Peripheral Injection of PAP Provides Local Pain Relief in Mice

Two years ago, researchers studying acupuncture in mice reported that needle stimulation relieves pain by eliciting adenosine release and adenosine receptor activation (Goldman et al., 2010). Now, Mark Zylka and Julie Hurt at the University of North Carolina at Chapel Hill, US, have discovered another way to boost peripheral adenosine levels and achieve analgesia: injection of the adenosine-producing enzyme prostatic acid phosphatase (PAP) at an acupuncture point, a strategy they dub “PAPupuncture.” The study, published online April 23 in Molecular Pain, shows that PAP application in mice produces local analgesia that mimics the effects of acupuncture but lasts far longer.

In previous studies, the Zylka group showed that PAP can relieve pain when injected into the spinal cord of mice. “But because spinal injections are invasive, there was growing interest to see whether we could inject PAP into other sites and achieve similar effects,” Hurt told PRF. “I think having a peripheral route of injection will fast-track the protein for use in humans.”

Prostatic acid phosphatase has long been examined in the context of prostate cancer, and secreted PAP serves as a marker for the disease. But recent studies revealed that a transmembrane form of the protein is expressed in many tissues, including dorsal root ganglia (DRG) neurons. Zylka’s work has established that PAP acts as an ectonucleotidase to convert extracellular adenosine monophosphate (AMP) into adenosine, which suppresses pain by activating adenosine A₁ receptors (A₁R) on DRG nerve terminals or postsynaptic neurons in the dorsal horn of the spinal cord. Injecting the protein into the spinal cord produces days-long analgesia in mouse models of inflammatory and nerve injury-induced pain (see Zylka et al., 2008, and subsequent studies).

Adenosine, acting through A₁R, has transient anti-nociceptive effects in the periphery, too. The study that implicated adenosine in acupuncture-induced analgesia also suggested that AMP dephosphorylation by endogenous enzymes including PAP might be the rate-limiting step for adenosine production. That finding, Hurt said, “opened a window of opportunity” to use the protein for localized pain relief.

To test peripheral PAP injection, Hurt and Zylka chose one of the locations used by acupuncturists—specifically, the Weizhong point in the popliteal fossa, a richly innervated area behind the knee that is also a common site for delivery of local anesthetics. In healthy mice, a single injection of human PAP in one knee decreased sensitivity to painful heat applied to the same-side paw. The effect lasted three days—much longer than the hour-long relief previously observed with acupuncture, and similar in duration to intraspinal PAP injection. PAPupuncture had no effect on sensitivity to mechanical pain, however. Additional experiments indicated that PAP-mediated anti-nociception required A₁R and downstream phospholipase C signaling.

The investigators went on to examine PAPupuncture in persistent pain models. In the complete Freund’s adjuvant (CFA)-induced inflammation model, injecting PAP into the Weizhong point reversed heat hypersensitivity and decreased mechanical sensitivity—an effect that lasted for three days. The treatment had similar effects on pain following spared nerve injury.

“The fact that we could identify such a long-lasting effect indicated that protein stability wasn’t going to be a problem,” Hurt said. “To take a human protein, and provide long-lasting pain relief—I think that’s pretty amazing.”

Importantly, PAP’s potential was not exhausted after three days. In healthy mice, a second injection extended the reduction in thermal sensitivity by several days, and a single, high-dose treatment produced a six-day effect.

Those findings led Hurt and her coworkers to dream of a scenario where weekly PAPupuncture visits would keep at bay chronic pain from conditions such as arthritis. The question remains whether PAP injection can relieve pain in people, and, if so, for how long. To pursue those questions, Hurt and Zylka have developed an expression system for generating large amounts of human PAP (Hurt et al., 2012). Their university has licensed the use of PAP for pain treatment to a local biotechnology company.

Meanwhile, Hurt is working to learn more about how, and where, peripherally injected PAP carries out its pain-relieving effects. If the protein stays near the injection site, that might eliminate systemic side effects. Indeed, “We believe that it is staying in the fossa because we observe very localized effects; when we measured contralateral effects, we saw nothing,” Hurt said.

Researchers are developing adenosine receptor agonists as pain relievers (for a review, see Zylka, 2011). But the findings with PAP suggest that a biological therapeutic may also be a good option for tapping into adenosine anti-nociceptive pathways.

Image: Cross-section of the leg of a mouse showing the Weizhong acupuncture point, where PAP was injected. Nerves (n), blood vessels (b), and muscle (m) are labeled. Credit: Hurt and Zylka, 2012.