3 drug molecules’ winding path out of the lab

Pfizer is the world’s largest drug company, and its visibility has only grown in the past 2 years after its launch of both a vaccine and a therapy for COVID-19. Together, those products will generate about $54 billion in sales for Pfizer this year.

ENT-01 AT A GLANCE

▸ Active ingredient: Squalamine phosphate hydrate

▸ Discovered: Early 1990s by Michael Zasloff in the livers of dogfish sharks

▸ Indication: Parkinson’s disease

▸ Mechanism: Displacement of α-synuclein deposits in intestinal nerve cells

▸ Status: Completed a Phase 2b clinical trial

Less well known is the company’s role as a contract manufacturer of other firms’ drug active ingredients and finished products. And known only to a select few is Pfizer’s special ability to synthesize complex steroidal molecules at its site in Kalamazoo, Michigan.

But it was just that ability that brought the tiny biotech firm Enterin to Pfizer’s door. Enterin has hired Pfizer CentreOne, the drug company’s contract manufacturing arm, to make squalamine, the active ingredient in a therapy called ENT-01 that the biotech is developing to treat Parkinson’s disease. Enterin announced the results of a successful Phase 2b study in January and is now planning a Phase 3 study. If that trial and others go as planned, Enterin could need hundreds of metric tons of squalamine from Pfizer in Kalamazoo.

Enterin’s roots are in research that Michael Zasloff, the firm’s chief science officer, conducted in the early 1990s when he was at the drug company Magainin Pharmaceuticals. He had discovered a family of antimicrobial peptides called magainins in the skin of the African clawed frog, and the search for the peptides in other animals brought him and his colleagues to the dogfish shark.

“We decided to look at a vertebrate that had a very primitive immune system but was also very hardy,” Zasloff recalls. While studying the shark, his team found another antimicrobial, but what the researchers thought would be a peptide turned out to be squalamine, a steroid with an unprecedented structure.

So began Zasloff’s 2-decade obsession with squalamine. The molecule’s polyamine tail gives it a positive charge. Over the years, he and his colleagues determined that when squalamine enters cells, it binds to negatively charged phospholipid membranes, displacing almost any positively charged protein that is electrostatically bound there.

Magainin tried to capitalize on this property of squalamine to treat cancer and wet macular degeneration, even after Zasloff left the company for an academic job, but it didn’t find commercial success, and it closed its doors in 2009.

A few years later, a relative of Zasloff’s was diagnosed with Parkinson’s disease. In researching its causes, Zasloff learned that the neurodegeneration associated with the disease is linked to α-synuclein, a positively charged protein that can aggregate in neurons. Dense clusters of α-synuclein, called Lewy bodies, are a hallmark of the brains of people affected by Parkinson’s.

Around the same time, Heiko Braak, a researcher in Germany, proposed an intriguing theory: that in people with Parkinson’s disease, α-synuclein first accumulates in neurons of the gastrointestinal tract before migrating to the central nervous system. That would explain why one of the earliest symptoms of Parkinson’s is constipation.

“The suggestion was that α-synuclein accumulates first in the gut, leads to the symptom of constipation, and is then followed by progressive accumulation in the spinal cord, the brain stem, and then ultimately the portion of the brain that interferes with movement,” Zasloff says.

Zasloff suddenly saw a new role for squalamine—as an oral drug that would treat Parkinson’s disease by displacing α-synuclein clumps in nerve cells in the gut.

So, in 2016, he formed Enterin with Denise Barbut, a neurologist and founder of several biotech start-ups, and William Kinney, a former colleague from Magainin, to develop squalamine for Parkinson’s. The following year, the company raised $12.7 million in series A financing from New Ventures III and other investors.

Kinney knows squalamine well. In the early days of Magainin, he helped Zasloff extract it from dogfish shark liver, eking out 100 mg for each 500 kg of liver. He also developed the original synthetic route to squalamine, a 16-step process that starts with bisnoralcohol, a raw material obtained by fermenting soybean sterols.

Back then, Kinney bought bisnoralcohol from Pfizer and hired two smaller contract development and manufacturing organizations (CDMOs) to conduct the synthesis. Ash Stevens (now part of Piramal Pharma Solutions) completed the first 9 steps, and Sipsy (now part of Central Glass) completed the last 7 steps.

The synthesis worked, but by the time Kinney was at Enterin, he knew it was not adequate to produce squalamine at scale. “The yield was not optimal. The process was complicated and needed column chromatography,” he recalls. “We knew we needed to improve.”

Pfizer, the bisnoralcohol source, was a logical scale-up partner. The company has a long history with steroidal compounds dating back to Upjohn, which pioneered the commercial manufacture of corticosteroids in Kalamazoo, and has long been a supplier of progesterone, testosterone, and other steroids to drug companies worldwide.

One of Pfizer’s contributions was a new method of creating squalamine’s polyamine tail and connecting it to the steroidal part of the molecule. The synthesis that Kinney developed used an azide to ensure the correct amine reacted with the steroid. But the azide was explosive and created a toxic by-product.

“We didn’t want to use an azide at scale at the plant,” says Carl Deering, a manager with 35 years of chemistry experience at Pfizer and predecessor firms. So the Pfizer team developed an alternative method involving protecting and deprotecting groups to ensure the proper coupling.

Kinney says he was also impressed by the work of Pfizer scientists to improve the recovery of squalamine, which was taking up to 2 weeks to crystallize and another week to filter. Because of the polyamine tail, squalamine “doesn’t know whether it’s a polar molecule or a greasy molecule,” he says, and a typical organic solvent-based crystallization results in an oily mess. Some water was needed.

The Pfizer team devised an improved solvent mixture and a humidified drying technique that “got to just the right hydration state to crystallize a beautiful crystalline solid,” Kinney says. “It was a simple solution but almost artistic in its beauty.”

In one more contribution, Pfizer is now preparing to bring the entire squalamine synthesis to the Kalamazoo site. “We want to bring the whole thing in-house,” Deering says.

At present, Pfizer turns bisnoralcohol into a key starting material. The company sends this material to another Michigan firm, Bridge Organics, for conversion into what is called Compound 34, which then goes back to Pfizer for the rest of the synthesis. Taking initiative outside of their contract with Enterin, scientists in Pfizer’s bioprocess development group engineered a fermentation to yield a new starting material that will allow the firm to eliminate 2 synthesis steps.

In all, Deering says, Pfizer has eliminated 3 of the 16 steps and shortened others to streamline the synthesis. And the firm continues to cut waste and improve efficiency. Its goal is to create a synthesis that will produce squalamine at a competitive cost if it is approved. “We are 100% confident we can scale it up,” Deering says.

Kinney and Zasloff say they have been pleasantly surprised by their Pfizer partners’ flexibility and responsiveness, not traits they had expected at the world’s largest drug company. They also like the direct contact they have with scientists working on their project. “Sometimes with CDMOs there’s no chemist-to-chemist communication,” Kinney says. “Not so with Pfizer.”

And they are happy they will have a heavyweight in their corner when the time comes to submit a new drug application (NDA) to the US Food and Drug Administration. “We want to feel confident that when an NDA is submitted, all the regulatory matters are done correctly,” Zasloff says. “We feel comfortable with the Pfizer team.”

It is estimated that 400 children worldwide have progeria, also known as Hutchinson-Gilford progeria syndrome. Children with this extremely rare disease, which causes genetic, premature aging, typically die of heart disease at an average age of 14.5 years.

ZOKINVY AT A GLANCE

▸ Active ingredient: Lonafarnib

▸ Discovered: 1990s by Schering-Plough

▸ Indication: Hutchinson-Gilford progeria syndrome

▸ Innovation: A farnesyltransferase inhibitor that blocks protein prenylation

▸ Status: US Food and Drug Administration approval in November 2020; European Medicines Agency approval expected this year

In November 2020, the California-based firm Eiger BioPharmaceuticals gained US Food and Drug Administration approval for lonafarnib, the first therapy for progeria. The drug, sold under the trade name Zokinvy, is not a cure, but it increases the life spans of people with progeria by an average of 4.3 years, and often longer, according to clinical trial results.

“We now have young adults on Zokinvy who started in our trials when they were 7, 8 years old, living into their early and mid-20s,” says Eiger’s CEO, David Cory. “One of them, Sammy Basso, just turned 26 and finished his master’s degree in genetics. Patients like that are an inspiration to all of us at Eiger.”

Eiger did not set out to develop lonafarnib for progeria, Cory says. The company licensed the compound in 2010 from Merck & Co., where it was in development as a cancer treatment. Eiger planned to repurpose the drug to treat hepatitis D, the most deadly form of human viral hepatitis.

In its clinical trials, Merck had dosed more than 2,000 people with cancer with lonafarnib and shown that it can block prenylation, the attachment of a lipid to a protein. The pharmaceutical giant was using the compound to target Ras proteins, which require prenylation and are frequently mutated in human cancers.

Eiger engaged with Merck because it wanted to explore using lonafarnib to block prenylation in the hepatitis D virus life cycle, Cory says. “During this process, we were introduced by Merck to the Progeria Research Foundation, which had found that this same prenylation pathway was involved in probably the most ultrarare disease known,” he recalls.

Cory thought Eiger could bring lonafarnib to market to treat this deadly disease, progeria, while continuing to develop it for hepatitis D. In progeria, a single-letter misspelling in a gene creates an abnormality in lamin A, a protein in the membrane surrounding the cell’s nucleus. The abnormal lamin A is called progerin. Lonafarnib blocks lamin A prenylation and thus reduces the production of progerin, which is what accumulates in cells and causes rapid aging.

Eiger was able to demonstrate the mechanism quickly. Gathering participants for trials of drugs for rare diseases can pose a challenge. But the Progeria Research Foundation oversaw efforts to bring children from around the world to Boston Children’s Hospital. Eiger was able to conduct clinical trials with 62 patients, and the results demonstrated a survival benefit in children with the disease.

In 2015, Eiger hired CordenPharma, a contract development and manufacturing organization (CDMO), to work on the registration, validation, and commercialization of lonafarnib, starting with the manufacturing of material for Phase 2 clinical trials.

The company was recommended as a good manufacturer for this particular molecule, says Christopher Kurtz, Eiger’s chief technical officer. One prerequisite was finding a firm that could handle the challenges of producing a so-called orphan drug and was willing to make kilogram quantities rather than the hundreds of metric tons often required for treatments for more common diseases.

Orphan drugs are not a large part of CordenPharma’s portfolio, says Brian McCudden, managing director of the Colorado site where the work on lonafarnib is taking place. But the CDMO has multipurpose manufacturing facilities that can handle production at any scale.

The challenges of making an orphan drug are multifold. Fewer batches means fewer changes to learn lessons that could increase process efficiency, McCudden says. “So it’s a slower ramp-up curve on the efficiency with an orphan drug than you would have with a drug that you make a lot of in a year.”

Still, Robert Busch, technical lead for CordenPharma Colorado, says his team was able to make a lot of process improvements along the way. “With pharmaceutical manufacturing there’s always challenges when you scale up and you go to new equipment trains,” he says. “We discovered more critical process parameters that were unknown to Schering-Plough,” which developed the compound in the 1990s before it was acquired by Merck.

Busch adds that he was impressed with Eiger’s thorough technology-transfer package and the collaborative approach the biotech firm took to working with the CDMO. “They’ve been rolling with all the punches that come with pharmaceutical manufacturing,” he says.

For Eiger, making a drug for a disease as rare as progeria also brought with it an economic challenge. “There’s a fixed cost associated with meeting all the regulatory requirements to get drugs approved that is kind of independent of the scale at which you’re doing it,” Kurtz says. “So it’s very, very expensive for a pharmaceutical company to bring a product to market for a really small number of patients.”

Eiger does not expect Zokinvy to become profitable, even though the firm is poised to win approval from the European Medicines Agency later this year and is eyeing expansion to countries including Israel, Japan, and Turkey. Every year, roughly one child with progeria is born in the US and one in western Europe. “It’s a small population but an unmet medical need that we believed was definitely our mission and vision,” Cory says.

Cory helped develop the rare-disease drug epoprostenol, the first treatment approved for pulmonary arterial hypertension, during a 12-year career at GSK. Cory says he grew to realize that there was a world of people with rare diseases who are underserved. He has been working in biotech companies developing drugs for rare diseases since 2000 and cofounded Eiger in 2008.

The biotech has built a pipeline of molecules with “breakthrough therapy” designation, a sought-after FDA label for drug candidates for unmet clinical needs. Besides Zokinvy, Eiger is developing molecules that tackle hepatitis D, COVID-19, congenital hyperinsulinism, and postbariatric hypoglycemia.

Developing lonafarnib for hepatitis D will be a much bigger commercial opportunity for Eiger. The disease affects approximately 12 million people globally. Eiger is testing lonafarnib on more than 400 people with hepatitis D and expects initial results by the end of this year.

Vanessa Zainzinger is a freelance writer who covers the chemical industry

Neuvivo, a 2-year-old firm with a drug candidate for amyotrophic lateral sclerosis (ALS) poised for Phase 3 clinical trials, has signed on Thermo Fisher Scientific to manufacture the active pharmaceutical ingredient. Selected from a field of seven potential service firms in part for its ability to take the project from clinical to commercial scale in one location, Thermo Fisher has begun manufacturing NP001 in Florence, South Carolina.

NP001 AT A GLANCE

▸ Active ingredient: Sodium chlorite

▸ Indication: Amyotrophic lateral sclerosis

▸ Use patent granted: 2005

▸ Innovation: Cold filtration precipitation process for making purified sodium chlorite crystals

▸ Status: Company awaits US Food and Drug Administration decision on accelerated approval of the drug.

Meanwhile, Neuvivo is petitioning the US Food and Drug Administration for accelerated approval of NP001.

This forward momentum reflects the high level of interest that any late-stage candidate targeting ALS, a devastating neurodegenerative malady also known as Lou Gehrig’s disease, would generate among patients for whom there is no cure or effective treatment available.

Patient advocacy groups are especially interested in Neuvivo’s claim that the therapy arrested the disease’s progress for a significant patient segment in a 6-month clinical trial. The two drugs currently approved by the US Food and Drug Administration to treat ALS, Rilutek (riluzole) and Radicava (edaravone), have shown only modest improvements—a slight increase in life expectancy with Rilutek and attenuated loss of physical function with Radicava.

Neuvivo has taken an interesting course to Phase 3 after two unsuccessful Phase 2 trials performed by Neuraltus Pharmaceuticals, a now-defunct company. And the active pharmaceutical ingredient itself—sodium chlorite—is intriguing in its simplicity.

Neuraltus was formed in 2009 by Ari Azhir, now CEO of Neuvivo, and Michael McGrath, chief scientific officer of Neuvivo. The company was based on a patent they secured for using sodium chlorite as a treatment for ALS. They also won a patent for a novel chlorite purification process.

The two were motivated by their theory that sodium chlorite can address inflammation as a root cause of the disease by regulating a disruption in the function of macrophages. Neuraltus folded in 2019, however, after disappointing data from Phase 2 trials in 2011 and 2017.

Azhir had left the company in 2011 before the first trial. McGrath, also a professor in the Departments of Laboratory Medicine, Pathology, and Medicine at the University of California, San Francisco, had stayed on as a consultant. Both remained intrigued with their macrophage regulation theory. They acquired the molecule, formed Neuvivo, and took a closer look at the trial data, focusing on subsets of patients involved.

“We found that patients younger than 65 years old had statistical improvements in a couple of end points,” including the ability to breathe, Azhir says. “In some subsets of patients with high inflammation, we actually stopped the progression of the disease.”

The results supported the partners’ initial determination that ALS has a component of inflammation. “It was clear to me there was inappropriate macrophage activation, and there were several ways I knew to try to regulate that activation,” McGrath says. “The one that really stood out was chlorite, or a material containing chlorite.”

Explaining the chlorite approach to treating the disease, McGrath describes a regulatory loop in which phagocytic cells switch back and forth every second or so from an activated, inflammatory state, in which they destroy bacteria, viruses, and other invasive elements, to a wound-healing state. Failure to switch back to the wound-healing state would be a cause for the inflammation component of ALS, McGrath says.

Sodium chlorite acts as a prodrug that stimulates the production of taurine chloramine, which triggers the switch from the inflammatory to the wound-healing state. Chlorite, McGrath says, is converted by heme iron into hypochlorite, which interacts with taurine’s sulfhydryl group to create taurine chloramine. Taurine chloramine cannot be administered directly, he says, because it is inactivated in human plasma.

The problem was arriving at an adequate purity of sodium chlorite. “The material that you buy, reagent grade . . . it’s got stuff in it,” McGrath says. Contaminants include chlorate, chloride, and sulfite. “In reviewing the literature, you can see that no one has purified chlorite because it tends to explode.” A standard approach involves mixing peroxide with concentrated bleach. A by-product of the exothermic reaction is chlorite.

Scientists at Neuraltus steered clear of that reaction, instead devising a cold filtration precipitation process that produces sodium chlorite crystals. “I thought it was pretty cool,” McGrath says. “Our chemist took the crystals and pounded on them to see if they would blow up. He lit a piece of paper to see if they would explode.” They did not. “So, Neuraltus, as the lead inventor, received a patent on sodium chlorite. Purified sodium chlorite. People look at it and say that can’t be true. I say, ‘Great, you try to make it, then.’ ”

As Neuvivo reworked the data from Neuraltus’s clinical trials, it narrowed the patient pool from 154 people aged 32–76 to 117 people aged 40–65. After two evaluations of the data, the company determined that the molecule showed strength in the subset, which comports with the fact that ALS is a heterogeneous disease, McGrath says. Results indicated a 36% slowing of decline in patient functionality compared with the placebo group.

The question now is whether the FDA will green-light access to the drug before the completion of a Phase 3 trial. “We are working with a couple of people that used to make such decisions at FDA,” Azhir says, noting that patient advocacy groups are very active in getting experimental drugs to people with ALS. “One group doesn’t even care about side effects of drugs,” given the lack of available treatments, Azhir says.

She says there is no telling if or when Neuvivo will get the go-ahead from the FDA. But she notes that President Joe Biden signed the Accelerating Access to Critical Therapies for ALS Act into law in December, which she sees as an indication of the government’s commitment to fast action in treating the disease.

The law calls for an investment of $100 million annually for the next 5 years to support research. It establishes a public-private partnership led by the FDA and the US National Institutes of Health to speed the development of therapies for ALS and other neurodegenerative diseases.

Azhir says the government remains under pressure from patient advocacy groups, which have been compared to AIDS advocates who succeeded in expediting access to drugs.

Neuvivo is going to keep the pressure on as well, she adds. It will bring the same determination to navigating a complicated regulatory process that it brought to clinical data analysis. “We are going to put our head down and just work with FDA,” she says.

Although the regulatory pathway is uncertain, Azhir says, Neuvivo is at least assured of adequate supply of purified sodium chlorite as soon as it’s needed. “These guys came top of the line,” she says of Thermo Fisher. “The engineers understood the technology very well; the questions they asked were very thorough; the turnaround was very good. We are scientists, and we are interested in what happens to our molecule.”