Drug Discovery Technology Of Abciximab Biology Essay

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To prevent thrombus formation in coronary artery or to inhibit platelet function during and after coronary artery procedures like angioplasty was one of the main area of concern. Earlier drugs such as Aspirin, Heparin, Ticlopidin and Clopidogrel were used as antiplatelet agents but had limited and weak antiplatelet effects thus discovery for more potent platelet inhibitors was in progress.(Tcheng, 1999)

Abciximab is fragment antigen binding(Fab) antibody fragment of the chimeric human-murine monoclonal antibody 7E3 which binds to platelet GP IIb/IIIa receptor.(Tcheng, 1999)

In 1985 in laboratory of Dr. Barry collar pathways leading in development of Abciximab was first initiated.(Tcheng, 1999)

In1994 US food and drug administration(FDA) approved first Glycoprotein(GP) IIb/IIIa inhibitor for platelet inhibition function was Abciximab as manufactured by Centocor and distributed by Eli lilly under trade name Reopro.(Tcheng, 1999)

Target Identification and validation:

There were numerous targets identified which were responsible for platelet activity but among all of the identified targets Glycoprotein(GP) IIb/IIIa receptor was identified as the key target which was responsible for platelet aggregation. On all activated platelets the expression of the GP IIb/IIIa receptor occurs without any consideration of the physiological activator. Platelet to platelet aggregation by binding to adhesion molecules like fibrinogen , von Willebrand factor etc. was found to be mediated by activated GP IIb/IIIa receptor therefore by blockade of this receptor thrombosis was effectively controlled. From which it was concluded that new platelet GP IIb/IIIa inhibitors can successfully prevent platelet aggregation by blockage of this final common pathway as shown in Figure 1.(Tcheng, 1999)

Study reported binding of abciximab to GP IIb/IIIa receptor and also major surface receptor responsible for different types of ligands involved in platelet function. Different variety of stimuli such as collagen, thrombin, Adenosine diphosphate(ADP) etc responsible for platelet to aggregate was blocked by binding of abciximab to GP IIb/IIIa receptor. Procoagulation and proliferative properties of platelets and vascular endothelies and smooth muscle cells respectively was found to be mediated by vitronectin receptors which was also blocked by abciximab thus resulting more effective in prevention of burst of thrombin generation and cell adhesion along with GP IIb/IIIa receptor binding.(Robert E. Jordan, 2002)

Lead Generation:

In an animal model anti-platelet effect of aspirin, heparin and c7E3 Fab antibody was used against glycoprotein IIb/IIIa receptor of arterial thrombosis to compare antithrombotic strategy with c7E3 Fab. For monitoring of heart rate, carotid artery flow velocity and arterial blood pressure animal model of cynomolgus monkeys were instrumented. In the study treatment was given with saline, heparin, aspirin, aspirin plus heparin and c7E3 Fab in 6 models for each group. After drug administration thrombus formation to the intimal surface of right carotid artery was initiated at 15 minutes via anodal electrolytic stimulation (100 muA) and was continued for 180 minutes. After drug administration electrolytic injury in the left carotid artery started at 210 minutes and was observed to continue for 180 minutes. Throughout the experimental protocol template bleeding time, whole blood cell counts, GP IIb/IIIa blockade, platelet aggregation and activated partial thromboplastin was analysed at different time breaks. Among different treatments at baseline hemodynamic and hematologic parameters were observed quite comparative but in the prevention of carotid artery thrombosis in animal models was proved ineffective when treated with aspirin, heparin or aspirin plus heparin. Inhibition of platelet aggregation was observed when treated with c7E3 Fab antibody with increase in bleeding time. As a result of this study prevention of primary thrombus formation was found to be safe and effective when treated with c7E3 Fab for inhibition of platelet GP IIb/IIIa receptor while it failed with other treatments in study in cynomolgus monkey.(Rote et al., 1994)

The large molecular weight abciximab and two small molecular weight GP IIb/IIia inhibitors Eptifibatide and Tirofiban were identified potent parenteral GP IIb/IIIa inhibitors though abciximab followed different pharmacodynamics as compared to low weight competitive antagonists. Use of abciximab was reported as more effective as compared to other two antagonists. Binding to the GP IIb/IIIa receptor by abciximab was like a cap thus receptor was covered and it also interacted with a secondary fibrinogen binding site that is lys-Gln-Ala-Gly-Asp-Val sequence on GP IIb/IIIa receptor while eptifibatide and tirofiban interact with only Arg-Gly-Asp(RGD) sequence on GP IIb/IIIa receptor binding pocket. Decrease in efficacy results from the abbreviated duration of inhibition of platelet function was major disadvantage with low molecular weight inhibitors. Best efficacy was reported by abciximab from trial results which was around 35-50% decrease in the primary composite endpoint of death, immediate revascularisation or myocardial infarction where as low molecule antagonists resulted in only 15-25% reduction.(Tcheng, 1999)

Lead optimisation:

80-90% inhibition of the GP IIb/IIIa receptor in the duration of treatment was successfully achieved by Intravenous 0.25 mg/kg bolus of abciximab at zero time and following continuous infusion of 10 ug/min for 12 hours. Rapid clearance of unbound drug from plasma by proteolytic breakdown and therefore plasma half-life was very short around 10-15 minutes. 50-60% of receptor blockade still existed at 24 hours after stopping abciximab Approximately 1.5 molecules of abciximab was required for each GP IIb/IIIa receptor. Just by stopping abciximab drug infusion platelet inhibition by abciximab was not reversed but by transfusion of platelets it was successfully reversed. Because of above considerations use of abciximab was declared safe and effective but the potential for uncontrolled bleeding and lack of direct antidote was kind of disadvantages with use of abciximab.(Tcheng, 1999)

Design and development:

Mouse derived monoclonal IgG antibody against the human GP IIb/IIIa receptor was first isolated by Dr. Barry Coller which was dubbed 7E3. The original 7E3 molecule was very large and because of antigenicity problems it was later re-engineered into a smaller molecules by combination of the constant region of a human IgG Fab to the variable region of 7E3 Fab portion resulting formation of chimeric moiety c7E3 Fab.(Tcheng, 1999)

Abciximab was designed from human-murine chimeric monoclonal antibody, chimeric 7E3IgG. Human constant domains and murine variable regions was present in chimeric 7E3IgG recombinant proteins. Continuous perfusion in mammalian cell culture was carried out to produce chimeric 7E3 antibody. Papain digestion of purified chimeric 7E3IgG resulting Fragment antigen binding(Fab) fragments and Fragment crystallisable(Fc) domain. By series of different steps like specific viral inactivation, column chromatography and different removal procedures 47.6 kDa Fab fragment was purified. Half of murine and half human sequences with 439 amino acids was present in final abciximab product.(Robert E. Jordan, 2002)

Figure.2: The figure is schematic diagram of murine 7E3 IgG on left, chimeric 7E3 IgG in centre in process the original murine domains was replaced by human constant domains and by proteolytic digestion of chimeric 7E3 IgG with papain chimeric 7E3 Fab was produced.(Robert E. Jordan, 2002)

Important Clinical Trials of Abciximab and outcomes:

In 1991 the first large randomized, double blind placebo controlled trial carried at 56 centres and involving 2099 patients was Evaluation of c7E3 Fab fragment for the prevention of Ischemic Complications trial (EPIC). EPIC trial was carried out during percutaneous transluminal coronary ballon angioplasty (PTCA) in patients. In EPIC trial patients were treated in three groups that is with placebo, abciximab bolus and abciximab bolus followed by infusion. Reports showed around 10 percent decrease in the rate of primary end point with only abciximab bolus as compared to placebo treatment. The subjects treated with abciximab bolus followed by infusion showed decrease in rate of primary end point or myocardial infarction around 35 percent but risk of bleeding was considerably more as compared to patients treated with only abciximab bolus.(1994)

For the improvement of results with abciximab for GP IIb/IIIa blockade and risk of bleeding a randomized, double blind, placebo controlled trial was carried out at 69 centres called as EPILOG trial started in 1995. In total 4800 patients were enrolled for trial and 2792 patients were treated as the stopping rule was covered in the first interim analysis trial was terminated. All selected patients were to undergo urgent percutaneous coronary revascularization .The patients were divided into three groups one group where treated with placebo with standard dose heparin, second group treated with abciximab with standard dose heparin and third group treated with abciximab with low dose heparin. In the risk of major bleeding no significant difference was reported among the groups but more frequent minor bleeding was reported among patients treated with abciximab with standard dose heparin. Therefore use of abciximab with weight adjusted, low dose heparin as GP IIb/IIIa inhibitor was markedly able to decrease risk of acute ischemic complication in subjects undergoing percutaneous coronary revascularization with satisfactory results in the risk of bleeding.(1997a)

To analyse whether abciximab was able to produce impressive outcomes in patients with refractory unstable angina who were undergoing PTCA a randomised, placebo controlled multicentre trial was carried out called as CAPTURE trial. 1265 patients with refractory unstable angina were treated in this trial. Before PTCA for 18-24 hours and after angiography subjects were treated with randomly assigned infusion of placebo or abciximab. The results showed that abciximab was impressively able to reduce the rate of thrombotic complications in specific reduce in rate of myocardial infarction during PTCA in patients with refractory unstable angina.(1997b)

In 1996 to evaluate safety of coronary stenting with use of platelet GP IIb/IIIa blockade a randomised, double blind, placebo controlled and ballon-angioplasty controlled trial called as EPISTENT trial was started. It was carried out at 63 centres in USA and Canada in total 2399 subjects with suitable coronary artery lesions and ischemic heart disease were randomly selected for trial and was divided in three groups one group assigned stenting with placebo(n=809), second group assigned stenting with abciximab (n=794) and third group with ballon angioplasty with abciximab(n=796). The major bleeding complications was reported in group assigned stenting with placebo also death and large myocardial infarction was reported while it was less with abciximab.(1998)

An international randomised, double-blind, placebo-controlled phase II trial was carried out to check the safety and potential efficacy of abciximab in patients with stroke after its success in treatment of acute cardiac ischemia called as Abciximab Emergents Stroke Treatment Trial (AbESTT). In total 400 subjects within six hours of onset of ischemic stroke were enrolled for trial. The results of rate of symptomatic Hemorrhage during five days after stroke was taken to conclude primary safety and using modified Rankin Scale the distribution of results at three months after stroke measured primary efficacy with use of abciximab in trial. The trial concluded that abciximab can be given intravenously with considerable degree of safety and results could prove better at three months after stroke but to test the efficacy of abciximab in ischemic stroke a larger trial was required.(2005)

From December 2003 to december 2005 an international randomised, double-blind, placebo-controlled phase III trial known as AbESTT-II was carried out to get more clear with the efficacy and safety with abciximab in ischemic stroke. In trial 1800 subjects were planned for enrollment but it was terminated after enrollment of 808 patients as recommended by independent safety and efficacy monitoring board because of unfavorable benefit risk profile. Primary safety and efficacy measurements were set same as previous trial but patients selected for trials were divided in two groups. In one group subjects were treated within five hours of onset stroke called as primary cohort and in second group persons were treated within five to six hours after stroke onset or within three hours of awakening with sign of stroke called as companion cohort. The trial results was not able to prove safety or efficacy of intravenous administration of abciximab in acute ischemic stroke treatment and increase in rate of symptomatic Hemorrhage in both groups was reported.(Harold P. Adams, 2008)

Use of Hemodyne analyser and Thrombelostrograph methods for close monitoring of anti-platelet effect with Abciximab:

On account of frequent incidence of bleeding in patients undergoing more surgical procedures receiving abciximab was found to be increased. Therefore requirement for near site technology or method to observe excessive concentrations, therapeutic efficacy and appropriate reversal of abciximab could result in significant decrease in morbidity rate related to use of abciximab. Therefore study was carried out with use of Hemodyne (Hemodyne, Inc, Midlothian, VA) analyzer and modified Thrombelostrograph (Haemoscope. Skokie, IL) to monitor antiplatelet effect with abciximab and results obtained were positive with clinical use of Hemodyne analyzer and modified Thrombelastograph with use of abciximab for its anti-platelet effect. The study highlighted few important parameters such as platelet contractile force measured with Hemodyne analyser and maximum amplitude for platelets as obtained from modified Thromboelastograph are responsive to the platelet inhibition effect of clinically appropriate concentration of abciximab and with addition of fresh Plasma rich platelets inhibition of platelet function can be easily reversed.Many such technologies were in clinical use for near site monitoring of antiplatelet drugs.(Greilich et al., 1999)

Reflective statement:

In all studies one common outcome reported was increase in risk of bleeding with abciximab that was only major drawback with the safe use of abciximab as GP IIb/IIIa inhibitor which was found to be decreased when treated with abciximab in combination with low-dose, weight adjusted heparin in EPILOG trial thus being from chemistry background i would emphasis more to search for similar anti-platelet chemical that can be given in combination with abciximab which can give more efficacy and safety with the use of abciximab as abciximab carries more potential in inhibiting platelet function and can produce best results as compared to other anti platelet agents.