Levamisole nonenzymatic breakdown products

The Backstory

Levamisole HCl has a great back story. We are including the tale of levamisole in our upcoming book and subscribers to our page will get the pre-print edition! For now, you should know that levamisole was created by design in the early 1960's by Paul Janssen. Dr. Janssen (1926-2003) was one of the 20th century's most innovative and inspiring pharmaceutical researchers. Levamisole was marketed for people as Ergamisol, and then it was removed from the human market but retained its popularity as a veterinary product. The drug fell out of favor as a single dewormer due to parasite resistance, but it is still used in some children with nephrotic syndrome for immune modulation.

The protagonist Paul Janssen

Dr. Janssen established an independent research company after studying physics, biochemistry, and medicine in Belgium. He became a physician and then went on to get a doctorate in pharmacology. Against all predictions at the time, he established Janssen Pharmaceuticals, and as is often the case, got a loan from Dad. Later in life he says his motivation was to show people he was right, he could succeed—and they were wrong! As luck would have it, the fifth molecule he synthesized was haloperidol. Haloperidol is used for psychosis and that first success launched his long career. He ultimately developed over 80 medications that are still deemed essential by health organizations.

Other drugs he developed that you may have heard of are fentanyl, Lomotil, levamisole,

miconazole, flunarizine, etomidate, mebendazole, loperamide, cisapride, risperidone and many more. He eventually merged Janssen Pharma with Johnson and Johnson and invented over 100 chemicals that were patented in his name. Janssen's contributions led to many breakthroughs in several fields of disease, including pain management, psychiatry, infectious disease and gastroenterology. He was a gifted and passionate scientist who revolutionized modern medicine.

From zero to current time

In the early 1960's Dr. Janssen set out to produce a dewormer for farm animals, his parameters were that it was novel, potent, safe, economical, injectable, and broadly active. He said at the time "it looked as if we were climbing trees to seek fish" because it wasn't until compound 2,722 that one worked. It didn't work well, but it turned the tide. He showed that three nematode species were removed from animals with the new compound. That it didn't work in all animals was not so great, but it was something. These results hinted about possible species differences in susceptibility to the drug. And this he says, was the beginning of a long story.

In hindsight, he reminisces about the intense activity which produced evidence to confirm their suspicion—that their initial molecule was a pro-drug. To work, the drug had to be metabolized by the liver into its active form. Only some species converted the drug into an active compound and all the laboratory derivatives he tested, except one, were inactive. They found that the active molecule had two drawbacks, high production costs and limited stability in water. Amazingly enough, in an alkaline solution hydrolysis produces an insoluble compound that was later found to be one of the main metabolites. He went on to produce levamisole.

Levamisole has many effects in the body and most are dose dependent. At very low doses, it inhibits alkaline phosphatases. It also inhibits other phosphatases. In mammalian biochemical pathways phosphokinases are controlled by "up-stream" regulators that are phosphatases and additional phosphokinases.

Serendipity again

As you probably guessed, the ability to manipulate powerful pathways in an animal are granted by phosphatases. Back then, Dr. Janssen didn't know he created a molecule with these capabilities. He was surprised when his molecule did more than kill nematodes when it was used in field studies. He saw a remarkable effect in beagles, a pre-weaning mortality rate dropped and distemper was eliminated from the colony. The field studies showed that in African school children the drug had a peculiar beneficial effect on the host-defense mechanisms. Cattle had an increased resistance to contagious bovine pleuropneumonia; sheep had a striking decrease in chlamydial infections; calves and pigs were surprisingly thrifty and free of parasitic, viral, bacterial or other infections. Ruminants and monkeys treated with levamisole were cured of herpes-like viral infections. The positive data kept coming in from around the world. They suspected the drug was responsible for immune modulation and possibly had some anti-viral effects.

Field observations weren't enough to demonstrate the amazing findings and the experiments to prove the observations started. Trial and error pointed to benefits that were related to normalizing an abnormal immune response. More experiments revealed that levamisole restores cell-mediated immune reactions by normalizing the function of phagocytes and T-lymphocytes when T-lymphocyte function is depressed. As word got out, by 1971 large-scale clinical research programs were started with the purpose of empirically testing the effect of levamisole on all of those human diseases that are definitely or conceivably caused or accompanied by anergy, or impaired cellular immune mechanism. Placebo based studies were conducted. Double blind, placebo controlled studies were done.

The moral of the story

We like what Dr. Janssen said when he opined about drug research, it is an interdisciplinary endeavor. The various disciplines of science are like the fingers on a hand-- of the same origin, but no longer in contact. He had some profound words but these are his most powerful: "Our striving to carefully distinguish those things that we think we know from those that we don't know, which is the essence of scientific thinking, should not stand in the way of serendipity. In drug research the interesting, unexpected new lead should be followed up at once, bypassing the many trivial and scientifically irrelevant bureaucratic hurdles"...he goes on. He talks about the future of levamisole, the second phase of discovery where obvious facts can no longer be denied, but the whole story isn't told yet. And the third and final phase, still to come (at the time) when the drug is described in text books, effective, and safe...widely used and promoted...but alas, it becomes an old story.

Had Dr. Janssen been alive today, I believe he would be pleased where we are taking his molecule. We are still learning about it and by experiment, continue to explain how, why and where it works. There is a possibility this molecule, with some tweaks, will give us a treatment for human neurodegenerative diseases. And other "incurable" infectious diseases.

Funny thing, we think, is that things he said are long forgotten. For example, he said that levamisole has limited stability in water, he said that over 60 years ago. "Expensive to make and unstable in water". Researchers have reported mixed results with levamisole and finally research showed it was due to non-enzymatic breakdown products. Breakdown products were produced as different researchers handled levamisole differently. That explains a lot.

Each non-enzymatic breakdown product had a different effect on lymphocyte proliferation. Proliferation was increased or decreased, or had no effect at all. That meant that the molecule is anti-inflammatory or inflammatory, immune modulating or immune stimulating and it only took a few hours to change the properties when levamisole is mixed with water. Refrigeration made no difference. Freezing might be unwise. Room temperature enhances various undesired molecules.

Pharmacists should know that a compounded formulation that is water based will not be the desired drug after just a few hours. The most important breakdown products are mercaptoethyl, phenyl imidazoidine (MPI), phenyl, dihydroimidazo, thiazole (PDT) and oxo, phenylimidazolidin, ethyl disulfide (OPED). The pH of the compounded formulation also matters! Compounding pharmacies don't worry about these things—they mix up a potion based on a request from a clinician that has not conducted an experiment. But this is one instance that they should. Compounding in water makes a useful drug a harmful one. We spent hundreds of thousands of dollars in manufacturing (to FDA standards) and many hundreds of thousands of dollars testing safety to make sure what we study is what we think it is.

Experiments are also conducted to make sure mixing drugs in an animal do no harm. Field studies are conducted to make sure what the owner may give under field conditions won't be detrimental to a treatment program. If you add drugs together, you must know the effects and the expected outcome. Sometimes people forget to consider what effect the drug may have on protozoa or the methods they use for host cell invasion. Our experiments are reported the results in our published papers.

Now you know why we spent so many research years, 24 and counting, and so much money on making a formulation that could be licensed for horses and didn't just mix things up in water. Those that use wet formulations are repeating mistakes long corrected in the literature. Those that guess at doses don't ascribe to the proven dose-dependent effects shown in the host, the disease state, and on the parasite, experiments conducted over 53 years and continue today. We are here at the bench because there is more to learn. We publish our experiments for those that are interested. You must read and understand our work to appreciate what we have done. Please be sure and read our papers as well as the background literature. Our work is built on that of Dr. Janssen and Dr. Gideon Goldstein, that's another story, for another day.

Levamisole 1

Levamisole 2