Erythropoietin: Scientific Wonder, Biotech Success, and Doping [Draft]

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Abstract

Erythropoietin was first hypothesized in the early 20^th century and within the same century its composition, function, mechanism, and synthesis were all discovered. This rapid rise in scientific knowledge parallels the rise in technological achievement. In 1906 at the turn of the century, Professor Carnot hypothesizes a compound named “hemopoietine” to be responsible for red blood cell proliferation in animals. Less than 100 years later, Amgen, a rising biotech company, produces a DNA recombinant version of erythropoietin available on the market as Epogen®. The tale behind the discovery of erythropoietin and its production into the one of the most successful biotech ventures in the world is further complicated by later abuse of erythropoietin recombinants by professional athletes. In the world of professional sports with millions on the line for contracts, some athletes would do anything to win. They turned to tampering with red blood cells production in their own body. However, as with many biological functions there exists a thin line between legitimate training and detrimental side effects.

Introduction

A glycoprotein of 165 amino acids and 4 sugar side chains in the blood at less than 10 picomolar concentration, erythropoietin appears, at first, quite insignificant in the body. Yet this one hormone plays a vital role in red blood cell formation as well as tells a fascinating story of both scientific accomplishment and drug abuse. On one hand you have the scientific miracle of one of the world’s most successful biotech companies. On the other you have a drug being used illegally by athletes for doping. It is a hormone found naturally in your body that keeps the red blood cells going. However, as is the case for most healthy body products, too much of a good thing can turn out to be detrimental to your health. This story of fame, fortune, and notoriety starts out at the turn of the 20^th century.

Discovery of Erythropoietin

The story of this fascinating molecule starts in the early 20^th century when Paul Carnot, Professor of Medicine at the University of Paris, and his colleague Madame Cl DeFlandre published their paper on increased red blood cell production in rabbits. They noticed that injecting serum collected from bleeding rabbits, increased red blood cell production in normal rabbits. They suspected a hormone that they dubbed “hemopoietine” increased bone marrow activity and was responsible for increasing red blood cell proliferation. However, there were issues with their paper. The experimental results reported were difficult to reproduce. While Carnot had to inject less than 10mL of serum into normal rabbits to experience red blood cell proliferation, other researchers had to inject substantially large quantities of serum. This discrepancy and other possible issues, lead to the paper being ignored for the following decades. Then in 1948, following a series of reticulocyte discoveries, Bonsdorff and Salavisto remained the suspected “hemopoietine” into the modern name, “erythropoietin.”

Mechanism of Action

What Paul Carnot originally hypothesized as hemopoietine is now known to be a vital hormone in red blood cell formation. The development of different cells found in blood falls under the process named hematopoiesis (also known as hemopoiesis). All cells in the blood original arise from a common ancestor in a manner similar to human beings. In the case of blood, a hematopoietic stem cell gives rise to myeloid or lymphoid. These two classes are further broken down into red blood cells, white blood cells, and platelets. For red blood cells, hematopoietic stem cells undergo development to become a common myeloid progenitor cells. These common myeloid progenitor cells later develop into proerythroblasts. Further maturation and chemical reaction transform proerythroblasts into mature erythrocytes, also known as red blood cells.

(Insert Hematopoiesis branching chart)

The process of red blood cell maturation and formation does not occur if erythropoietin is absent. Why does the process red blood cell production halt when erythropoietin is not available? Erythropoietin is needed to bind to the aptly named erythropoietin receptor in order to set off a series of kinases and other secondary messengers. These secondary messengers and associated transcription factors are needed to activate red blood marrow and start the erythrocyte stem cell differentiation process. In addition to stem cell differentiation, erythropoietin also promotes the survival of existing red blood cells by protecting these cells from apoptosis.

Further Development of Erythropoietin

In the present day, we have the benefit of hindsight to be able to see the function of erythropoietin as well as its chemical composition. However, researchers of the past had to gather the pieces of information through various experiments. In 1950 Reissman, K. reported the link between oxygen consumption, erythropoietin production, and erythropoiesis. Interest in the compound increased as scientist collaborated to figure out the details of erythropoietin. In 1953 Erslev, A performed a similar rabbit experiment to Professor Carnot, only this time taking plasma and adjusting for more possible error. The plasma from the bleeding rabbits, when injected into the normal rabbits resulted in increased hematocrit and reticulocyte counts. This was followed in 1955 by the development of the first quantitative and specific assay for erythropoietin. Now scientists could test to determine the hormone’s concentration in various samples. The specific production site of erythropoietin was later discovered in 1961 when Kuratowska

 et al. isolated erythropoietin in isolated dog kidneys. Enough information had been compiled for erythropoietin by that point for an entry in the 1966 international reference standard. Within a span of about six decades, erythropoietin has gone form a purely speculated hormone to one with a specific index and reference.

Science continued to race ever forward as the 1970s brought another round of vital discoveries. In 1973 erythropoietin stimulation was linked to prostaglandins a diverse family of cell signaling molecules. Just four years later erythropoietin was extracted from over 1,000L of human urine through pain staking work. That same year scientists reported that the liver was the primary erythropoietin production sites for fetuses opening new door in the research of human development. With knowledge of where the hormones were produced as well as the purified compound itself; work could be started towards applying erythropoietin towards medical treatments.

Erythropoietin Involvement in Medical Treatments

Erythropoietin plays a vital factor in red blood cell formation and losing the ability to regenerate red blood cells eventually leads to anemia. This is especially serious in patients suffering from severe chronic kidney disease (CKD), as the primary producer of erythropoietin is the renal cortex.

Stages of Chronic Kidney Disease (CKD)
Stage 1	Kidney damage with normal kidney function (estimated GFR ≥90 mL/min per 1.73 m2) and persistent (≥3 months) proteinuria.
Stage 2	Kidney damage with mild loss of kidney function (estimated GFR 60-89 mL/min per 1.73 m2) and persistent (≥3 months) proteinuria.
Stage 3	Mild-to-severe loss of kidney function (estimated GFR 30-59 mL/min per 1.73 m2).
Stage 4	Severe loss of kidney function (estimated GFR 15-29 mL/min per 1.73 m2).
Stage 5	Kidney failure requiring dialysis or transplant for survival. Also known as ESRD (estimated GFR <15 mL/min per 1.73 m2).

Directly taken from government website: https://www.niddk.nih.gov/health-information/health-statistics/kidney-disease

If kidney function is limited or nonexistent, as is the case for patients undergoing dialysis[*] there will be limited to no erythropoietin being produced. The lack of erythropoietin in turn causes issues during hematopoiesis resulting in halted production of red blood cell production from red bone marrow. With no new red blood cells to replace damaged and old red blood cells, anemia kicks in. The body starts to suffer from reduced oxygen capacity as the number of mature red blood cells present to capture oxygen via hemoglobin is significantly reduced. The decrease in oxygen induces hypoxemia, a state of low arterial oxygen supply. Activities linked to oxygen such as the electron transport chain (ETC) during adenosine triphosphate (ATP) production are significantly hindered, as oxygen is not available to be reduced. Expected symptoms are weakness, fatigue, headaches, paleness, dizziness, shortness of breath, and chest pain. If severe enough, the body tissues of certain vital organs will suffer from weakness that could impede function (especially the heart).

With the rise in medical technology, patients with chronic conditions such as CKD continue to increase. Considering the fact that more patients continue to live with chronic kidney disease as well as kidney transplants, erythropoietin was needed more than ever. Unfortunately, only minute quantities of the hormone could be purified from human urine of patients suffering from aplastic anemia. This process was too inefficient for mass medical consumption. The answer to the erythropoietin shortage was recombinant DNA production.

Biotech Success Amgen and Epogen®

In 1980 a new biotech company AMGen (Applied Molecular Genetics Inc.) was founded with an ambitious CEO George B. Rathmann. The company had one specialized focus, the application of recombinant DNA technology. Recombinant DNA technology takes human genes needed for production of certain proteins and incorporates them into bacterial genes. The goal is to have the bacteria produce the human hormones as their growth is exponential and more efficient than humans.

Using recombinant DNA technology as their base model, the company applied their specialty production towards many different possible business ventures ranging from oil extraction to the production of indigo dye. Eventually they focused on medical treatments, specifically the production of necessary human hormones. They set their sights on erythropoietin and its responsible genes. In 1985, they succeed in isolating the human gene responsible for erythropoietin. By 1989, they produced their first medication Epogen®.

Medical Drug Turned into Illegal Doping

Although originally intended to treat patients suffering from anemia due to CKD, Epogen and other red blood cell proliferation inducing medications became a source of illegal sports doping.

Away From Blood Transfusions and Medication and Towards Altitude Training

With most forms of blood doping through the use of medications and transfusions now banned by international sports communities, attention was shift to a more “natural” way to induce higher levels of red blood cells in the body. The natural ways focused more on carefully planned oxygen deprivation through use of high elevation locations or artificially generated atmospheres. 

 

Erythropoietin Legacy

Despite the negative reception of erythropoietin medication or procedure abuse, the

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[*] The process of removing waste and toxins, such as urea, from the body. Usually involves hooking a patient up to a machine that will diffuse toxic solutes out of the blood.