Scientists at Scripps Research have demonstrated in preclinical experiments that an antibody in single-chain fragment variable (scFv) structure that binds to the potent opioid carfentanil can reverse indications of carfentanil overdose.
A synthetic opioid with around 100 times the potency of fentanyl, carfentanil is a derivative. It is frequently combined with illegal substances like heroin and cocaine to increase its euphoric effects, along with fentanyl and other fentanyl analogues, leading to several fatal overdoses.
In the study, published in ACS Chemical Neuroscience on August 3, 2023, the researchers developed a human antibody that binds very tightly to carfentanil, fentanyl and other fentanyl variants.
They demonstrated in animals that the most harmful of the variations, carfentanil, can cause a potentially fatal respiratory depression that can be reversed by providing an antibody solution soon after an overdose. According to the findings, compared to currently available treatments for synthetic opioid overdose, the antibody may be more effective and persist longer.
“We expect this antibody to be a valuable new weapon for fighting the opioid crisis,” says study senior author Kim D. Janda, Ph.D., the Ely R. Callaway, Jr. Professor of Chemistry at Scripps Research.
The study’s first author was Lisa Eubanks, Ph.D., a senior staff scientist in the Janda laboratory.
Mu-opioid receptors are neuronal receptors that opioid medications, whether they are synthetic or derived from the opium poppy, bind to and activate. Because these receptors are found on several kinds of neurons throughout the human nervous system, opioid medicines have a variety of effects, including pain alleviation and euphoria as well as respiratory depression, which causes slower and shallower breathing. The immediate cause of death in the tens of thousands of fatal opioid overdoses that take place each year in the United States is respiratory depression.
We expect this antibody to be a valuable new weapon for fighting the opioid crisis.
Kim D. Janda
Carfentanil, after fentanyl, is the next-most common synthetic opioid found in illicit drugs in the U.S. Once available legally as a tranquilizer for large animals, it was pulled from the market by the FDA in 2018 because of its potential for misuse and its potential lethality at doses measured in micrograms.
Carfentanil is so potent that the U.S. government regards it as a possible chemical warfare agent; the Janda lab’s early work on the new antibody was aimed at finding antidotes to such weapons.
The mu-opioid receptor blockers naloxone and naltrexone are currently used to treat overdoses of fentanyl and carfentanil, however even at high doses, these therapies are occasionally ineffective against synthetic opioids.
Moreover, the benefits of these treatments typically last for less than an hour after dosing potentially allowing respiratory depression from fentanyl or carfentanil (which persist much longer in the body) to resume.
Janda and his team set out to develop an anti-fentanyl antibody that would have three basic features: Firstly, it should bind with very high affinity to fentanyl and its derivatives, pulling them out of the bloodstream and thereby causing them to diffuse out of the brain as well; secondly, it should persist in the body so as to provide reasonably long-term protection; and thirdly, it should be able to get quickly into the bloodstream and be delivered by a simple intramuscular injection, which requires no special training.
To obtain antibodies, Janda and his team vaccinated rodents with a molecule they designed that would elicit antibodies against carfentanil, fentanyl and variants. The rodents were engineered to produce human antibodies (rather than rodent antibodies, which would trigger an unwanted immune response if administered to humans).
The researchers were able to isolate several of the antibodies that bind to carfentanil with extremely high affinity and bind to fentanyl and a number of other fentanyl derivatives quite robustly. They then selected the most potent of these antibodies, modified it to be more lightweight (so that it would get quickly into the bloodstream), and further altered it so it would persist in the blood for days.
Tests in rodents showed that the optimized scFv, dubbed C10‐S66K, did indeed have a powerful effect at reducing carfentanil’s actions on the brain reversing carfentanil-driven respiratory depression when injected 15 minutes after a heavy carfentanil exposure. The effect after about 40 minutes was stronger than naloxone’s, was still increasing after two hours, whereas naloxone’s peaked at 30 minutes, and swiftly declined.
As part of the study, the collaborating laboratory of Ian Wilson, Ph.D., Hansen Professor of Structural Biology at Scripps Research, used X-ray crystallography to determine the near-atomic resolution structures of carfentanil and fentanyl-bound C10‐S66K.
The antibody should bind to several fentanyl variants well, according to these structural findings, without interfering with the actions of other helpful opioid compounds like naloxone and naltrexone.
The U.S. Food and Drug Administration (FDA) has approved a full length IgG version of this antibody termed CSX-1004 for clinical trials, slated to begin this month for the prevention of fentanyl overdose.
