Blood Clotting and Benzamidine
Now that I don't have to be graded on my memorization of the blood clotting cascade, let me just step back for a second and express awe for the physiological process of hemostasis. Think about this: we have to maintain our blood in a freely circulating form 99.999% of the time but as soon as there is a breach in the system, we have to quickly mobilize a cascade of events to prevent catastrophic blood loss. Pharmacological fiddling with this exquisitely controlled process is bound to have complications. But we have learn to walk this tightrope with drugs to prevent blood clots following stroke, orthopedic surgery, vascular stent implant, and other procedures and disorders that might cause such emboli to travel to our lungs, heart, or brain.
News yesterday from the American Heart Association meeting in Chicago brought our attention to a new class of anticoagulant drugs that may finally knock the coumarin, warfarin (Coumadin, racemic mixture), off its lofty pedestal as the oral antithrombotic drug of the last six-plus decades. Yes, the standard of care for about 65 years. The large, ROCKET AF trial presented yesterday had investigated the efficacy and side effect profile of warfarin relative to rivaroxaban (Xarelto, Bayer/J&J), a once-daily, oral inhibitor of the clotting factor, Factor Xa (part of a complex that converts prothrombin to the endogenous coagulation protein, thrombin).
To understand why this new drug is noteworthy, we have to consider how warfarin works. Warfarin acts upstream of Factor Xa by inhibiting Vitamin K epoxide reductase (VKOR), a crucial enzyme in the activating gamma-carboxylation of several clotting factors, not just Factor Xa. Vitamin K is the name for a group of 2-methyl-1,4-naphthoquinones that transition through a hydroquinone and epoxide in this reaction cycle.
The primary disadvantages of warfarin are the need for frequent monitoring of bleeding time (or INR, international normalized ratio), the interaction with foods containing vitamin K, and the risk of bleeding (particularly intracranial bleeding). Beyond the food-drug interaction, mostly with green leafy vegetables, polymorphisms exist both in the VKOR target and CYP2C9, the primary enzyme responsible for metabolizing warfarin.
Benzamidine, humble servant to many a successful Western blot, has given rise to one of two exciting developments in cardiovascular medicine.
Rivaroxaban, an oxazolidinone, and the recently-approved dabigatran (Pradaxa, Boehringer-Ingelheim), a benzamidine, bypass many of these problems via their own distinct mechanisms. Xarelto/rivaroxaban directly targets the serine protease activity of Factor Xa. Pradaxa/dabigatran is a direct thrombin inhibitor targeting the serine protease activity of this downstream coagulation factor.
The Thomson Reuters article yesterday by Ransdell Pierson and Debra Sherman on the 14,000+ patient ROCKET AF trial noted that both Xarelto and Pradaxa have now been shown to be roughly equivalent to warfarin in efficacy, albeit in different patient populations, but much better with regard to spontaneous bleeding complications. Which drug wins out in the estimated $15 billion US market is a toss-up, with another Factor Xa inhibitor, apixaban (Pfizer/BMS), currently about a year behind in similar trials.
Leeching the best of German science
I'm always interested in where these drugs come from in the first place and I now realize that I, and a multitude of other biologists, work routinely with the source of these exciting drugs: the competitive serine protease inhibitor, benzamidine. Anyone who has prepared cell or tissue lysates for further biochemical analysis or enzyme purification will often include a cocktail of protease inhibitors in the mix, a precaution made easy now by the sale of protease inhibitor tablets whose composition flusters my students at oral examination time. (Durn kit mentality of these kids; in my day, we made up stocks of various small molecule and peptide protease inhibitors such as benzamidine and aprotinin - and we liked it.).
The story of benzamidine traces back to a then-leading group in coagulation studies at Erfurt, Germany, led by Fritz Markwardt and Peter Walsmann, where the compound was first shown to be a serine protease inhibitor. This 1968 paper in Experientia by the pair was the first description of thrombin inhibition by benzamidine and the discovery gave rise to the use of benzamidine-agarose to purify thrombin.
Interestingly, the same group that worked on benzamidine was also responsible for the molecular studies of hirudin, the anticoagulant protein of medicinal leeches (Hirudo medicinalis) that had been used in crude form for over 100 years and is now available in the injectable recombinant product lepirudin (Refludan, Berlex). A 2007 review of hirudin by Nowak and Schrör (PDF) provides a great perspective on the history and development of the peptide and is certainly deserving of its own blogpost at another time.
A little known historical fact inherent in the term "Vitamin K" speaks to the contribution of German science to the study of hemostasis: the K refers to Koagulationsvitamin. In fact, About.com chemistry blogger, Dr. Anne Marie Helemenstine, last week commemorated the birthday of the American co-discoverer of vitamin K, Edward Doisey, who with Henrik Dam shared the 1943 Nobel Prize in Physiology or Medicine for this work.
Reading about the rich history of this field fills me with immense respect for the work of those who came before me, particularly since they were most certainly working with a small fraction of the technology available to me today. But as is common with science history, the structures and environments that gave rise to these discoveries often fall by the wayside over the passage of time. This bittersweet reality is reflected in the hirudin review mentioned above which closed with this acknowledgement of the work of Markwardt and Walsmann:
Dabigatran, the active metabolite of Pradaxa, a benzamidine prodrug.
This short review acknowledges 30 years of successful hirudin research by the scientists working at the Institut für Pharmakologie und Toxikologie of the former Medizinische Akademie (Medical Academy) Erfurt. This group made many important contributions regarding the pharmacology of hirudin and design of hirudin-like compounds as antithrombotic drugs. This institute does not exist anymore as a consequence of the termination of the Medical Academy Erfurt. This contribution is in memory of this unique research unit, directed by Fritz Markwardt, and is dedicated to him and Peter Walsmann for their outstanding contributions to hirudin research together with the best wishes to Peter Walsmann on occasion of his upcoming 80th birthday.