The Man Who Gave Us Epo
I learned over the weekend via this tweet from Serena Stockwell at Oncology Times that Eugene Goldwasser passed away on Friday at age 88. The biochemist and renal physiologist who spent most of his career at the Argonne National Cancer Hospital and Department of Biochemistry and Molecular Biology at the University of Chicago (web page) demonstrated that a hormone made in the kidney could increase the number of new red blood cells, a major advance in physiology. His laboratory purified the protein hormone, erythropoietin (or Epo), from sheep in 1971 and from humans in 1977.
While a remarkable discovery of its own, Goldwasser’s partnership with then-Applied Molecular Genetics (Amgen today) led to the first- and second-generation recombinant biopharmaceuticals, erythropoietin and darbopoietin, and other versions such the the PEGylated EPO, Mircera. These drugs have transformed the lives of kidney dialysis and cancer patients, raised a furor in competitive sports – cycling in particular – and have been central to some of the most robust legal wrangling and medical costs discussions of our generation.
Serena pointed me toward this obituary by Merrill Goozner, who devoted an entire chapter to Goldwasser in his 2004 book, The $800 Million Pill - sounds as though I have another book to go on my Christmas list. Goozner noted that Goldwasser, a Brooklyn native, attended the University of Chicago in the biological sciences and worked during World War II on scientific approaches against chemical warfare agents. I could find only one publication on Goldwasser’s work in this regard – not a surprise as much of the work is likely to have been classified. This 1947 paper appeared in the Journal of Pharmacology and Experimental Therapeutics (1947; 89:1-13) describing work in 1942 and 1943 on the use of 2,3-dimercaptopropanol (dimercaprol) against the vesicant (blistering) war agent, Lewisite – chlorovinyldichloroarsine or 2-choloroethenyldichloroarsine (an arsenic compound having nothing to do with this month’s arsenic adventures.).
Goozner notes that Goldwasser completed his doctoral work at Chicago after the war and worked at the Argonne Cancer Research Hospital, a US Atomic Energy Commission-supported institution on the University of Chicago campus. This 1953 NEJM editorial notes that the hospital was headed originally by Leon O. Jacobson, a physician who together with Goodman and Gilman at Yale popularized the use of one of the first synthetic chemotherapy drugs, nitrogen mustard, a therapeutic derivative of chemical warfare agents that exhibited efficacy against leukemias and lymphomas. (As an aside, Goldwasser wrote a beautiful history (PDF) of his mentor and collaborator, “Jake,” for the National Academies Press after Jacobson’s death in 1992.).
Goldwasser began investigating the regulation of the blood-forming elements and demonstrated in this 1956 Nature paper with Jacobson and colleagues that a factor existed in the bloodstream that could enhance production of new red blood cells. The studies built on work initiated by turn-of-the-century French researchers Paul Carnot, C. Deflandre, and colleagues.
To test this hypothesis, the investigators took plasma from rats that had been made anemic by bleeding (to a hematocrit <25) and transfused samples of their plasma to rats that could not make new red blood cells because of removal of their pituitary gland (hypophysectomy). The bled rats had in their plasma a transferrable factor that was able to restore the ability of the recipient animals to make new red blood cells as measured both by counting and incorporation of the radioisotope iron-59 into newly-synthesized hemoglobin. In fact, the group showed that anemic plasma from other species – dogs, rabbits, or humans – could also increase iron-59 uptake in these rats.
The sensitivity of the iron-59 uptake method allowed for better quantification and the investigators seized upon an observation made in 1929 that cobalt chloride could stimulate erythropoiesis similar to bleeding (triggered by causing destruction of heme protein, we later learned). But where was the factor coming from?
From their subsequent 1957 Nature paper:
Previous attempts to determine the site of formation of erythropoietin by surgical removal of organs and by organ extracts have been unsuccessful. With the sensitive assay methods available and the demonstration that a single dose of cobaltous chloride or an acute massive haemorrhage greatly elevates plasma erythropoietin level in rats and rabbits within 10-12 hr, we have been able to study this problem more thoroughly. We have reported that rats which have been subjected to hypophysectomy, thyroidectomy, splenectomy, adrenalectomy, and gonadectomy retain the capacity to respond to repeated phlebotomy with an increase in the plasma content of erythropoietin which is comparable with that observed in similarly bled normal animals. In addition, we have found that removal of seven-eighths of the liver, or removal of the adrenals, spleen, pancreas, stomach, or intestines, from the rat does not lessen the response to an injection of cobalt.
Having exhausted almost all known endocrine organs, the investigators removed both kidneys from a group of rats. The nephrectomized rats were then stimulated with cobalt chloride as before. When their plasma was transferred into other rats, no new erythropoiesis was observed. Either the kidney made this hormone or converted it to an active form after being made somewhere else.
After more than a decade of investigating how erythropoietin affected the maturation of red blood cell precursors, Goldwasser and colleagues succeeded in purifying the hormone from anemic sheep plasma in 1971. Then in 1977, Goldwasser, Takaji Miyake, and Charles Kung were able to purify human erythropoietin from 2,250 liters of urine from Japanese patients with aplastic anemia as detailed in this 1977 paper in the Journal of Biological Chemistry. We normally think of urine as being devoid of protein but glomerular filtration capacity will let small proteins through such as metallothionein under normal conditions and larger proteins under pathophysiological conditions (Epo is 34,000 daltons).
As Goozner reports, this world’s supply of pure erythropoietin allowed Goldwasser to provide samples to a small California biotechnology company, Applied Molecular Genetics, now Amgen. Several groups cloned the cDNA and a rich story awaits another post regarding the legal battle for erythropoietin licensing. Because the protein was most active as a glycoprotein, it had to be expressed in a mammalian cell line rather than bacteria (as had been done for recombinant insulin and granulocyte-colony stimulating factor). Amgen’s recombinant erythropoietin was approved in 1989 by the US FDA.
Goozner also pointed out several interesting points – first, that pharmaceutical companies in the the Chicago area had no interest in the hormone, leading him to search out biotechnology companies. Moreover, the University of Chicago did not seek to patent the discovery despite disclosure by Goldwasser – of course, this was before the Bayh-Dole Act which granted ownership of discoveries to investigators of federally-funded projects.
The work of Goldwasser ultimately relieved the suffering and improved the quality of life of millions of patients with cancer or anemia from other causes, particularly kidney disease. The hormone was also at the center of several blood doping scandals, particularly in cycling, and continue to shadow some of the best in the sport.
I look forward to more discussion of the significance of Goldwasser’s work in the coming days as his career is remembered.