of tPA alone proved to be of limited efficacy in restoring

coronary blood flow after thrombotic occlusion (33). Reperfusion was

successfully acheved, however, when 7E3 F(ab’)2 was adrmnistered prior to

fibrinolytic therapy. Another study whch employed the same model showed

that 7E3 irhbited the development of thrombosis in the everted grafts for a

period extending beyond the duration of blockade of platelet aggregation (34).

Similarly, in studies using the electrolytic model of vascular injury,

administration of 7E3 F(ab’)2 prior to fibrinolytic therapy accelerated

thrombolysis (35), prevented reocclusion and reduced infarct size (36), and



44



Robert E. Jordan, Marian T. Nakada, Harlan F. Weisman



improved 5-day survival as compared with animals pretreated with aspirin or

hrudm (29, 37).



2.4



Animal Toxicology Studies



Toxicology studies in monkeys indicated that abciximab, administered in

bolus doses as hgh as 8 mgkg, has a favorable safety profile. Monitoring of

the animals for 2 weeks following abciximab treatment revealed only mild,

transient mucocutaneous bleeding, such as gingival bleeding, epistaxis, and

bruising. However, these signs were believed to be largely the result of

laboratory restraint procedures and frequent blood collections. Administration

of a bolus dose of up to 0.6 mg/kg, followed by a continuous 96-hour infusion

of 0.8 mg/kg/min, was likewise well tolerated, with no toxicity noted either

during the mfusion or during 3 to 6 weeks of observation thereafter.

In an effort to more closely simulate the clinical situation in whch

abciximab likely would be applied, monkeys were treated with a bolus dose of

0.3 mg/kg, followed by a continuous 48-hour infusion at 0.45 or 0.5

mg/kg/min, in combination with aspirin, heparin and either tPA or

streptokinase. This study confirmed that pairing an abciximab bolus and

infusion with standard antiplatelet, antithrombotic, and fibrinolytic therapy

was well tolerated both acutely and for at least 3 weeks following treatment.

The combination regimen was not associated with any signs of acute adverse

reactions, such as hypersensitivity, hemorrhage, or thrombocytopenia.



2.5



Other Actions of Abciximab



2.5.1



Inhibition of Granule Release



Abciximab exerts a dose-dependent ihbitory effect on the release of

platelet granule constituents (38). At a dose that produced 100% inhibition of

platelet aggregation, abciximab resulted in 81% ihbition of the release of

adenosine triphosphate (ATP) from dense platelet granules. Abciximab also

acted on the release of constituents from platelet a-granules, inhlbiting the

release of P-thromboglobulin (P-TG) by 87% and the release of plasminogen

activator irhbitor 1 (PAI-1) by 81%. These findings suggested an additional

mechanism through whch abciximab might counter the formation of mural

thrombin and reduce mitogen release at the site of vascular injury.

2.5.2



Inhibition of inflammatory processes



The IgG form of 7E3 was shown to bind to the leukocyte integrin receptor

Mac-1 (39), although with apparent lower affinity than has been shown for



Abciximab: The First Platelet Glycoprotein Ilb/IIIa Receptor

An tagonis t



45



GPIIbmZa or avp3. Nevertheless, t h l s binding pointed to a potential

irhbitory effect of abciximab on the recruitment of inflammatory cells to

injured blood vessel walls. Incursion of excessive numbers of inflammatory

cells such as monocytes and macrophages likely contributes to intimal

hyperplasia and restenosis at sites of vessel injury. Recent in vitro

observations confirmed that relatively hgh concentrations of abciximab

effectively blocked monocyte and neutrophd adhesion to vessel wall ligands

fibrinogen and ICAM-I (40). In related observations in treated patients,

abciximab reduced the activated-platelet mediated activation of leukocytes

and the upregulation of Mac-1 on those cells in circulation (41). Thus, both a

direct and indirect inhibition of Mac-1-mediated inflammatory pathways and

the potent antithrombotic action of abciximab may converge to aid in the

regulation of vascular repair and to sustaining clinical benefit.

2.5.3



Inhibition of platelet-mediated thrombin generation



Activated platelets are catalytic centers for the generation of thrombin, the

enzyme responsible for causing fibrin deposition withln the thrombus. The

anticoagulant heparin inhlbits thrombin and slows the coagulation process. In

the EPIC trial (42) noted that patients receiving abciximab had longer

activated clotting times (ACT) than placebo patients receiving similar doses

of heparin but no abciximab. T h ~ ssuggested an anticoagulant action of

abciximab that was confirmed in a direct in vitro study in whch abciximab

was shown to block the generation of thrombin on activated platelet surfaces

(43). Thls effect was a direct consequence of the blockade of both platelet

GPIIbma and avp3. Thus, the anti-thrombotic benefits of abciximab may

contain an anticoagulant component in addition to the ihbition of platelet

aggregation,



3.



CLINICAL PHARMACOLOGY



3.1



Dose-Response Studies in Humans



Studies in healthy volunteers and in patients with stable coronary artery

disease confrmed that an abciximab bolus dose of 0.25 mdkg was sufficient

to block at least 80% of GP I I b m a receptors and virtually abolish ADPinduced platelet aggregation (44, 45). Although the onset of action of

abciximab is immediate, the above effects were reported at 2 hours after

administration. Concomitant with a decrease in receptor blockade to below

80% at 4 to 6 hours, the degree of platelet inhibition began to wane gradually.



46



Robert E, Jordan, Marian T. Nakada, Harlan F. Weisman



At 24 hours following the bolus dose, the level of GP IIb/IIIa receptor

blockade had fallen to 50% to 60% and platelet aggregation was inhibited by

only 60%.

Since it is likely to take more than 8 hours for an injured atherosclerotic

blood vessel to undergo passivation (24), that is, to become nonthrombogenic

and nonplatelet-reactive, it was suspected that the duration of platelet

irhbition achieved with a single bolus dose might not be therapeutically

adequate. A bolus dose followed by a continuous infusion proved to be more

effective in producing sustained platelet ihbition. Studies conducted in

patients with stable coronary artery disease and in patients undergoing hghrisk percutaneous transluminal coronary angioplasty (PTCA) showed that a

bolus dose of 0.25 mglkg, followed by a 24-hour infusion of 10 mdmin,

yelded greater than 80% blockade of GP IIb/IIIa receptors and virtually

complete abolition of platelet aggregation for the entire 24-hour infusion

period (Figure 5) (46, 45). (As discussed below, an abciximab r q m e n

consisting of a 0.25 mdkg bolus and a 12-hour infusion of 10 mg/min was

later to be deployed for large-scale clinical trials in patients undergoing

percutaneous intervention). Partial recovery of platelet aggregation, to 50% of

baseline levels, was seen withm 6 hours of cessation of the infusion. However,

full recovery of platelet function, in parallel with the fall-off in GP IIbAlIa

receptor blockade, is much more gradual. Abciximab remains bound to and

homogeneously distributed among platelets for prolonged periods of up to 2

weeks (47, 48). Since the duration of GPIlbRUa binding exceeds the average

platelet life span, it appears that abciximab is continuously redistributed and

re-equilibrated among all circulating platelets, including those newly entering

the circulation from the bone marrow. The duration of platelet irhbition

following abciximab treatment is also prolonged and varies depending on the

particular stimulus of platelet aggregation. Recovery of platelet function after

abciximab in response to a strong agonist such as 20 mM ADP takes 2 days,

but when aggregation is induced by weaker stimuli, such as lower doses of

ADP or certain types of shear force, full recovery of platelet function takes at

least 7 days (49).



Abciximab: The First Platelet Glycoprotein IIb/IIIa Receptor

Antagonist



47



100



80

60



40

20

0



Time (Hr)

Figure 5. Blockade of GP IIb/lIIa receptors and inhibition of ex vivo platelet aggregation in

patients with stable coronary artery disease who received abciximab, in a bolus dose of 0.25

mglkg followed by a 24-hour infusion of 10 mg/min. From Jordan RE, Wagner CL,

Mascelli MA, et al. Preclinical development of c7E3 Fab; a mousehuman chimeric

monoclonal antibody fiagment that inhibits platelet function by blockade of GPIIbmJa

receptors with observations on the immunogenicity of c7E3 Fab in humans. In: Horton MA,

ed. Adhesion Receptors as Therapeutic Targets. Boca Raton, Fla: CRC Press; 1996:281305.



In another recent study using blood that was collected from patients

undergoing angoplasty and then subjected ex vivo to laminar shear stress in a

cone-and-plate viscometer, a bolus injection of abciximab, 0.25 mgkg,

rapidly and almost completely blocked GP I l b A a receptors and inhibited th~s

type of shear-induced platelet aggregation by 50% (50). In addition,

abciximab completely eliminated the formation of large platelet aggregates.

Platelet aggregation recovered partially within 2 days but remained inhibited

to some degree for 1 week, at whch time abciximab still blocked 35% of

GPIIbKUa on circulating platelets.

In contrast to the prolonged circulation of platelet-bound abciximab,

plasma levels of unbound abciximab disappear rapidly from the circulation

following a bolus dose. The initial half-life of free abciximab is about 30



Tài liệu bạn tìm kiếm đã sẵn sàng tải về

1 The GP IIb/IIIa Receptor as a Target for Antithrombotic Therapy

Tải bản đầy đủ ngay(0 tr)

×