Daraprim: The 62 Year Old $750 Pill

In September 2015, the CEO of a new company called Turing Pharmaceuticals gained instant notoriety when he jacked up the price of a little known drug called Daraprim from $13.50/pill to $750/pill. Turing is not the first company to raise drug prices to astronomical levels, but the Daraprim situation is unique for a number of reasons. First, Turing inflated the cost of Daraprim over 50-fold overnight, making it one of the largest jumps in a drug’s price ever. Second, the company did not invent or improve upon Daraprim; this drug is 62 years old and all Turing did was pony up $55 million to acquire the rights to it.

Martin Shkreli, who unapologetically increased the cost of Daraprim by 5,500%, has been dubbed “Pharma Bro” and “the most hated man in America” by various media outlets.

Third, the company justified the huge price increase by stating that it will use a portion of the profits to invest in generating better drugs for toxoplasmosis, the neglected disease that Daraprim treats. This reverses the polarity of drug development:  most pharmaceutical companies use money they’ve already raised or earned to develop a new drug. But Turing already has the drug and claims that it will develop a better drug with the profit made from overcharging current patients. Many would argue that Turing should have used the $55 million to develop a new toxoplasmosis drug in the first place, rather than gouge current patients and further strain the US health care system. Moreover, the patients who are forced to pay for this research will not get their money back if Turing fails to deliver, which is often the case in drug research.

The world had Daraprim before Elvis had a song played on the radio!

In addition to highlighting problems with the nation’s policy on drug pricing, the situation has also taken toxoplasmosis from the shadows and into the limelight. This disease is caused by a single-celled parasite called Toxoplasma gondii. The parasite infects nearly anything with a backbone, but animals (including people) that have a healthy immune system keep the parasite in check, locking it into a latent, encysted form. If immunity deteriorates due to disease, such as HIV/AIDS, or chemotherapy, such as that given to fight cancer, the latent Toxoplasma parasites will start replicating again, leaving massive tissue destruction in their wake. If you want to learn more about Toxoplasma parasite, you can do so here.

In the center of this heart tissue is a tissue cyst filled with latent Toxoplasma parasites. These parasites can start growing again if immunity is compromised, causing rapid damage to the heart. The parasite cysts also reside in the brain, creating serious neurological problems if the disease is reactivated.

Toxoplasmosis can be life-threatening if not treated promptly. So how does Daraprim help save lives?

Daraprim is the trade name for pyrimethamine, the chemical structure shown here. $750 will buy you a single pill. A standard round of treatment for toxoplasmosis is usually a month, putting the cost at ~$22,500. Unfortunately, this drug only puts the disease into remission, so the patient typically needs additional courses throughout his or her lifetime.

Daraprim is an antifolate drug; as you may surmise, taking it leads to a depletion of the B-vitamin called folate. Folates are absolutely required by all living cells to make genetic material such as RNA and DNA. Everyone knows we need folate in our diet or else we may suffer from anemia, digestive issues, cognitive defects, or growth problems. But what you may not know is that all living things need folate – bacteria, fungi, and parasites like Toxoplasma.

Pyrimethamine targets an important enzyme in the folate metabolic pathway called DHFR (dihydrofolate reductase). By inhibiting DHFR, pyrimethamine stops the conversion of dihydrofolic acid to tetrahydrofolic acid, the latter of which is needed to make purines and thymidylate, molecules that are required for DNA and RNA synthesis. If germs can’t make DNA or RNA, they simply cannot grow anymore.

Like trimethoprim, pyrimethamine is an inhibitor of the enzyme DHFR. Sulfa drugs (sulfonamides) are also added to the treatment regimen as they serve to block the upstream step of folate metabolism, delivering a one-two punch to the pathogen.

Hopefully you can now see why Daraprim kills parasites like Toxoplasma, but you may be wondering why the drug doesn’t also kill the patient. Truth is, at high enough concentrations for a long enough time, Daraprim can kill a person. But the risk is offset for two main reasons. One, pyrimethamine binds to the parasite form of DHFR much better than to the human form of DHFR. Both Toxoplasma and human DHFR function the same way, but there are subtle differences in the enzyme’s structure between the pathogen and the patient that make it bind to the drug with different affinity. Said another way, if you consider Daraprim to be like a piece of metal, then the parasite DHFR enzyme is like a stronger magnet than the human DHFR enzyme. Two, a patient taking Daraprim is usually given folinic acid (leucovorin), which converts to tetrahydrofolic acid without the need for DHFR.

Pyrimethamine was developed by Nobel-Prize winning scientist Dr. Gertrude Elion in the early 1950s. Dr. Elion was also instrumental in the development of acyclovir for herpes viruses. I wonder what she would say to Mr. Shkreli.

The discovery of pyrimethamine as a potent anti-parasitic agent many years ago marked a great advance in the medical sciences. It is a shame we let the clay from the best minds get molded by the worst hands.

Contributed by:  Bill Sullivan, Ph.D.

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Roos DS (1993). Primary structure of the dihydrofolate reductase-thymidylate synthase gene from Toxoplasma gondii. The Journal of biological chemistry, 268 (9), 6269-80 PMID: 8454599

Sullivan WJ Jr, & Jeffers V (2012). Mechanisms of Toxoplasma gondii persistence and latency. FEMS microbiology reviews, 36 (3), 717-33 PMID: 22091606

From Herb Garden To Medicine Cabinet: Developing A New Drug for Malaria

We live on a lush planet filled with over 290,000 species of plants. Herbs are a particular type of plant that lack a wooden stem, and humans have often sampled them with hopes of finding a new food or flavoring. Sometimes ingestion of an herb produces unwanted effects, such as death. But other herbs have medicinal qualities, such as the alleviation of fever.

Dichroa febrifuga, a medicinal herb that has been historically used to treat fever, is named for its active ingredient, febrifugine.

Dichroa febrifuga is one of the most important herbs in traditional Chinese medicine, used for millennia to treat ailments such as malaria. Malaria is caused by a unicellular parasite called Plasmodium that is transmitted by mosquitoes, and a high fever is one of the trademark symptoms.

 

Malaria has a complex life cycle. After the parasites (sporozoites) are injected via mosquitoes, they travel to the liver (merozoites) and then infect red blood cells. In blood cells, they gobble up the hemoglobin as a nutrient source for replication and development into sexual stages (gametocytes) that can be taken up by another mosquito, thereby spreading the parasite to a new victim.

Malaria continues to be a devastating disease, killing up to 1 million people each year, most of whom are children under the age of five in sub-Saharan Africa. There is an urgent need for new treatments since the parasite has developed resistance to most of our anti-malaria drugs.

While effective against malaria, febrifugine is not tolerated well. What is needed is a better understanding of how febrifugine works:  how does it kill the malaria parasite? If the natural product’s mechanism of action against malaria could be identified, it would pave the way for the development of refined derivatives that are more specific against the parasite and less detrimental to patients. Alas, this is not an easy task. Over 2000 years in the making, scientists have now identified an enzyme in the parasite that is inhibited by febrifugine. That enzyme is called prolyl-tRNA synthetase.

Prolyl-tRNA synthetase is critical for the production of proteins in a cell, a process known as translation. As shown in the figure below, messenger RNA (mRNA), which serves as the “middle man” conveying the information in genes to build proteins, is read by molecular machines called ribosomes. Another type of RNA molecule called transfer RNA (tRNA) recognizes specific nucleotide sequences in the mRNA, bringing the corresponding amino acid to the ribosome so it can be added to a growing protein sequence.

The production of proteins in the cell. Proteins are composed of amino acids (the colored balls) that are connected together in a specific order, as directed by the gene coding for it. The chain of amino acids then typically folds into a three-dimensional shape so that the protein can do its job in the cell.

Aminoacyl-tRNA synthetase enzymes are needed to “charge” the tRNA; in other words, they attach the correct amino acid to the correct tRNA. When prolyl-tRNA synthetase is blocked by febrifugine, the amino acid proline does not get attached to tRNA. This leads to a buildup of “uncharged” tRNA, which is interpreted as a sign of starvation by the cell (or by the single-celled malaria parasite in this case). Proline is a common amino acid needed to build many proteins, and when prolyl-tRNA synthetase isn’t able to do its job, protein production grinds to a halt.

Even better, this enzyme is required in multiple stages of the parasite’s life cycle, knocking out both the liver and the blood forms. But as mentioned above, humans do not tolerate febrifugine very well, probably because we also have a version of prolyl-tRNA synthetase and perhaps other proteins that febrifugine poisons. Having identified this drug target is helping researchers develop derivatives of febrifugine, such as halofuginol, that act more strongly against the parasite’s prolyl-tRNA synthetase with less toxicity in humans.

Halofuginol is chemically similar to febrifugine (see above), having potent activity against malaria but less adverse effects on the host.

So how did scientists figure out that febrifugine targets prolyl-tRNA synthetase? There are several ways to identify the molecular mechanism of drug activity. In this case, the group cultured malaria in the presence of drug, forcing the parasites to evolve or die. Those that lived were less sensitive to febrifugine, meaning that they accrued a genetic change (one or more mutations in their DNA) that allowed them to persist despite the presence of the drug. This process is very analogous to the development of penicillin-resistant bacteria.

Parasites that were able to grow better in febrifugine had their genomes sequenced. Such a feat would have taken years and millions of dollars not long ago, but today it has become routine. The genome sequence of the febrifugine-resistant parasites contained a common mutation in the gene encoding prolyl-tRNA synthetase, which signaled that this enzyme plays a critical role in the drug’s action. Understanding how the parasite develops resistance also helps scientists design compounds that act on the target differently. As you may surmise, we are in a constant “arms race” with these insidious microbes, but this discovery is a step towards a victory for us.

 

Two independent parasite lines that were resistant to febrifugine, HFGR I and II, contained mutations in their prolyl-tRNA synthetase gene. In drug-sensitive parasites (Dd2), an amino acid called leucine (leu) is present at position 1444, but in the mutant parasites, a DNA change led to a different amino acid that conferred resistance to the drug.

 

Contributed by:  Bill Sullivan
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Herman JD, Pepper LR, Cortese JF, Estiu G, Galinsky K, Zuzarte-Luis V, Derbyshire ER, Ribacke U, Lukens AK, Santos SA, Patel V, Clish CB, Sullivan WJ Jr, Zhou H, Bopp SE, Schimmel P, Lindquist S, Clardy J, Mota MM, Keller TL, Whitman M, Wiest O, Wirth DF, & Mazitschek R (2015). The cytoplasmic prolyl-tRNA synthetase of the malaria parasite is a dual-stage target of febrifugine and its analogs. Science translational medicine, 7 (288) PMID: 25995223