Anesthesia’s Awakening

It was business as usual for Brian Weiser. It had been several minutes since he’d administered propofol, a potent general anesthetic, and one of his charges just refused to go under. Most didn’t budge in response to a quick tap. But one quickly darted away.

“He’s slowing down,” Weiser told his supervisor, University of Pennsylvania Perelman School of Medicine anesthesiologist Roderic G. Eckenhoff. “But he’s still got a startle reflex.”

So Weiser made a few adjustments, filling a widemouthed glass pipette with a solution of propofol in pond water and adding some to his charges’ plastic dish. “There’s always one resistant tadpole,” he said, gently tapping the petri dish on his laboratory benchtop.

Medicinal chemists are used to using model species to test their compounds. Some go with mice, others rats, still others zebrafish. But for people like Eckenhoff and Weiser, a Penn pharmacology graduate student, the search for drugs is, and requires, a different animal. Such hunts typically start with a biological target known to be implicated in a disease. But in anesthesia, those kinds of target-based approaches are in their infancy, which is part of the reason for the tests on tadpoles. In anesthesia, target discovery and drug discovery are two sides of the same coin.

Each year, more than 40 million patients in North America alone undergo a procedure that requires anesthesia, according to the American Society of Anesthesiologists. The earliest general anesthetics, ether and chloroform, have given way to newer agents. Anesthesia is the safest it’s ever been. But that doesn’t mean anesthetics are risk-free drugs. “One of the problems with anesthetics is that they have a number of side effects,” Eckenhoff says. “That’s why you see a specialty of anesthesiology, for managing the side effects, for monitoring for them, for keeping patients safe.”

Highly trained people administer anesthetics because they affect breathing, blood pressure, and heart function. A growing body of evidence suggests anesthetics could also have cognitive effects long after they have left the body. In particular, children who have been under general anesthesia multiple times by the age of four appear to carry a higher risk of developing learning disabilities. In animal tests several teams have shown that commonly used anesthetics boost aggregation of amyloid-β, the peptide thought to be the culprit behind Alzheimer’s disease.

“Safety is an interesting issue with anesthetics,” says R. Adron Harris, a pharmacology expert at the University of Texas, Austin. One way to assess drug safety is by calculating what’s called the therapeutic index, he says. “That’s the ratio of the dose that would kill you to the dose that gives you the desired effect.”

For most drugs that number is 10 or greater, with some antibiotics having ratios of more than 100. But anesthetics clock in lower, with several below 5. “By that measure, they’re quite dangerous,” Harris says. “But in fact mortality due to anesthesia is pretty low. That speaks to the skills of anesthesiologists,” he says. He thinks a new anesthetic with a better therapeutic index would be especially welcome in regions where the cost of having many highly skilled anesthesia professionals is prohibitive.

“Anesthetics are imperfect,” says Edmond I. Eger, a professor of anesthesia at the University of California, San Francisco Medical Center. “But it’s possible they can be improved.”

Back at Penn, the stubborn tadpole finally went under. Satisfied, Weiser got ready to reverse the propofol’s effect. With the pipette, he removed as much of the propofol solution as possible and disposed of it, being careful not to disturb the immobile amphibians. Then, with a whir of the pipette’s electric motor, he added fresh pond water to the dish and once again began to wait.

Tadpoles have been a mainstay of anesthesia research for more than a century. They are relatively cheap as far as lab animals go, and their response to anesthetics is an excellent mimic of humans’. In fact, tadpoles helped bolster one of the earliest theories about how anesthesia worked. As late as the 1980s, most practitioners thought anesthetics interacted nonspecifically with the lipids in nerve cell membranes. This theory grew out of the work of researchers Charles Ernest Overton and Hans Horst Meyer, who at the turn of the 20th century independently observed that anesthetics that were more soluble in oil had greater anesthetic power in tadpoles.

This nonspecific mechanism inspired a decidedly empirical approach to the search for new anesthetics, says Neil L. Harrison, a pharmacologist at Columbia University. “Rather than anesthetics being rationally designed, they were discovered semi-serendipitously,” he says. “People in drug companies used to get some mice and inject them with whatever happened to be sitting on the shelf.”

But in 1984, Imperial College London biophysicists Nicholas P. Franks and William R. Lieb learned something that opened new avenues for anesthesia drug discovery. The pair found that at clinically relevant concentrations, anesthetics inhibit the glowing action of the firefly luciferase protein, suggesting anesthetics exert their effects by interacting with proteins (Nature, DOI: 10.1038/310599a0). As with many results that run counter to convention, the work didn’t catch on at first. “It was the beginning of quite a struggle,” Franks says. “It really took many years thereafter before the work was thoroughly accepted.”

It wasn’t until the 1990s that the tide began to turn, and today the majority of researchers and physicians think proteins are key to anesthetic action, Eger says. “Nick Franks really caused a sea change in the thinking about how anesthetics work,” he adds.

After Franks’s and Lieb’s work, more researchers started looking for the protein targets of anesthetics. It’s an important quest en route to better anesthetics, explains Richard Olsen, a pharmacologist at the University of California, Los Angeles. “Students of anesthesia know that you can define five or six different aspects of the anesthetized state,” including immobility, amnesia, analgesia, unconsciousness, and sedation, Olsen says. Each of these effects could be due to the anesthetic interacting with a different protein. If that were the case, it might be possible to tune anesthetics for different applications simply by tweaking affinities for target receptors, Olsen adds. Olsen consults for multiple companies developing sleep aids.

Researchers can’t yet do what Olsen suggests because they still don’t fully understand how anesthetics work. For the injectable anesthetics propofol and etomidate, the picture is reasonably clear. They largely act at one target, the γ-aminobutyric acid type A (GABA_A) receptor. GABA_A is a chloride ion channel with more than a dozen subtypes in different parts of the nervous system. When the receptor responds to the neurotransmitter GABA, it admits chloride ions and prevents the neuron from generating an electrical signal. Anesthetics enhance the action of GABA, in essence quieting brain function.

Knowing that the injectable anesthetic alphaxalone acts on GABA_A is helpful for anesthesiologist Alex S. Evers and chemist Douglas F. Covey of Washington University in St. Louis. They are working toward next-generation versions of alphaxalone, a steroid anesthetic pulled from the market in the 1980s because of allergic reactions to its lipid formulation. Steroid anesthetics have several advantages over other classes—for example, they don’t lower blood pressure or suppress breathing, Evers says.

The St. Louis team has had a long-standing collaboration to explore the chemical features of steroids that lend anesthetic activity while developing water-soluble steroids that will be easier to formulate. “Recently we think we’ve learned enough to make a better steroid anesthetic,” Covey says. Covey and Evers both advise Sage Pharmaceuticals, a start-up with an interest in anesthetic steroids.

The pair’s most advanced alphaxalone analog, a sterol with a nitrile group on its D ring, has a structure consistent with criteria that Covey and Evers have learned are important for inducing anesthesia (J. Med. Chem., DOI: 10.1021/jm2002487). It also has comparable activity to alphaxalone in mice. “We’ve filed a provisional patent application on the compound,” Covey says.

Much of the development of new anesthetics centers on the injectable class. That’s because the mechanistic picture is far more complicated for inhaled anesthetics. Most physicians and researchers who spoke with C&EN say that between five and 20 proteins may be targets of inhaled anesthetics. Those targets may include GABA, but also certain ion channels that respond to the neurotransmitter glutamate and the amino acid glycine, as well as certain potassium ion channels. “Some people will say they know what’s going on” with inhaled anesthetics, Eger says. “But I don’t believe they really do.” Eger disclosed a consulting relationship with Baxter Healthcare, a maker of inhaled anesthetics.

Eckenhoff and his collaborators at Penn are just one of a list of teams trying to make sense of anesthetics’ mechanisms. Penn chemist William P. Dailey’s group provides light-activated versions of inhaled or injectable anesthetics, such as m-azipropofol, to Eckenhoff, who can then use ultraviolet light to covalently link the anesthetic to any proteins it binds, whether in vitro or in a live tadpole (J. Med. Chem., DOI: 10.1021/jm1004072). Every target a light-activated anesthetic detects is subjected to follow-up tests to confirm that they are relevant to the mechanism of anesthesia, Eckenhoff says.

Fishing for targets is one thing, but using that knowledge gained to search for new anesthetics, whether injected or inhaled, is harder than it seems. Little three-dimensional structural data on anesthesia-receptive binding pockets exist. One exception is an X-ray crystal structure of an ion channel from blue-green algae, a homolog of a channel thought to mediate anesthesia in humans, published earlier this year (Nature, DOI: 10.1038/nature09647; C&EN, Jan. 24, page 31). What’s more, most of the ion channels that anesthetics act on are tough to obtain in purified form, which makes it challenging to develop the screens drug companies traditionally use to find leads.

But Eckenhoff pressed on anyway. In 2009 he reported that the horse version of the protein apoferritin, which is cheap and commercially available, could mimic anesthetic binding to at least one receptor, GABA_A (J. Biol. Chem., DOI: 10.1074/jbc.M109.017814). But to measure compound binding to apoferritin, his team was using a time-consuming isothermal titration calorimetry technique that was unlikely to find use in high-throughput screens.

Enter Ivan J. Dmochowski, a Penn chemist with expertise in both ferritin and fluorescence. Together, he and Eckenhoff identified the first fluorescent general anesthetic, 1-aminoanthracene (Proc. Natl. Acad. Sci. USA, DOI: 10.1073/pnas.0810590106). The molecule immobilizes tadpoles reversibly, and with fluorescence microscopy, the team can see its locations in a tadpole body. What’s important for screening is that the molecule’s fluorescence goes up dramatically when it binds ferritin and goes down when another molecule, such as an anesthetic, outcompetes it for ferritin’s binding site, he adds. This means that by monitoring 1-aminoanthracene’s fluorescence level, researchers can tell when another molecule binds tightly to ferritin and can single it out for further testing.

To make a truly high-throughput screen, the Penn team started working with the National Institutes of Health Chemical Genomics Center (NCGC) in Rockville, Md. There, specialists miniaturized the screen so it would run on plates with 1,536 wells apiece and made sure the screen worked as intended by testing it on a commercially available collection of 1,280 pharmacologically active compounds (PLoS One, DOI: 10.1371/journal.pone.0007150). “That collection is meant for validating screens,” says David Maloney, a chemistry team leader at NCGC. “If you don’t get hits from that, you’re not likely to get hits at all.”

The NCGC team has now hunted for anesthetics among 375,000 compounds in NIH’s Molecular Libraries Small Molecule Repository. Maloney says one family of small molecules is under additional investigation but declined to disclose the molecules’ structures. Promising compounds from NCGC’s medicinal chemistry effort get shipped across the Capital Beltway and up Route 95 to Penn, where Eckenhoff’s team tests them directly in tadpoles to verify that the hits truly mediate anesthetic action. The best of those compounds go into mice. “Our approach is unusual in that we’re lacking the standard in-house structure-activity relationship tests,” says Anton Simeonov, NCGC’s chief of biology. “Our SAR assay is tadpoles.”

“It’s a little like going back to the old-school way of doing drug discovery,” Maloney adds.

But old school doesn’t mean outdated, Simeonov says. The assay has the potential to uncover anesthetics with completely new chemical scaffolds, he says. “We might even be able to find a target that’s been overlooked,” he adds.

Sometimes, it isn’t finding a target that elicits anesthesia that leads to a new drug candidate. Biophysicist Keith W. Miller, anesthesiologist Douglas E. Raines, and colleagues at Harvard Medical School and Massachusetts General Hospital (MGH) learned that finding the target responsible for a side effect is just as important. They’ve developed a next-generation version of the intravenous anesthetic etomidate that is currently in preclinical studies.

Etomidate, first marketed by Janssen Pharmaceuticals, is a product of the serendipity school of anesthetic discovery, Raines says. Etomidate is an imidazole, and Janssen “made dozens of these compounds because many imidazoles are antifungal agents.

“Of these dozens of compounds, several of them caused the rats to go off to sleep,” Raines says. The company ultimately chose to develop etomidate as an anesthetic because of its high therapeutic index, he explains.

Etomidate is part of the standard anesthesia tool kit, but it’s not always the best option because of a side effect: It interferes with steroid synthesis, Miller says. “That’s bad news for certain patients, especially patients in intensive care,” who tend to be on narcotic painkillers that exacerbate the steroid imbalance, leading to complications or even deaths, he adds.

Miller, Raines, and their collaborators have studied etomidate for years, with techniques including light-activated analogs and animal assays. “One of our hopes was to design out that unfavorable action while retaining action at GABA_A,” etomidate’s main anesthetic target, Miller says.

Biochemists knew that etomidate blocks the action of 11β-hydroxylase, a cytochrome P450 enzyme that’s critical for making multiple steroids. Its interaction with etomidate is not well understood, “but there are X-ray structures of other members of that superfamily of enzymes with imidazole-containing drugs bound,” Raines says.

Experts in computer modeling helped the Harvard-MGH team see what the etomidate-hydroxylase interaction might look like. They realized that the drug’s basic nitrogen was likely coordinating to the hydroxylase’s heme group, which might have been leading to the blockade of steroid synthesis.

That result pointed to a clear course of action, Raines says. “We switched out that nitrogen and put in a carbon. That dramatically lowered the affinity for the hydroxylase but maintained affinity for anesthetic targets,” he says. The team called their resulting compound carboetomidate, and the molecule is now in preclinical studies (Anesthesiology, DOI: 10.1097/ALN.0b013e3181cf40ed).

Tweaking etomidate’s ester moiety to make it break down more quickly in the body also solves the steroid synthesis problem (Anesthesiology, DOI: 10.1097/ALN.0b013e3181ae63d1). The resulting molecule is called MOC-etomidate, Miller says.

To move carboetomidate or MOC-etomidate to the point where drug companies might want to spend millions to get them to bedside, Raines has cofounded a company called Annovation BioPharma, together with the Partners Innovation Fund, a venture capital group launched by MGH and Brigham & Women’s Hospital.

Raines thinks the lessons of the etomidate story could be widely applicable in anesthesia research. “As we learn more about what the targets are for side effects—for instance, the effect of suppressing breathing—we might be able to tweak molecules to reduce binding to those targets,” he says.

Though the efforts at Harvard, Penn, and Washington University have uncovered drug leads, the discovery programs are small. Few pharmaceutical or biotech companies have anesthetics in their late-stage pipelines. “We’re not talking about the search for cancer drugs here,” Franks says. “The drive to improve anesthetics is nowhere near as strong.”

But some argue that research in anesthetic discovery and target discovery remains important because it could provide more than new general anesthetics. For example, it could lead to drugs for related applications, including memory drugs, sleep aids, and sedatives, says Beverley Orser, an anesthesiologist at the University of Toronto. New molecules that induce a state of anesthesia also have potential as tools to learn more about the mind, Orser adds. “I would love to see more chemists working on this problem,” she says.

“The next big set of questions will be about what brain regions and networks of circuits are involved in producing anesthesia,” agrees Columbia’s Harrison. “The ‘how’ of anesthesia may be partially solved, but certainly not the ‘where,’ ” he says.

Eventually, Weiser’s tadpoles were swimming in their dish as though nothing had happened. What’s most remarkable about that is the fundamental mystery that it exemplifies, Dmochowski muses. All of biology, from plants to tadpoles to people, can be reversibly immobilized with just about the same concentration range of drug, he explains. “It’s almost a defining characteristic of life that we can be anesthetized,” he says. Because of these similarities, anesthetic target discovery might someday point researchers to the proteins and processes behind consciousness itself. “It’s bigger than anesthesia,” he says. “It’s one of the last frontiers in pharmacological research.”