“It was pretty embarrassing,” said Gary Bennett of McGill University, Montreal, Canada. It was 2005, and his group had been working on a rat model of paclitaxel-induced peripheral neuropathy. Building on the pioneering animal studies of Guido Cavaletti, University of Milano-Bicocca, Monza, Italy, and Jon Levine, University of California San Francisco, US, Bennett was using the animals to try to replicate the debilitatingly painful sensory neuropathy that leads so many cancer patients to limit or discontinue chemotherapy.
Bennett and coworkers were looking for lesions in the animals’ nerves that would explain the pain, but they could not find any. “So we got desperate, and we did quantitative electron microscopy of the nerves,” he said.
“We pasted [the pictures] together, in a montage that was three feet across,” Bennett continued. “While counting the fibers, [then-postdoctoral fellow] Sarah Flatters noticed that something was wrong with the mitochondria. Lots of them were swollen, some had vacuoles, and some had a disrupted membrane and looked like they’d blown up.
“We knew this [result] was real,” he said, and not an artifact of tissue fixation, because the mitochondria in the adjacent Schwann cells were normal. And, when rats were left to recover and their pain disappeared, so did the abnormal-looking mitochondria (Flatters and Bennett, 2006).
Bennett followed up the discovery of the damaged mitochondria with studies showing that exposure to paclitaxel and oxaliplatin caused energy production to drop precipitously in the neurons and to remain chronically low for weeks after chemotherapy had stopped (Zheng et al. 2011).
Once mitochondria cease producing ATP, axons depolarize, because there is not sufficient energy to power the sodium/potassium pump, explained Bennett. In his experiments, depolarization was followed by spontaneous discharge in 20 to 30 percent of sensory fibers (compared to near zero in normal animals; see Xiao and Bennett, 2008), which could account for the pain of chemotherapy-induced neuropathy.
Further supporting the case for mitochondrial dysfunction, the researchers found that very low doses of any of three mitochondrial poisons would worsen the pain and the spontaneous discharge in chemotherapy models (Xiao and Bennett, 2012). And Bennett and others showed that olesoxime or acetyl-l-carnitine, two drugs that protect mitochondria, could prevent or reverse neuropathy in animals exposed to chemotherapeutic drugs (Xiao et al., 2011).
Bennett’s work, along with that of others, has cast mitochondria in a central role in the genesis of chemotherapy-induced peripheral neuropathy (CIPN; for a recent review, see Bennett et al., 2014). Still, researchers and clinicians face many unanswered questions about the cause of, and potential solutions to, this debilitating side effect of cancer treatment. Despite success at forestalling CIPN in animal models, there are currently no effective preventive strategies in clinical use, and only one moderately efficacious treatment for pain. But if Bennett’s theory is correct, some new mitochondria-targeted therapies in the pipeline might not only mitigate pain far more effectively than current treatments do, but also might prevent and reverse the neuropathies.
Neuropathy is more than a bothersome side effect of cancer treatment—it may be life threatening: CIPN is often painful enough to force patients to reduce dosing or discontinue treatment, even in the face of death.
Nerves at risk
CIPN, most commonly associated with pain in the feet and hands, can cause constant or intermittent stabbing or shooting pain, as well as burning, tingling, numbness, and cold or mechanical sensitivity. The classes of chemotherapeutic drugs associated with CIPN include platinum analogs, anti-tubulins, and proteasome inhibitors. Different treatments evoke slightly different constellations of symptoms, but pain is usually the most troubling for patients. Moreover, the pain and other symptoms can be permanent, even when cancer is cured.
Under aggressive chemotherapy, the rate of neuropathy with some agents approaches 100 percent. Standard doses are a compromise between killing tumors and avoiding neuropathy. Anywhere from 20-70 percent of patients will develop painful neuropathy, depending on the drug used, doses given, and exact treatment regimen. For the most part, the drugs affect sensory nerves, while motor neurons and Schwann cells appear to be unscathed (Xiao et al., 2011), a selectivity that is not completely understood.
Bennett believes that the cascade that leads to CIPN begins when chemotherapy drugs damage enzymes in the electron transport chains within the mitochondria in the sensory nerves. The resulting energy deficit leads to nerve discharge and a selective degeneration of distal nerve endings that shows up as a loss of intra-epidermal nerve fibers. That initial damage also leads to formation of superoxide and other reactive oxygen species (ROS), which can further impair the mitochondria and attack other parts of the cell as well.
Paul Fernyhough, University of Manitoba, Winnipeg, Canada, suggests that other forces also operate in CIPN to cause neuropathy. For example, the anti-tubulin drugs cause the network of microtubules in neurons to become inflexible, he said. This inflexibility could slow delivery of neurotransmitters, growth-associated proteins, and “a whole slew of [other] factors” to their destinations at the nerve endings, he noted.
“All kinds of mechanisms have been postulated,” said William Robinson of Tulane University School of Medicine, New Orleans, US. “Some suspect that there is a direct toxic effect from the platinum drugs.”
“Cisplatin, like oxaliplatin, gets into the cell bodies of the sensory nerves in the posterior sensory ganglia and can react with DNA in the nuclei of these cells,” said Stephen B. Howell of Moores UCSD Cancer Center, University of California, San Diego, US. “It is damage to these nerve cell bodies that is thought to account for much of the sensory neuropathy produced by these drugs, although other mechanisms may contribute as well.”
Patrick Dougherty of the University of Texas MD Anderson Cancer Center, Houston, US, presented evidence for his own hypothesis of chemotherapy-induced neuropathy at the 2013 Society for Neuroscience meeting. His work suggests new details about how chemotherapy drugs instigate degeneration of intra-epidermal nerve fibers and cause pain.
According to Dougherty, the cascade that leads to production of mitochondrion-damaging ROS begins when chemotherapy triggers innate immunity via interactions with toll-like receptors. These receptors normally become activated when they detect toxins from bacteria and viruses. “We have new data suggesting that if you block the toll receptor signaling, you don’t see the superoxide response or the generation of neuropathy,” he said, adding that he has shown this using paclitaxel, oxaliplatin, and bortexomib, and has indirect evidence for vincristine, while others have shown the same effect for cisplatin. The results with paclitaxel were recently published (Li et al., 2014).
Other cells besides neurons may participate, too. Most recently, endothelial cell activation and immune cell infiltration into nerves were implicated in ROS production—and pain—in an animal model of vincristine-induced neuropathy (Old et al., 2014).
Treatments: lots of smoke, not much fire
Work in animal models and in the clinic has generated many hypotheses, lots of potential targets, and many clinical trials, but so far no successes in preventing or reversing CIPN in large, controlled trials in people. In recently published clinical guidelines, the American Society for Clinical Oncology recommends no agents for the prevention of CIPN (Hershman et al., 2014).
Consistent with an important role for reactive oxygen species in CIPN, antioxidants reduce pain in animal models and prevent the mitochondrial damage seen in the early experiments (Ghirardi et al., 2005; Flatters et al., 2006; Xiao and Bennett, 2008). However, antioxidants, notably the nutritional supplement acetyl carnitine, did not prevent CIPN, and may have even made it worse, in a large, randomized trial of women with breast cancer treated with paclitaxel (Hershman et al., 2013).
Bennett suggests that the problem is not with the theory, but may lie with the potency of acetyl carnitine. “In humans the doses are several grams a day—an amount that is challenging for intestinal uptake,” he said.
Tricyclic antidepressants and anticonvulsants, historically used to treat neuropathic pain conditions, have lacked efficacy against CIPN and have side effects that interfere with prolonged use (Kaley and Deangelis, 2009)
The only drug approved to treat CIPN pain is the anti-depressant, duloxetine, which was shown effective in a Phase 3 clinical trial (see PRF commentary on Smith et al., 2013). Reduced pain was seen in 59 percent of treatment arm patients versus 38 percent on placebo.
Study co-author Ellen Lavoie-Smith of the University of Michigan School of Nursing, Ann Arbor, US, called the results “practice changing,” and added, “I would absolutely prescribe this drug for oxaliplatin-induced CIPN.”
“It's the first positive result for CIPN, and that is important,” said Bennett. “But the degree of pain relief is not overwhelming.
In the pipeline
A cornucopia of drugs in development for preventing or reversing neuropathy, and for controlling pain, includes antioxidants, neuroprotectants, dietary interventions including fish oil and amino acid supplements, anticonvulsants, cannabinoids, opiates, and other analgesics, an antibiotic, cryotherapy, acupuncture, and more.
Daniela Salvemini of St. Louis University, Missouri, US, and collaborators have been developing highly selective adenosine A3 receptor agonists that block and reverse both pain and neuropathy, including CIPN, in animal models (Chen et al., 2012; and see PRF related conference report). Further, the A3 agonists do not interfere with anti-tumor activity of the chemotherapeutic agents, said Salvemini.
In fact, the adenosine agonists have the added advantage, in the case of cancer, of having their own anti-tumor activity, and they are being tested by other investigators in Phase 2 trials against cancer (as well as against arthritis), said Salvemini.
Salvemini also said the compounds can synergize with current anti-pain medications, such as morphine, amitriptyline, and gabapentin.
Mechanistically, “We have evidence that A3 agonists block, and protect against mitochondrial dysfunction,” thus supporting the mitochondrial hypothesis of CIPN, said Salvemini.
She and coworkers have also been investigating a different potential agent, targeting a major reactive nitrogen species, peroxynitrite (reactive nitrogen species are damaging in much the same way as are ROS). In two recent papers, the researchers showed, by administering a compound that catalyzes peroxynitrite’s decomposition, that peroxynitrite is involved in the development of mechanical hypersensitivity normally induced by bortezomib, oxaliplatin, and paclitaxel. In addition, they went on to implicate, for the first time, peroxynitrite as “one of the key players in the development of mitochondrial dysfunction in all three settings,” (Doyle et al., 2012; Janes et al., 2013).
Meanwhile, Ahmet Hoke, Johns Hopkins University, Baltimore, US, is searching for new drugs against CIPN by adding taxol to cultured nerve cells and then testing a wide variety of compounds to see if they can prevent degeneration of neuronal cells in vitro. Ethoxyquin, a quinolone-based antioxidant that has been used as a preservative and as a pesticide, prevented damage in the nerve cells and in animal models about 70 percent of the time, he said.
An investigation of its mechanism of action showed that ethoxyquin inhibited binding of two proteins to heat shock protein 90 (HSP90) (Zhu et al., 2013). Hoke said he is working on figuring out how the two proteins mediate taxol’s toxicity in a quest for more—and possibly more specific—drug targets.
In a small, 100-patient pilot clinical trial, Dougherty and his collaborators recently tested a compound, minocycline, that blocks inflammatory cytokines, which are also implicated in CIPN (Wang et al., 2012).These cytokines are released by the innate immune system following interactions between chemotherapy drugs and toll-like receptors. Dougherty has not completed analysis of the results of co-treatment with minocycline and bortezomib, but he said that he and his colleagues observed a reduction in numbness and tingling in treated patients relative to controls.
Another recent small trial looked at mangafodipir, a compound with antioxidant and neuroprotective activity that is approved for use in people as a magnetic resonance imaging contrast agent. After obtaining positive results in a mouse model of oxaliplatin-induced neuropathy, the investigators tested mangafodipir in 22 patients with pre-existing neuropathy. The treatment resulted in improvement in neuropathy in some patients even after additional cycles of chemotherapy (Coriat et al., 2014). In a commentary accompanying the study, Deirdre Pachman and colleagues at the Mayo Clinic, Rochester, Minnesota, US, reiterated the poor track record for translating results of small Phase 2 trials into larger, controlled trials in CIPN but nonetheless allowed that the preliminary mangafodipir results warrant additional study (Pachman et al., 2014).
Challenges in the clinic
In the absence of good options for preventing or treating CIPN, oncologists are left to try to balance the benefit of life-saving cancer treatments with the risk of long-term pain and disability. But not everyone who undergoes chemotherapy develops CIPN, and the problem is that there is no way to know who is at risk. “If we want to make prophylactic therapies a … clinical reality, we need to find who is susceptible,” said Sarah Flatters, now an independent investigator at King’s College London, UK.
Last year, Dougherty and coworkers published research showing that cancer patients who present before chemotherapy with subclinical neuropathy—including reduced baseline innervation and subtle symptoms such as a mildly impaired sense of touch—end up having more treatment-related symptoms than patients with normal nerve function at baseline (Boyette-Davis et al., 2012; Vichaya et al., 2013). Such patients generally are unaware that they have deficits in sensory function; nonetheless, fine-grained testing is revealing of such, said Dougherty.
Such patients can be helped by spreading their chemotherapy doses out over time, or “customizing their therapy to try to avoid toxicities.... Instead of cisplatin, you could use carboplatin,” he said.
Managing drug regimens to avoid CIPN requires clinical data, some of which is now being collected. For example, in one large international collaborative study, researchers are looking at 12,000 colon cancer patients worldwide to ask whether shortening the six-month oxaliplatin treatment to just three months alters survival and disability (André et al., 2013). These kinds of trial data are necessary to guide clinical practice that aims to balance anti-tumor and neurological effects of drugs.
One new approach that may greatly facilitate the diagnosis of neuropathy in general and aid in the management of CIPN is the non-invasive imaging of nerve fibers in the cornea via confocal microscopy. The cornea is densely innervated with nociceptive fibers, which can serve as surrogates for peripheral nerve fibers in the skin. Recent work in a mouse model of paclitaxel-induced neuropathy indicated that corneal imaging or skin biopsy gave comparable readouts of nerve damage (Ferrari et al., 2013). This method, if validated in people, could offer a replacement for technically demanding and time-consuming skin biopsies, potentially reducing the time and expense of clinical trials and hastening drug development.
Despite all that has been learned about painful neuropathy and the progress that has been made toward developing drugs to prevent and possibly reverse neuropathy, some fundamental questions still need to be answered to advance the field.
Flatters calls for a better understanding of causal mechanisms. The different drugs that cause painful neuropathy have different mechanisms of anti-tumor action, and it is not clear whether all pathways to neuropathy and pain are convergent on mitochondria, as Bennett argues, or whether they have divergent actions as well, she said. Her own recent work showed that ROS are causal to the development and maintenance of paclitaxel-induced pain (Fidanboylu et al., 2011), but whether mitochondrial damage induced by paclitaxel causes ROS production, or vice versa, remains to be determined.
Questions remain about the genesis of the pain itself. In Bennett’s animal models, the pain correlates with the appearance of spontaneous discharge in nerve fibers (Xiao and Bennett, 2008). That discharge results in activation of secondary neurons in the spinal cord, which convey information from the nociceptive primary afferents to the brain and create a chronic state of hyperexcitability in the spinal cord neurons.
However, other work suggests that additional mechanisms besides the energy deficit may promote pain: ROS can directly activate afferent pain-sensing neurons (Trevisan et al., 2013) and drive central sensitization in spinal cord neurons in neuropathic pain models (see PRF related news story; Kim et al., 2011; Kim et al., 2004). Cytokines have also been implicated (Bennett, 2010).
On the clinical side, another quandary is, How much neuropathic pain is due to trauma, versus chemotherapy, versus vasculolytic neuropathy caused by radiation? Still another question involves coasting, the phenomenon in which the pain begins anywhere from a few weeks to a few months after the commencement of chemotherapy. Why the wait?
The lack of development of pain in some patients suffering from neuropathy is similarly mysterious. Complicating the search for answers, there are no pain-free animal models of chemo-induced neuropathy, although Bennett, and perhaps others, he said, have attempted to develop such.
But Dougherty said his theory that chemotherapy drugs trigger innate immunity could explain both the lag time and the lack of pain in some patients. What starts out in response as a small influx of activated macrophages infiltrating the dorsal root ganglion causes a bit of damage therein, eliciting more inflammatory responses in dorsal root ganglion neurons and in supporting cells, he said. This draws still more macrophages in a growing, vicious cycle, until “the damage finally reaches some critical point where sensations become altered, and pain is generated.”
As for the mystery of the lack of pain in some patients, Dougherty suggests that sometimes the vicious cycle may peter out before the critical point is reached.
One sure thing is that this story is far from over. Investigators repeatedly, and unbidden, waxed eloquent concerning all that remains unknown and uncertain in the field of CIPN. Nonetheless, these same investigators are optimistic that the prognosis for patients is on the cusp of major improvements.
David C. Holzman writes on science, medicine, energy, and the environment from Lexington, Massachusetts, US.
Image credit: ©iStockphoto.com/ninuns