History of Folates


The history of research into the effects of folates, which are derivatives of folic acid or vitamin B9, goes back almost 80 years to 1931. A scientist by the name of Lucy Wills identified folates as a nutrient that was needed to prevent anemia during pregnancy. Dr. Wills demonstrated that anemia could be reversed with brewer’s yeast, and in the late 1930s scientists identified folates as the corrective substance. The crystalline form of folates was first isolated from spinach leaves in 1941, and in 1943 folates were isolated in a pure crystalline form. Early on it was recognized that the active metabolites of folates were essential for numerous bodily functions, such as the ability to stimulate cell growth.


In 1957, the world-renowned scientist Dr. Charles Heidelberger synthesized the cytotoxic 5-FU. Today 5-FU is still a core component of several cancer treatment protocols, including colorectal cancer, which is the third most commonly diagnosed type of cancer today. As the side effects of 5-FU were severe, Heidelberger started work on trying to mitigate these effects without lowering the anti-tumor effect of the cytotoxic. 21 years later, in 1978, two young scientists working for Heidelberger, discovered that by mixing 5-FU with a reduced folic acid called leucovorin, the adverse events of 5-FU could be decreased and at the same time the tumour-reducing effect of 5-FU could be increased. This discovery, based on combining 5-FU with a folate, was contrary to what scientific expertise believed possible at the time, and therefore became the subject of much dispute. However, the combination gradually gained acceptance, and became the dominating combination treatment against colorectal cancer worldwide.


The pair that made the discovery of the synergistic effects by combining leucovorin with 5-FU in 1978 were Bengt Gustavsson, a cancer surgeon and scientist working at the University Hospital of Sahlgrenska in Sweden, and Paul Spears, a scientist at the University of Southern California. Paul Spears later left the work on the effects of folates on cytotoxics to concentrate on cancer mathematics. Bengt Gustavsson however continued his research on the possibility of combining folates with cytotoxics to reduce their side effects and to increase their efficacy.


Gustavsson continued his research with several eminent researchers at Sahlgrenska University, and showed that the 5-FU-LV combination was only effective on about 30% of the patients treated. The team managed to identify the biologically active key metabolite of leucovorin. The metabolite facilitates the synthesis of DNA in human cells and it is generated from leucovorin through three enzymatic reactions. Variations among individuals in the structure of these enzymes are believed to be the reason why leucovorin only has an effect on about 30% of all patients. The remainder simply cannot transform leucovorin into the active metabolite, hence there is no positive effect in 5-FU-LV treatment. Gustavsson also found that even in the patients that did react positively to leucovorin, the metabolic processes strongly decreased the effect of the compound. His conclusion was therefore that if one could synthetically produce and administrate the active metabolite directly, one would not only be able to provide a combination treatment with 5-FU for all those who cannot metabolize leucovorin, but also strengthen the effect in the 30% that could make use of leucovorin.


To successfully synthesise the active metabolite, Gustavsson started collaborating with a small company producing folates in Lugano, Switzerland. This later lead to close research collaboration with the world leader in folate synthesis, Merck & Cie (Formerly Merck Eprova), located in Schaffhausen, Switzerland, which is a subsidiary of Merck. This close collaboration has been ongoing for over 15 years and it forms one of the cornerstones in Isofol’s development. Early in the 1990’s this collaboration resulted in the production of a so called racemic form of the active metabolite. This racemic form comprised of equal amounts (50/50) of two compounds which were mirror images of each other, i.e. they were optical stereoisomers. Pre-clinical studies made by Gustavsson showed that the racemic form of the active metabolite in certain cases proved to increase toxicity of 5-FU. Overall however, the racemic form substantially increased the positive effects on 5-FU when compared with leucovorin. Gustavsson then conducted Phase I and Phase II clinical trials which proved the racemic form indicated a significantly increased positive effect on humans. After a decade of research and hard work, the results culminated in the identification of arfolitixorin.

A non-racemic form of the active metabolite was now strongly desired. After intensive research, Gustavsson identified the isomer which had a positive biological effect in humans and wanted to exclude the other, less effective and potentially toxic, isomer. Until a few years ago, it was considered impossible to generate a stable isomer of metabolite due to the compound’s high sensitivity to oxidation by air. Having spent over a decade of research and USD 35 million in total development costs, a breakthrough was finally reached in 2005. Almost 30 years after Gustavsson participated in the discovery of the positive effects on 5-FU by leucovorin, the collaboration between Merck & Cie and Gustavsson resulted in a synthetically produced non-racemic form of the active metabolite. The novel pharmaceutical substance was given the name arfolitixorin.


Bengt Gustavsson founded Isofol Medical in 2008 and Isofol Medical today holds pending patent protection for arfolitixorin across all major markets. In 2009, Isofol Medical entered into an exclusive global license-, production- and cooperation agreement with Merck & Cie to develop the use of arfolitixorin for a wide range of cancer treatments. Merck & Cie also holds the trademark and the intellectual property rights for the production process of arfolitixorin.


Pre-clinical trials have demonstrated that arfolitixorin makes 5-FU significantly more effective both in comparison with 5-FU-LV and in comparison with the racemic active metabolite combined with 5-FU. Early studies also show the same synergistic effects when combining arfolitixorin with antifolates, another class of cytotoxics frequently used in cancer treatment.

Isofol has initiated a pivotal phase 3 AGENT-study to prove the efficacy of arfolitixorin in colorectal cancer patients. Read more >>