Addition of newly identified compound makes naloxone more potent and longer lasting, mouse study finds

The current opioid epidemic in the United States kills tens of thousands of people each year. Naloxone, sold as Narcan, has saved countless lives by reversing opioid overdoses. But newer, more powerful opioids continue to emerge, and first responders are finding it increasingly difficult to revive people who have overdosed.

Now, researchers have found an approach that could extend the life-saving power of naloxone, even in the face of increasingly dangerous opioids. A team of researchers from Washington University School of Medicine in St. Louis, Stanford University and the University of Florida has identified potential drugs that make naloxone more potent and longer-lasting, capable of reversing the effects of opioids in mice at low doses without worsening withdrawal symptoms. The study is published July 3 in Nature.

“Naloxone is a lifesaver, but it’s not a miracle drug; it has its limitations,” said Susruta Majumdar, Ph.D., co-senior author and professor of anesthesiology at the University of Washington.

“Many people who overdose on opioids need more than one dose of naloxone before they are safe. This study is a proof of concept that we can improve the effectiveness of naloxone (make it work longer and be more potent) by giving it in combination with a molecule that influences opioid receptor responses.”

Opioids like oxycodone and fentanyl work by slipping into a pocket on the opioid receptor, which is found primarily on neurons in the brain. The presence of opioids activates the receptor, triggering a cascade of molecular events that temporarily alter how the brain functions: they reduce the perception of pain, induce a feeling of euphoria, and slow breathing. It is this suppression of breathing that makes opioids so deadly.

The molecular compound described in the paper is a negative allosteric modulator (NAM) of the opioid receptor. Allosteric modulators are a hot area of ​​research in pharmacology because they offer a way to influence how the body responds to drugs by adjusting the activity of the drug receptors rather than the drugs themselves. Co-author Vipin Rangari, Ph.D., a postdoctoral researcher in Majumdar’s lab, performed the experiments to chemically characterize the compound.

Naloxone is an opioid, but unlike other opioids, its presence in the binding pocket does not activate the receptor. This unique characteristic gives naloxone the power to reverse overdoses by displacing problematic opioids from the pocket, thereby deactivating the opioid receptor.

The problem is that naloxone loses its effect before other opioids. For example, naloxone works for about two hours, while fentanyl can stay in the bloodstream for eight hours. Once naloxone leaves the binding pocket, any fentanyl molecules still circulating can reattach to the receptor and reactivate it, causing overdose symptoms to return.

The research team, led by co-senior authors Majumdar; Brian K. Kobilka, Ph.D., professor of molecular and cellular physiology at Stanford University; and Jay P. McLaughlin, Ph.D., professor of pharmacodynamics at the University of Florida, set out to find NAMs that enhance naloxone by helping it stay in the binding pocket longer and more effectively suppress opioid receptor activation.

To do this, they screened a library of 4.5 billion molecules in the lab for molecules that bind to the opioid receptor with naloxone already lodged in the receptor pocket. Compounds representing several molecular families passed the initial screen, with one of the most promising compounds being compound 368.

Further experiments in cells revealed that in the presence of compound 368, naloxone was 7.6 times more effective in inhibiting opioid receptor activation, in part because naloxone remained in the binding pocket at least 10 times longer.

“The compound itself doesn’t bind well without naloxone,” said Evan O’Brien, Ph.D., lead author of the study and a postdoctoral researcher in Kobilka’s lab at Stanford. “We think naloxone has to bind first, and then compound 368 can step in and lock it in place.”

Better yet, Compound 368 enhanced naloxone's ability to reverse opioid overdoses in mice and allowed naloxone to reverse the effects of fentanyl and morphine at 1/10 the usual doses.

However, people who overdose on opioids and are resuscitated with naloxone can experience withdrawal symptoms such as pain, chills, vomiting, and irritability. In this study, although the addition of compound 368 increased the potency of naloxone, it did not worsen the mice's withdrawal symptoms.

“We still have a long way to go, but these results are really exciting,” McLaughlin said. “Withdrawal from opioids probably won’t kill you, but it’s so severe that people often go back to taking opioids within a day or two to stop the symptoms. The idea that we can save patients from overdose by reducing withdrawal could help a lot of people.”

Compound 368 is just one of several molecules that show potential as an opioid receptor NAM. The researchers have filed a patent on the NAMs and are working to refine and characterize the most promising candidates. Majumdar estimates that it will be 10 to 15 years before a naloxone-enhancing NAM is brought to market.

“Developing a new drug is a very long process, and in the meantime, new synthetic opioids will continue to emerge and become more and more potent, which means more and more deadly,” Majumdar said. “We hope that by developing a NAM, we can preserve the power of naloxone as an antidote, no matter what type of opioids emerge in the future.”