Editor’s note: This is part two of a two-part RELIEF feature article. See part one here.
Different responses to opioid drugs
Anne Murphy, a neuroscientist at Georgia State University in Atlanta, was among the first researchers studying sex differences in pain. Like many others, she began quite by accident, when the lab she was working in ran out of male rats.
“So I grabbed a female. She wouldn’t go under anesthesia, she behaved totally differently from the males, and I had this epiphany: I realized this isn’t the same animal, when it comes to the response to opiates.” Since then, Murphy has spent her career revealing the different ways that males and females respond to opioid drugs, and why.
Traditionally, researchers had thought that opioid drugs worked better to control pain in women than in men—a conclusion based on data about self-administered morphine consumption in the hospital.
“The question was, ‘who pushes the pump more?’ Because males pushed more, that was always interpreted as the medications not working as well in men, that they need more to produce analgesia,” Murphy said, referring to relief from pain. “It’s a grossly erroneous assumption.”
It turns out that the majority of morphine’s negative side effects are exacerbated in females. “So it’s not that women stop consuming opioids because they are working so well at alleviating pain, it’s because they’re making us sick.”
In fact, opioids are not as effective in females. Murphy went on to show, in a variety of models of chronic pain, that female rats require twice the dose that males do to achieve the same level of pain relief.
“There are no sex differences in basal pain sensitivity in these animal models,” Murphy explained, referring to how much pain the animals have before being subjected to an experimental manipulation. “So this is about opiate efficacy—how well morphine is working,” she said.
To find out why the drugs worked differently in the sexes, Murphy measured levels of opioid receptors in males and females. Opioid receptors are proteins to which morphine and other opioids bind to achieve their effects.
“It was a simple question,” said Murphy, “and nobody had looked outside the hypothalamus,” a brain region that mainly regulates bodily functions.
She tested levels of opioid receptors in the periaqueductal gray (PAG), a hot zone for production of the brain’s own natural, or endogenous, opioids. “We looked in the PAG, and sure enough, males had more receptors.”
She found that this explained the improved pain relief from opioids in males. Using genetic engineering, Murphy said, “we took opioid receptors out of the PAG in male animals, gave them morphine, and they behaved like females. So, here you have a brain region, the PAG, that’s critical for opioids to block pain” and it differs in males and females.
About the same time of Murphy’s work, other researchers showed that some side effects of morphine might be due to microglia. From that research, she said, “now we know that microglia oppose the analgesic effects of opioids. So our next question was, ‘I wonder if there are sex differences in microglia in the PAG?’”
Although male and female animals showed the same number of microglia in the PAG, in female rats, morphine caused the cells to take on an “activated” state that prevented the drug from working. But, Murphy said, “we shut down microglia signaling, and morphine worked beautifully in females.” Here, the dose required for pain relief went down dramatically.
“They looked just like the males in their response to opioids, just by shutting down microglia,” according to Murphy.
Sex differences in the body’s own opioids
Not only do opioid drugs work better in males, but according to some studies, male mice and men also seem to get pain relief from endogenous opioids.
For instance, Nicole Scheff, a researcher at New York University, used a mouse model of oral cancer to test this idea. To assess pain in the animals, she and her colleagues measured the time mice spent gnawing on a dowel made of resin; gnawing is a behavior that mice are naturally drawn to do.
The mouth movements required are “similar to talking or eating in humans,” Scheff said. How long mice take to gnaw through the resin serves as an indicator of pain-related behavior—that is, behavior that might reflect a painful sensation coming from the mouth.
“Similar to human patients, males showed less pain-related behavior than females despite similar lesion severity, location of the lesion and the degree of invasiveness. Their tumors were not different, but their pain behaviors were. Males were much faster on the task,” according to Sheff. (A lesion is an area of abnormal tissue.)
Scheff next found that sensory neurons surrounding the oral cancer from both males and females were electrically hyperexcitable, suggesting they had been altered by something in the environment around the tumor (known as the tumor microenvironment, which includes blood vessels as well as different cells and molecules). Yet, only females demonstrated heightened pain behaviors.
“We thought there must be something in the microenvironment inhibiting that excitability” in males, Scheff said. She suspected that endogenous opioids might be the culprit.
So, Scheff injected cancer-free mice with fluid isolated from a petri dish of cancer cells. She suspected this fluid contained something released by cancer cells that caused pain but also caused the release of endogenous opioids in males.
She treated males with an injection of the fluid and naloxone, a drug that blocks the pain-relieving effects of endogenous and medical opioids alike. Those mice displayed much longer gnaw times than those that received the cancer fluid alone. This indicated greater pain behavior; now the endogenous opioids no longer provided pain relief.
“So that means there are already some opioids on board in males, which was unexpected,” Scheff said. “Where are they coming from?”
Further experiments led her to a type of immune cell called neutrophils. “Surprisingly, there were a lot more neutrophils infiltrating the lesion in males compared to females—a lot more, like three to four times more.” When Scheff used an antibody to inactivate all the neutrophils in male mice, their gnaw time was significantly extended, indicating their pain level had gone up.
“That sealed the deal on the idea that neutrophils could be responsible for the endogenous pain relief mechanism in males,” she said. Scheff and her colleagues hypothesize that neutrophils are activated by the oral cancer and release opioids into the cancer microenvironment to shut off pain-sensing neurons—but only in males.
While endogenous opioids might seem like an advantage for male patients with oral cancer, it might not be, said Scheff. “In males they have this mechanism on board to quiet the nerve down.” In the long run, that could mean that men are going longer without knowing they have cancer, allowing it to reach a later stage before getting diagnosed and treated.
“It’s always pain that brings people into the doctor’s office,” Scheff said. In that case, “pain is a good thing—it’s protective.”
Better pain drugs?
When it comes to sex differences in pain, said Ted Price, a pain researcher at the University of Texas at Dallas, “it totally blows my mind that males and females are this different,” particularly when the endpoint—pain perception—is remarkably similar in both sexes. In all patients, Price said, “there’s a terrible impact on quality of life, but there are completely different drivers” of the pain.
But what’s the upshot of knowing this?
“It’s going to help us to come up with drugs that are not going to continue to fail clinical trials,” said Jeffrey Mogil, a researcher at McGill University in Montreal who studies sex differences in pain.
Because new drugs have been developed based on biological mechanisms discovered in male animals, it’s conceivable that the drugs could actually work in men but not women. “We don’t know how much that has already happened,” according to Mogil. But men and women are usually grouped together in clinical trials, so the data don’t show whether drugs might actually work in one sex or the other, even when they don’t show benefits in the whole population.
“No one even looks to see if that’s why the trials have failed,” Mogil said.
Jessica Ross, a postdoctoral pain researcher studying sex differences in pain at Stanford University in Palo Alto, said that opioids are just one example of “a treatment we already know affects the sexes differently: in males it’s more potent but females have more side effects. How do we find pain treatments for women that are just as effective as in men without endangering them?”
According to Price, the important question in the pain field is, “‘Why are we failing so badly in our ability to treat chronic pain in patients?’ My view is that these biological sex differences play a huge role in driving especially neuropathic pain, and until we understand them, and use that understanding in a meaningful way for drug development and clinical trial design, we’re in danger of continuing to fail. And given what we know, there’s no good reason for us to continue to fail.”
“We can prevent this from happening,” Mogil said. “The more we follow up and find these biological differences, at some point you’ll have analgesics that will literally be prescribed for women and others prescribed for men. That’s crazy, because there’s exactly zero precedent.”
“The NIH was on the right track when they began asking us to use male and female mice in our work,” said Scheff. It may take a bit of extra effort and expense up front, “but it’s only going to help patients in the long run.”
Stephani Sutherland, PhD, is a neuroscientist and freelance journalist in Southern California. Follow her on Twitter @SutherlandPhD