PACAP Goes Its Own Way in Migraine

A new study reports that the PACAP signaling pathway is independent from the CGRP pathway, confirming the promise of PACAP as a drug target for migraine treatment.

The migraine field has seen its share of recent successes in the treatment realm, particularly with drugs targeting either calcitonin gene-related peptide (CGRP), a peptide elevated in the serum during migraine attacks, or the CGRP receptor. Yet only about half of patients improve with anti-CGRP therapies, underscoring the need for new drug targets.

Could pituitary adenylate cyclase-activating peptide (PACAP), a related peptide that leads to migraine attacks in patients and is under clinical development, fit the bill? By addressing the relationship between CGRP and PACAP molecular pathways, a new study confirms that drug development focusing on PACAP is on the right path.

Using animal models of migraine-like pain, researchers led by David Kristensen at the Danish Headache Center, Rigshospitalet (Copenhagen University Hospital), provide evidence for the independence of the PACAP pathway from that of CGRP. The investigators show that genetically modified mice that don’t respond to CGRP nonetheless respond to PACAP, exhibiting an increase in hind paw and periorbital hypersensitivity, as well as dilation of the carotid arteries. Giving wild-type mice PACAP along with an anti-CGRP antibody produced similar results.

“This is a very significant and exciting paper from a group that does very rigorous science,” said Andrew Russo, a migraine researcher at the University of Iowa who was not involved in the new research.

Russo was particularly impressed with the study’s use of two different experimental approaches – genetic and pharmacological – that converge to bolster the idea that CGRP and PACAP work via different mechanisms.

“This strongly supports the continued development and testing of PACAP-based antibody drugs. This research would predict that maybe those antibodies will work on patients who are not helped as much by the CGRP antibodies,” Russo said.

The current research builds on previous mouse work from Russo’s lab, published last year in the Journal of Neuroscience. That work provided evidence that light aversion in response to PACAP worked by a mechanism independent of that underlying light aversion from CGRP.

The new study appeared online February 7, 2022, in Brain.

Clinical observations point to a research path forward

The impetus for the new research came from observations others had made about the effectiveness of anti-CGRP therapy in migraineurs.

“A couple years back, we saw papers about antibodies targeting CGRP and the CGRP receptor,” said Kristensen. “What was quite interesting was that, if you look through the papers, at that point only about 50%-60% of patients responded to these drugs. Coming from a biological perspective, we began to discuss how this could be. The non-responders must be using something other than CGRP for getting their migraines or having their pain. I had been working with knockout animals for many years and thought it could be interesting to figure out whether or not we could create a migraine-like state in an animal without CGRP signaling,” continued Kristensen.

On the path toward that goal, the researchers, including first author Charlotte Ernstsen, a PhD student at the Danish Headache Center and University of Copenhagen, first looked at the effects of PACAP-38 (one of the two isoforms of PACAP) in wild-type mice with normal CGRP signaling.

They saw that, compared to controls receiving vehicle injection, each of three different doses of PACAP-38 injected under the skin led to hypersensitivity in response to tactile stimulation of the hind paw. This hypersensitivity was greatest one hour after the animals had received PACAP-38 but continued for up to two hours. Tactile stimulation in the region above the eyes and between the ears also produced hypersensitivity.

Looking to the knockouts

Now it was time for the investigators to turn to their knockout animals. These mice were engineered to lack Ramp1, a protein necessary for the binding of CGRP to its receptor; without this protein, CGRP signaling is impaired. As expected, the knockouts did not respond to CGRP, showing much less touch hypersensitivity and blood vessel relaxation, compared to wild-type animals.

With this validation of the authors’ knockout approach, the stage was set to address the question of whether the PACAP and CGRP systems depend on different underlying mechanisms.

This was indeed the case, as knockout animals receiving PACAP-38 showed hind paw and periorbital hypersensitivity, and carotid artery relaxation, to the same degree as wild-type animals. The knockouts were also protected from the hypersensitivity that glyceryl trinitrate, a substance that leads to migraine in a CGRP-dependent fashion, caused in the wild-type animals.

Another line of evidence to support the independence of the PACAP and CGRP pathways came from experiments where wild-type animals received a CGRP antibody one day before receiving PACAP-38. Here, there were no differences in cutaneous hypersensitivity between animals that had received the antibody and mice that received a control antibody.

The authors would also show that PACAP signaling was independent of signaling by Trpa1, an ion channel implicated in migraine. And, PACAP signaling only partially depended on other channels called ATP-sensitive potassium channels, which have also been identified as important players in migraine.

Russo said that the consistency of the new findings with those from his lab makes for a convincing case.

“The similar results with tactile sensitivity from the current paper and light aversion from our work really supports the conclusion that these two peptides should be considered as independent targets for drug therapy.”

Speaking of targets

But which part of the PACAP system should be the focus of drug development? PACAP binds to three different receptors, including PAC1, which was the target of an antibody developed by the biotechnology company Amgen. However, as reported last year, this drug failed in a Phase 2 clinical trial for the prevention of migraine.

Kristensen said that in light of the unsuccessful clinical trial, along with a lack of knowledge about how the different receptors work, targeting PACAP itself would be a better approach. This is exactly the avenue that Lilly and Lundbeck are now pursuing, with each company working on antibodies, now in Phase 2 trials for migraine prevention, against PACAP.

From a basic science perspective, Russo said that he is keen to learn from future research not only which receptors PACAP is acting on, but in which cells – with neurons, mast cells, and vascular smooth muscle cells all legitimate possibilities. This could best be accomplished, he said, by mutating or knocking out the different receptors in specific cell types of interest.

Kristensen said his group is now working to uncover which receptor PACAP is targeting. Looking at additional study endpoints and strains of mice, as well as at different species like rats, will also be important for future research. The study authors hope that other labs will be able to replicate their new findings by using such approaches.

The “dream scenario”

From a clinical perspective, Kristensen hopes that the research of the sort his group is doing will one day result in personalized medicine – what he calls the “dream scenario” in which doctors could determine which patients are most likely to respond to PACAP, and which to CGRP, before embarking on long-term treatment.

“So instead of a patient being put on a CGRP antibody for a long duration, then finding out it doesn’t work, and then being put on a PACAP antibody, maybe a clinician can begin to find out much earlier whether or not a patient would respond to one or the other,” said Kristensen. “Maybe in 10 years’ time we’ll have this test, to see which antibody you should be put on,” he added.

In the meantime, the authors hope their work highlights the importance of a productive back-and-forth between the clinic and the lab.

“The drugs we’re using in preclinical research – we know them from the clinic,” first author Ernstsen said. “Then we test them in animals – we can do knockouts and test for mechanisms – and the results can maybe be translated back to patients; clinicians get opportunities to see things that are extremely relevant to patients and take that into how they treat them. That cross-communication at multiple steps along the way is so important.”

Neil Andrews is a science journalist and executive editor of the Migraine Science Collaborative.

Reference:

The PACAP pathway is independent of CGRP in mouse models of migraine: possible new drug target? Ernstsen et al. Brain. 2022 Feb 7;awac040.