The Long Road to Direct Factor Xa Inhibitors: A History of Anticoagulation
Marjorie Burnsworth

Tacoma Community College

July 24, 2014

Abstract
This paper explores the history of the chemical research and development of anticoagulant medications over the years and how this has lead to the development of direct factor Xa inhibitors (fXa inhibitors). Since ancient doctors first used leeches to treat their patients, people have been aware of the properties of anticoagulants and anti-thrombolytics, but the development of Heparin almost 100 years ago signified the start of a century of chemical discovery and development in the field of anticoagulation that has peaked in the last 25 years. Following the development of Heparin, scientists next developed Vitamin K Antagonists (VKA's), such as Coumadin, which gave an oral option for anticoagulation but they were not without side effects. Nearly 30 years passed before chemists came up with the next step forward in anticoagulation, low-molecular-weight-heparin (LMWH). It was LMWH that first opened scientists eyes to factor Xa and the possibility of considering it as a possible target for future anticoagulation. In order to proceed with development, however, they had to look back to the past. Back to leeches and heparin, as these would prove hold the keys to the future development of direct factor Xa inhibitors.

The Long Road to Direct Factor Xa Inhibitors:
A History of Anticoagulation
The concept of anticoagulants, or "blood-thinners", as most people know them have been around since the ancient times and can be traced back to Sanskrit writings of the ancient Indian physicians Caraka and Suśruta, or the Greco-Roman physician Galen (10) who used leeches to treat peoples ailments. We have come a long way in anticoagulant and anti-thrombolytic therapy since then, but no time has been more exciting than the last 25 years with the development of direct factor Xa (10a) inhibitors. This "new type" of anticoagulant existed in theory long before it was developed by chemists and proved to be effective. The antithrombolytics that were developed prior to the direct factor Xa (fXa) inhibitors, such as Coumadin and Heparin, are still used today for treatment and management of a variety of conditions, including venous thromboembolism (VTE) and long-term prevention of ischemic stroke in patients with atrial fibrillation, but have multiple food and drug interactions, varying metabolism from patient to patient, and require close and frequent monitoring to maintain a narrow window of therapeutic levels so that patients are not at risk for bleeding or clotting, both of which can be life-threatening. Still these medication are effective for preventing clot formation and were stepping stones on the path toward the development of fXa inhibitors. The fXa inhibitors that have been developed have one specific target in the coagulation cascade and work like a laser effecting only that on target at a key junction, as opposed to it's predecessors that acted on multiple sites, like the spray from a shotgun blast, likely contributing to their mutliple interactions. How did we get from leeches to a medication with such a specific target? Is has been a long road for chemists over the last 100 years with each developed treatment leading to the next up to where we are now. The first thing that needs to be understood when discussing the development of anticoagulants is how the human blood clots so that how these different agents work can be understood. A simplified explanation is that blood coagulates or clots through a series of events and activations referred to as the "Coagulation Cascade". The coagulation cascade is initiated by either trauma to tissue or to a blood vessel. When trauma occurs, platelets are called to the scene to form a temporary clot by an activator called "tissue factor" that is released from cell membranes to set everything into motion. Once activated by the tissue factor, there are a series of factors (proteases) and co-factors that are present in the blood and each one activates the next one in the series or "cascade" with the end result being the creation of fibrin that stabilizes the clot that was initially made by the platelets (1, 3, 4). There are two pathways that lead to the same goal of a creating fibrin and they come together in a "Y" shape (see figure 1) and end in one common pathway. The junction of these two pathways occurs when factor X is activated and converted to factor Xa(1). Essentially, factor Xa is the gatekeeper at the start of common coagulation pathway and this is what makes the development of fXa inhibitors so promising and exciting. The first medication that was developed as an antithrombolytic was Heparin in 1916 (see figure 2). It was prepared using a dog liver and is still made today from bioligical agents such as porcine intestine or bovine lung. In fact the word "heparin" is from a Greek word meaning "liver". Heparin, also known as Unfractionated Heparin or UFH, did not come to be used medically as an anti-thrombotic agent until the 1930's. Heparin is a heterogeneous mixture of polymers that include a variable number of sugars (2). Heparin molecules themselves are negatively charged and therefor bind to the positively charged patches on antithrombin (AT). By bonding with the antithrombin the heparin accelerates the rate at which the AT inactivates the thrombin (factor IIa), factor Xa, and multiple other upstream antigoaculant enzymes (1). Although heparin is an effective anticoagulant, it is not without its problems. It is not well absorbed and absorption rates vary from person to person, it has a short half-life, requires frequent blood tests for monitoring, and it could also cause a potentially life threatening side effect called HIT, or Heparin Induced Thrombocytopenia. As reserachers continued to look for other kinds of anticoagulants the next development was a class of drugs called the Vitamin K Antagonists (VKA), which were first developed in 1941. The vitamin K antagonists includes the "holy grail" of anticoagulants, Coumadin, that was developed in 1953 and is still used today (Figure 2). Vitamin K is essential for the production and synthesis of multiple proteins and clotting factors in the coagulation cascade, such as Factors II (prothrombin), VII, IX and X, as well as protein C and protein S. VKA's interfere with the conversion of Vitamin K into its two epoxide forms that each contain a three-membered ring involving an oxygen atom and two carbon atoms. These forms of Vitamin K are necessary for the carboxylation reaction that turns glutamate residues into γ-carboxyglutamates (Gla), and the γ-carboxyglutamates are necessary to activate the proteins in the coagulation cascade(12). Although VKA's are commonly used today, they work indirectly affecting multiple sites (table 3) and have many drawbacks that include many interactions with foods, especially those that contain vitamin K, alcohol and other medications, as well as they frequent monitoring. The next development in anticoagulation did not occur until 1980 when low-molecular-weight-heparin (LMWH) was developed (1). LMWH was an updated and improved version of the old heparin. Chemists discovered that they could use chemicals or enzymes to separate and purify UFH and make it a much more homogenous mixture than the Heparin from which it originated (2). This purification also gave the new LMWH less inactive ingredients which lead to a decreased risk of HIT. What was also discovered about these LMWH's was that because of their smaller size, they worked more on an allosteric mechanism, forming non-covalent bonds that caused inhibition by changing or blocking the active site without actually filling it. Since Factor Xa is mostly inhibited by allosteric mechanisms as well, the LMWH were found to greatly enhance the inhibition of fXa with lesser inhibition of antithrombin and other factors that Heparin influenced (2). The discovery of the LMWH's inhibition of fXa lead anticoagulation research in a new direction as it brought attention to the possibility of fXa specifically being a new target for anticoagulation (1). The process of developing and targeting fXa specifically for inhibition made the scientists look back to the past. Specifically, they looked back to leeches and other hematophagous organisms that feed on blood. Antistasin was a polypeptide that was isolated from the salivary gland of the Mexican Leech, Haementeria officialis, in 1987 and TAP (Tick Anticoagulant Peptide) was extracted from the soft tick, Ornithodoros moubata, in 1990. Both of these proteins were specific for inhibiting fXa and were found to have constant inhibition values (Ki) in the range of 0.3 to 0.6 nmol/L. Research then showed that TAP, a direct fXa inhibitor, was more effective than heparin and LMWH which are indirect fXa inhibitors (1). A 1989 study of antistasin showed that during a kinetic analysis of the inhibitory interaction between antistasin and Factor Xa a mixed, primarily competitive type of inhibition was seen (5) as opposed to the allosteric mechanism that was seen in the LMWH's. Despite the promising research done on antistasin and TAP, those two specific enzymes were never taken to human trials. Instead, researchers were focused on developing a synthetic version of a fXa inhibitor. The first attempts at creating a synthetic inhibitor all contained a common anti-parasitic agent called isoxazoline (C3H5NO) or isoxazoline derivatives such as benzamidine (C7H8N2), the strong, polar base of guanidine (HNC(NH2)2.), or napthylamidine (C10H8 ) which are all thought to mimic the sequence amino acids in prothrombin that is recognized by factor Xa (Glu-Gly-Arg) (1). This research lead to the development of DX-9065a, a small-molecule, direct, selective, and reversible factor Xa inhibitor. DX-9065a was shown in studies to inhibit fXa and also reduce the generation of thrombin and fibrin formation among patients with stable coronary artery disease (6). Development of DX-9065a was halted because the oral bioavailability of the drug in humans was only 2-3% because of the presence of an amidine group which is highly basic and polar (1). Scientists used the partial success with DX-9065a as a starting point for the continued research and development of synthetic factor Xa inhibitors. They turned more to a structure-based approach and started by creating a molecular model of fXa using a crystallographic structure of the molecule (1). The structure-based, molecular modeling technique was helpful in trying to develop fXa inhibitors because it's complex structure. Factor Xa has a complex, folded tertiary structure that shields it active site (the catalytic triad Ser195-His57-Asp102) and two essential subsites, S1 and S4 (7), making it difficult to develop a drug that could inhibit its functions. The fXa molecular model that was created was then combined with structure models from the first generation fXa inhibitors that had been developed, such as DX-9065a, and a quantitative structure-activity relationship analysis was done. The analysis of this structure-based, molecular modeling technique showed that a basic amidine group was located in the first, or P1, position of the first generation fXa inhibitors and it utilized isoxazoline derivatives that lined up with the essential S1 subsite on fXa. The amidine in the P1/S1 site is supposed to be involved in 2 key reactions. The first is an electrostatic interaction between positive and negative charges (a so-called ion pairing) in the S1 pocket or subsite of the fXa molecule. The second component is a molecule/functional group that binds to a central metal atom using Hydrogen bonds at 2 different sites. The scientists discovered that by eliminating either of these interactions, the constant amount of fXa would increase by one to two orders of magnitude (1). Since the amidine groups are highly basic in nature and have a low bioavailability, they were found to be a poor choice to be located at the essential P1/S1 position. The reasearchers built on this information by replacing the basic amidine group in the P1 site with a non-basic variation to see it this would increase the oral bioavailability. Various elements were also tried in the inhibitors P4 position which was located directly opposite of S4, the other essential subsite of fXa. This was an attempt to maximize interactions at the S4 site to overcome the subsequent loss in potency caused by the removal of amidines from the P1 position (1). While the continued research on DX-9065a and the structure based modeling was occurring, other chemists were working on the development of a drug that would be called fondaparinux. Fondaparinux was the bridge and the proof of principle that fXa inhibitors would work for anticoagulation. It was developed in the year 2000 (figure 2) and obtained FDA approval at the end of 2001. Technically fondaparinux is considered a Heparin derivative and a "selective" fXa inhibitor (13). It is a homogeneous, synthetic pentasaccharide that mimics the site on heparin that binds to antithrombin and only inhibits factor Xa indirectly through it's effects on the antithrombin in the coagulation cascade. The first step toward developing fondaparinux went back several years to when scientists discovered that a pentasaccharide molecule was the smallest sequence of heparin that would still activate antithrombin in order to produce the inhibition of factor Xa. Next, they discovered that there is a specific sequence of sulfate groups that need to be placed on the polysaccharide chain in order to optimize it's binding to the antithrombin (13). Although fondaparinux is not a direct factor Xa inhibitor, it's success was further proof that showed factor Xa inhibition was a valid target for the future of anticoagulation. This was exciting news to researchers and pushed them even harder to try to develop an oral, direct fXa inhibitor. Decades of research, development and some unsuccessful drug trials, all led to the successful synthesis of an oral, synthetic, direct fXa inhibitor in 2005 called Rivaroxaban (trade name Xarelto) followed by other medications such as Apixaban (trade name Eliquis) and others that are still in development and trials. One of the things that all of the successfully developed fXa inhibitors have in common is that they all utilize the information previously gathered in the molecular structure-models and have non-basic groups located in their P1 and P4 positions. They also have a mostly "L-shaped" bonding structure which allows these groups better access to the fXa active and subsites for inhibition (7). Rivaroxaban was approved for use in 2008 and is metabolized in the liver through a series of oxidative and hydrolytic pathways that are catalyzed by the liver enzyme CYP (9). Rivaroxaban is an oral, direct factor Xa inhibitor that inhibits free and clot-bound factor Xa and factor Xa in the prothrombinase complex (a complex that forms on the negatively charged phospholipid rich membrane of Platelets and leads to thrombin activation). It works by direct and specific competitive inhibition of factor Xa (see figure 3) and was actually found to select for factor Xa >10,000 times more than other proteases (14). It has also been estimated that one molecule of factor Xa can catalyze the production of approximately 1000 molecules of thrombin because of the amplification inherent in the coagulation cascade (14), so by targeting and inhibiting this one lone factor instead of casting a broad net, thrombin production can be significantly decreased while medication side effects and interactions are minimized as well. This is what makes Rivaroxaban, and drugs like it that directly inhibit fXa, so ideal. Looking back over the last 100 years, you can see the long way that that chemists and the scientific community has come in terms of anticoagulation therapy. There has been constant research with intermittent breakthroughs, but nothing has been more exciting than the developments of the last 25 years. The world learned significantly from the development of Heparin almost 100 years ago, discovering how it worked as they continued to understand more and more how coagulation occurred. The development of Vitamin K antagonists were also a significant advance in the outpatient treatment of clots and thrombus prevention, but both medications act vaguely on multiple areas of the coagulation cascade in addition to having multiple interactions and requiring frequent blood tests and monitoring. Entering the new age of direct factor Xa inhibitors shows great promise with an anticoagulant that has one specific target, minimal interactions and requires no routine monitoring. The future is even more promising as scientists continue to improve on these medications that were once only theoretical but now actually exist, continuing to decrease there interactions and increase there efficacy and tolerability. It is an exciting time in both the scientific and health care community.

Figure 1 (4).

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Figure 2

Timeline of development of parenteral and oral anticoagulants. LMWH indicates low-molecular weight heparin; fXa, factor Xa; TAP, tick anticoagulant peptide; VTE, venous thromboembolism; AF, atrial fibrillation

Figure 3 (7)

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