Direct thrombin inhibitors (DTIs) are a class of anticoagulant drugs that can be used to prevent and treat embolisms and blood clots caused by various diseases. They inhibit thrombin, a serine protease which affects the coagulation cascade in many ways. DTIs have undergone rapid development since the 90's. With technological advances in genetic engineering the production of recombinant hirudin was made possible which opened the door to this new group of drugs. Before the use of DTIs the therapy and prophylaxis for anticoagulation had stayed the same for over 50 years with the use of heparin derivatives and warfarin which have some well known disadvantages. DTIs are still under development, but the research focus has shifted towards factor Xa inhibitors, or even dual thrombin and fXa inhibitors that have a broader mechanism of action by both inhibiting factor IIa (thrombin) and Xa. A recent review of patents and literature on thrombin inhibitors has demonstrated that the development of allosteric and multi-mechanism inhibitors might lead the way to a safer anticoagulant.

History

Anticoagulation therapy has a long history. In 1884 John Berry Haycraft described a substance found in the saliva of leeches, Hirudo medicinalis, that had anticoagulant effects. He named the substance ‘Hirudine’ from the Latin name. The use of medicinal leeches can be dated back all the way to ancient Egypt. In the early 20th century Jay McLean, L. Emmet Holt Jr. and William Henry Howell discovered the anticoagulant heparin, which they isolated from the liver (hepar). Heparin remains one of the most effective anticoagulants and is still used today, although it has its disadvantages, such as requiring intravenous administration and having a variable dose-response curve due to substantial protein binding. In the 1980s low molecular-weight heparin (LMWH) were developed. They are derived from heparin by enzymatic or chemical depolymerization and have better pharmacokinetic properties than heparin. In 1955 the first clinical use of warfarin, a vitamin K antagonist, was reported. Warfarin was originally used as a rat poison in 1948 and thought to be unsafe for humans, but a suicide attempt suggested that it was relatively safe for humans. Vitamin K antagonists are the most commonly used oral anticoagulants today and warfarin was the 11th most prescribed drug in the United States in 1999 and is actually the most widely prescribed oral anticoagulant worldwide. Warfarin has its disadvantages though, just like heparin, such as a narrow therapeutic index and multiple food and drug interactions and it requires routine anticoagulation monitoring and dose adjustment. Since both heparin and warfarin have their downsides the search for alternative anticoagulants has been ongoing and DTIs are proving to be worthy competitors. The first DTI was actually hirudin, which became more easily available with genetic engineering. It is now available in a recombinant form as lepirudin (Refludan) and desirudin (Revasc, Iprivask). Development of other DTIs followed with the hirudin analog, bivalirudin, and then the small molecular DTIs. However, such DTIs were also having side effects such as bleeding complications and liver toxicity, and their long-term effects were in doubt.

Mechanism of action

Blood clotting cascade

When a blood vessel ruptures or gets injured, factor VII comes into contact with tissue factors which starts a process called the blood coagulation cascade. Its purpose is to stop bleeding and repair tissue damage. When this process is too active due to various problems the risk of blood clots or embolisms increases. As the name indicates the cascade is a multi-step procedure where the main product thrombin is made by activating various proenzymes (mainly serine proteases) in each step of the cascade. Thrombin has multiple purposes, but mainly it converts soluble fibrinogen to an insoluble fibrin complex. Furthermore, it activates factors V, VIII and XI, all by cleaving the sequences GlyGlyGlyValArg-GlyPro and PhePheSerAlaArg-GlyHis, selectively between Arginine (Arg) and Glycine (Gly). These factors generate more thrombin. Thrombin also activates factor XIII that stabilizes the fibrin complex and therefore the clot and it stimulates platelets, which help with the coagulation. Given this broad action of thrombin it stands as a good drug target for anticoagulant drugs such as heparin, warfarin and DTIs and antiplatelet drugs like aspirin.

Binding sites Thrombin is in the serine protease family. It has 3 binding domains in which thrombin-inhibition drugs bind to. Those proteases have a deep narrow gap as an active binding site that consists of two β-barrel subdomains that make up the surface gap which binds substrate peptides. The surface in the gap seems to have limiting access to molecules by steric hindrance, this binding site consists of 3 amino acids, Asp-102, His-57 and Ser-195. Thrombin also has two exosites (1 and 2). Thrombin is a little different from other serine proteases as exosite 1 is anion-binding and binds to fibrin and other similar substrates while exosite 2 is a heparin-binding domain.

DTIs inhibition . DTIs inhibit thrombin by two ways; bivalent DTIs block simultaneously the active site and exosite 1 and act as competitive inhibitors of fibrin, while univalent DTIs block only the active site and can therefore both inhibit unbound and fibrin-bound thrombin. In contrast, heparin drugs bind in exosite 2 and form a bridge between thrombin and antithrombin, a natural anticoagulant substrate formed in the body, and strongly catalyzes its function. But heparin can also form a bridge between thrombin and fibrin which binds to exosite 1 which protects the thrombin from inhibiting function of heparin-antithrombin complex and increases thrombin's affinity to fibrin. DTIs that bind to the anion-binding site have shown to inactivate thrombin without disconnecting thrombin from fibrin, which points to a separate binding site for fibrin. DTIs aren't dependent to cofactors like antithrombin to inhibit thrombin so they can both inhibit free/soluble thrombin as well as fibrin bound thrombin unlike heparins. The inhibition is either irreversible or reversible. Reversible inhibition is often linked to lesser risk of bleeding. Due to this action of DTIs they can both be used for prophylaxis as well as treatment for embolisms/clots.

Active site's pockets

DTIs that fit in the active binding site have to fit in the hydrophobic pocket (S1) that contains aspartic acid residue at the bottom which recognizes the basic side chain. The S2 site has a loop around tryptophan which occludes a hydrophobic pocket that can recognize larger aliphatic residues. The S3 site is flat and the S4 site is hydrophobic, it has tryptophan lined by leucine and isoleucine.

Nα-(2-naphthyl-sulphonyl-glycyl)-DL-p-amidinophenylalanyl-piperidine (NAPAP) binds thrombin in the S1, S2 and S4 pockets. The amidine group on NAPAP forms a bidentate salt bridge with Asp deep in the S1 pocket, the piperidine group takes the role of proline residue and binds in the S2 pocket, and the naphthyl rings of the molecule forms a hydrophobic interaction with Trp in the S4 pocket. Pharmaceutical companies have used the structural knowledge of NAPAP to develop DTIs. Dabigatran, like NAPAP binds to S1, S2 and S4 pockets. Benzamidine group on the dabigatran structure binds deep in the S1 pocket, the methylbenzimidazole fits nicely in the hydrophobic S2 pocket and the Ile and Leu at the bottom of the S4 pocket binds to the aromatic group of dabigatran.

Drug development

Hirudin derivatives

Hirudin derivatives are all bivalent DTIs, they block both the active site and exosite 1 in an irreversible 1:1 stoichiometric complex. The active site is the binding site for the globular amino-terminal domain and exosite 1 is the binding site for the acidic carboxy-terminal domain of hirudin. Native hirudin, a 65-amino-acid polypeptide, is produced in the parapharyngeal glands of medicinal leeches. Hirudins today are produced by recombinant biotechnology using yeast. These recombinant hirudins lack a sulfate group at Tyr-63 and are therefore called desulfatohirudins. They have a 10-fold lower binding affinity to thrombin compared to native hirudin, but remain a highly specific inhibitor of thrombin and have an inhibition constant for thrombin in the picomolar range. Renal clearance and degradation account for the most part for the systemic clearance of desulfatohirudins and there is accumulation of the drug in patients with chronic kidney disease. These drugs should not be used in patients with impaired renal function, since there is no specific antidote available to reverse the effects. Hirudins are given parenterally, usually by intravenous injection. 80% of hirudin is distributed in the extravascular compartment and only 20% is found in the plasma. The most common desulfatohirudins today are lepirudin and desirudin.

Hirudin In a meta-analysis of 11 randomized trials involving hirudin and other DTIs versus heparin in the treatment of acute coronary syndrome (ACS) it was found that hirudin has a significantly higher incidence of bleeding compared with heparin. Hirudin is therefore not recommended for treatment of ACS and currently it has no clinical indications.

Lepirudin

Lepirudin is approved for the treatment of heparin-induced thrombocytopenia (HIT) in the USA, Canada, Europe and Australia. HIT is a very serious adverse event related to heparin and occurs with both unfractionated heparin and LMWH, although to a lesser extent with the latter. It is an immune-mediated, prothrombotic complication which results from a platelet-activating immune response triggered by the interaction of heparin with platelet factor 4 (PF4). The PF4-heparin complex can activate platelets and may cause venous and arterial thrombosis. When lepirudin binds to thrombin it hinders its prothrombotic activity. Three prospective studies, called the Heparin-Associated-Thrombocytopenia (HAT) 1,2, and 3, were performed that compared lepirudin with historical controls in the treatment of HIT. All three studies showed that the risk of new thrombosis was decreased with the use of lepirudin, but the risk for major bleeding was increased. The higher incidence of major bleeding is thought to be the result of an approved dosing regimen that was too high, consequently the recommended dose was halved from the initial dose. As of May 2012 Bayer HealthCare, the only manufacturer of lepirudin injections, discontinued its production. They expect supplies from wholesalers to be depleted by mid-2013.

Desirudin Desirudin is approved for treatment of venous thromboembolism (VTE) in Europe and multiple phase III trials are presently ongoing in the USA. Two studies comparing desirudin with enoxaparin (a LMWH) or unfractionated heparin have been performed. In both studies desirudin was considered to be superior in preventing VTE. Desirudin also reduced the rate of proximal deep vein thrombosis. Bleeding rates were similar with desirudin and heparin.

Bivalirudin