Stuart R. Gallant, MD, PhD
This November will be the 35th anniversary of the approval of tissue plasminogen activator (tPA, alteplase) a leading treatment for heart attack, ischemic stroke, and severe pulmonary embolism—and the first licensed treatment to be produced in Chinese hamster ovary (CHO) cells. During all three of these events (heart attack, ischemic stroke, and pulmonary embolism) a fibrin clot blocks blood flow, often leading to severe and potentially fatal damage to the person’s heart or brain or dysregulation of the person’s circulatory system.
To remove the clot, a clot-busting drug such as tPA is administered leading to clot lysis:
The clot buster cleaves plasminogen into plasmin, and plasmin degrades the clot into fragments, leading to the restoration of blood flow. This is the mirror image of hemophilia (an inherited disorder discussed in a previous post [1]). In hemophilia, the patient cannot form a clot and uncontrolled bleeding ensues. I mention that post because the clotting cascade was discussed in that post.
Clot Busters
In PharmaTopo’s earlier discussion of Factor VIII deficiency and the development of recombinant Factor VIII, we saw how Genentech benefited from decades of work on hemophilia [1]. Clot lysis was a similar situation. At the time Genentech first cloned tPA in 1982, three molecules were known to activate plasminogen to plasmin, triggering clot lysis:
- Streptokinase, discovered in 1933 from beta-hemolytic streptococci, was used to treat a number of coagulation related conditions, including heart attack [2]. While it was an effective treatment, streptokinase had two disadvantages: it tended to produce immune reactions in the patients who received it, and large clinical trials had never been completed until the mid-1980s, so high quality medical information on the best utilization of streptokinase was not available until those trials were completed.
- Urokinase (urine plasminogen activator), discovered in human urine in 1947, was produced by Abbott Laboratories and approved by the FDA in 1978 for treatment of heart attack and pulmonary embolus [3, 4]. As an activator of plasminogen, urokinase has some properties that make it more suited for peripheral clot lysis (for example, deep vein thrombosis). Interestingly, the Abbott urokinase was produced using tissue culture of human neonatal kidney cells (more on that below).
- tPA (tissue plasminogen activator), discovered in 1946 [5], was selected by Genentech as its development target. This third clot buster had certain advantages, as a pharmaceutical product. First as a human protein, it does not activate the human immune system, leading to immune reactions and immune clearance the way that bacterial streptokinase does. Second, it is bound to a fibrin clot, improving its ability to achieve open blood flow in a blocked blood vessel. (Though, the second advantage wasn’t clearly understood at the time that Genentech started the project.)
A Tale of Two Kinases—First Chapter: Urokinase
To me, there are really two interesting clot busters in this story: urokinase and tPA. Certainly, streptokinase has been important to many patients, but it is no longer sold in the US market. It had its time, and it was displaced by two products (urokinase and tPA) with better efficacy and fewer side effects. (If you are interested in streptokinase, see Sikri and Bardia [2].)
The stories of urokinase and tPA make an interesting and useful contrast in development, manufacturing technology, and clinical efficacy. So, rather than merely concentrate on one clot buster, PharmaTopo is going to present both stories. First, urokinase…
In the human body, urokinase plasminogen activator is produced predominantly in the kidney as a 55 kD protein (22 kD light chain and 33 kD heavy chain) which is enzymatically processed to a low molecular weight form (33 kD) [6]. Abbott Laboratories chose to produce urokinase in tissue culture of HNK cells (human neonatal kidney tissue donated from newborns who died from some other cause than infectious disease—donors who passed away from infection were not accepted to prevent infectious agents from being carried into the urokinase product).
| Tissue Culture In tissue culture a tissue sample is treated in the following manner: 1) dissection, followed by chopping, 2) enzymatic treatment of the cells to create a suspension of individual cells, 3) exchange into cell growth media, 4) growth in suspension, or more often, attached cell culture. Because tissue cultured cells have not been immortalized, only a finite number of passages are possible with the cells continuing to divide. Cell Culture in Recombinant DNA Technology In cell culture, a cell line is “transformed” by exposure to certain chemicals, radiation, certain viruses, or by spontaneous mutation. Transformed cells often grow at higher densities and without need to attach to surfaces (compared to normal untransformed cells), and critically, they are immortal (with no limit on the number of possible passages). Transformed cell lines are then “transfected” in a process that inserts multiple copies of the gene for a protein to be manufactured into the transformed cell. Transformed and transfected cell lines can then be banked and characterized—creating a higher degree of control, reproducibility, and productivity than is possible in tissue culture. |
The HNK cells were produced by a separated company (BioWhittaker), contracted by Abbott as a supplier—the HNK cells supplied to Abbott laboratories would ultimately cause two very interesting problems from which we can learn:
Problem 1: You Are Only as Good as Your Raw Material Suppliers
Following a November 1998 inspection of Abbott Laboratories’ manufacturing facility, FDA personnel raised concerns the HNK cells used to produce urokinase [4]. At that time, the cells were purchased from a tissue bank in Cali, Columbia. The FDA inspectors: 1) observed gaps in documentation and testing and 2) episodes of contamination detected by in-process (though not final release) testing. While no known infections had occurred in patients treated with the Abbott product, the FDA pointed out, “the likelihood that cases of infectious diseases caused by Abbokinase (urokinase), if any, would have been recognized as such and reported to FDA is probably very low. Therefore, the actual risk to patients of developing an infectious disease as a result of using Abbokinase is unknown.” Abbott was forced to withdraw the product from the market prior to making $50 million in process modifications and improvements, only returning to the market in 2003. One result of this episode is that the FDA banned importation of HNK cells.
Problem 2: US Antitrust Law and Its Limits
Abbott Laboratories’ urokinase patent expired in 1993. A small Canadian company, Microbix Biosystems, decided that the urokinase business looked like a good investment, and began a development program to launch its own urokinase product. Of course, HNK cells were critical to the entire enterprise, and Microbix approached BioWhittaker about supplying cells to the project—which it was initially happy to do. However, Abbott learned of this potential 2nd straw in the Abbott Laboratories milkshake and took action signing an agreement with Whittaker to become an exclusive supplier for Abbott. Legal discovery would later show that the purpose of the supply agreement was to “assure that other groups could not utilize the cells for production” of a completing product.
Unfortunately, the Microbix project fell apart, and the company took action under the Sherman Antitrust act against Abbott Laboratories for the contract which locked Microbix out of the BioWhittaker HNK supply. Microbix ultimately lost in court. Although they were able to show the purpose of the exclusive contract, they were not able to demonstrate that the failure of their project was directly due to Abbott’s actions, nor what amount of profits had been lost (if any) due to Abbott’s plan to exclude Microbix.
Urokinase Market Today
With all the mishegoss around urokinase production, the market has been fragmented considerably. As of 2012, the following clot busters were licensed in the US [7]:
Recently, Microbix Biosystems has been looking for investment to relaunch urokinase into the central venous catheter clearance ($330 million annually), pulmonary embolism ($90 million annually), peripheral thrombosis ($760 million annually), and stroke ($580 million annually) markets [8]. However, as of the Microbix 2021 annual report [9], Microbix had not obtained necessary funding to redevelop the product. We can think more about the clot lysis market after discussing tPA in the 2nd part of this post.
[1] pharmatopo.com/index.php/2022/03/22/pharmaceutical-design-focus-factor-viii-1-of-2/
[2] Sikri, N. and Bardia, A. “A History of Streptokinase Use in Acute Myocardial Infarction,” Texas Heart Institute Journal, Volume 34, Number 3, 2007.
[3] Degryse B. “The urokinase receptor system as strategic therapeutic target: challenges for the 21st century,” Current Pharmaceutical Design 17 (19) 1872–1873 (2011).
[4] Hartnell, G.G. and Gates, J. “The Case of Abbokinase and the FDA: The Events Leading to the Suspension of Abbokinase Supplies in the United States,” JVIR 11:841–847 (2000).
[5] Astrup T. and Permin P.M. “Fibrinolysis in Animal Organism,” Nature 159:68 1–2 (1947).
[6] Vetterlein, D. and Carlton, G.J. “Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain,” Thromb. Haemostas 49 (1) 24-27 (1983).
[7] Sidawy, A.N., et al. Rutherford’s Vascular Surgery and Endovascular Therapy, 9th Edition.
[8] Microbix. KINLYTIC. Non Confidential Information Presentation, Nov 2019.
[9] Microbix Systems Inc. Annual Report 2021, version 7.
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