This is the final installment of a four-part report on selected talks from the 10th IASP Research Symposium, The Genetics of Pain: Science, Medicine and Drug Development, held 7-9 February 2012 in Miami, Florida, US. See also Part 1, Part 2, and
Part 3, or download a PDF of the entire report.
The following is an updated version of a story originally published on 16 March 2012.
There are two main reasons to track down genes involved in pain. One is to help identify people at risk for pain, and the other is to illuminate new mechanisms and, ultimately, novel targets for therapy. The translational value of genetic research as a pathway to drug discovery was on display in several presentations throughout the meeting.
Clifford Woolf, Children’s Hospital Boston, US, showed the twists and turns that ensue in moving from gene to potential treatment. In his case, a novel gene led researchers to consider a new use for an old drug.
The novel gene is GCH1, which encodes GTP cyclohydrolase, the rate-limiting enzyme in the synthesis of tetrahydrobiopterin (BH4). BH4 is a cofactor required for enzymes that produce catecholamines (dopamine, epinephrine, norepinephrine), serotonin, and nitric oxide. Woolf and colleagues discovered GCH1 in a genomewide expression profiling of genes that were upregulated in rat models of neuropathic pain, and went on to show that the enzyme, and BH4, control neuropathic and inflammatory pain in rodents. That finding translated to humans, where they showed that a haplotype for human GCH1 that caused decreased enzyme activity was associated with a lower risk of chronic pain, and with reduced sensitivity to pain (Tegeder et al., 2006; Tegeder et al., 2008). This result has since been replicated (Campbell et al., 2009; Lötsch et al., 2010; Kim et al., 2010).
BH4 synthesis is complicated, involving de-novo, salvage, and recycling pathways. Woolf mentioned unpublished data indicating that genes involved in all three pathways show changes in expression in neuropathic pain models in rodents. Based on the accumulation of human genetic and animal results, Woolf suggested that BH4 levels could serve as a biomarker of pain: People who make more BH4 have a greater risk of chronic pain.
Are there targets for new analgesics in the BH4 synthetic pathway? Recently, an independent group using a yeast screen to detect novel targets for approved drugs identified sulfasalazine, a mixed antibiotic and salicylate, as an inhibitor of the last enzyme in the BH4 synthetic pathway, sepiapterin reductase (SPR) (Chidley et al., 2011). The drug, which is over 70 years old, is widely used as an anti-inflammatory to treat rheumatoid arthritis (RA) and inflammatory bowel disease.
Rheumatologists view sulfasalazine as an anti-inflammatory and disease modifier, not as an analgesic, but clinical trials have shown it does reduce pain in patients with RA. In addition, the compound blocks allodynia in diabetic rats (Berti-Mattera, 2008).
“We think this is pretty encouraging,” Woolf said, noting that he and his colleagues are working to make novel SPR inhibitors. Using a crystal structure of the enzyme to guide drug design, they have synthesized an inhibitor with μM potency in cells. And, they are in the planning stages of studies that will look at the activity of sulfasalazine itself in people with neuropathic pain.
Woolf speculated that SPR inhibitors might have dual anti-inflammatory and analgesic actions. GCH1 and BH4 upregulation was recently documented in T cells of the immune system (Chen et al., 2011), a cell type that Woolf and colleagues showed contributes to neuropathic pain (Costigan et al., 2009; see also coverage of Michael Costigan’s talk, below).
“The data encourage us to think that BH4 has activity on neuronal excitability and on immune cells, and thus that reducing BH4 will provide new tools to treat pain and inflammation,” Woolf concluded.
In another talk, Josef Penninger, Austrian Institute of Molecular Biology, Vienna, spoke about alternative models for gene discovery. Penninger collaborated with Woolf and others to identify pain genes in the fruit fly Drosophila melanogaster using avoidance of noxious heat as a high-throughput behavioral screen. In all, they found 580 candidate genes. Among them was straitjacket, a gene encoding a calcium channel subunit that the researchers went on to show is required for central processing of pain signals in flies and in mice (Neely et al., 2011; and see PRF related news story). Variants in the human straightjacket homolog CACNAD2D3 were associated with the risk of chronic pain in people (Neely et al., 2011; and see PRF related news story). The screen also identified the fly transient receptor potential channel A1 (TrpA1) as a mediator of painful heat sensation in flies (Neely et al., 2011) whose nociceptive function is conserved in humans. Penninger mentioned unpublished results on another gene, the phosphoinositide-3 kinase γ subunit (PI3Kγ), which also appears to function in mice (the knockout is hypersensitive to thermal stimuli and capsaicin), and humans (a single nucleotide polymorphism [SNP] in the human gene for PI3Kγ gene is associated with risk of chronic pain).
The results raise the question of how to evaluate the hundreds of other candidate pain genes from the fly screen. Knocking out each gene in a higher organism like the mouse takes a long time. Is there a quicker way? One reason that mammalian genetics take so long is that cells are diploid, containing two copies of each gene. Detecting recessive, loss-of-function effects requires removing both gene copies. Penninger asked if it was possible to develop a mammalian haploid cell that could be used for rapid genetic screens.
The answer, perhaps surprisingly, is yes. As they recently reported, Penninger and colleagues have succeeded in isolating haploid pluripotent embryonic stem cells (ESCs), and have developed a method to do saturating mutagenesis in the cells for genomewide screening for recessive traits (Elling et al., 2011). To derive the cells, the researchers induced parthenogenic cell division in unfertilized mouse oocytes, and then used standard methods to produce blastocysts, from which they isolated haploid ESCs. The two lines they established could be differentiated into all three germline tissues, and, Penninger said, to astrocytes and neurons. The lines could also be used to make chimeric mice. Similar results have been reported by another group (Leeb and Wutz, 2011).
Penninger and colleagues also developed a viral vector that they could use to induce reversible insertional mutations genomewide. With a single infection, they achieve what Penninger believes is near-saturation mutagenesis of every gene, producing up to 10 million independent mutations, one per cell. They used the mutants to identify for the first time a protein essential for cell death induced by ricin, a plant toxin and potential bioterrorism weapon.
Yeast genetics in a pluripotent embryonic stem cell background will allow the construction of a genomewide mutant ESC bank, Penninger said. Rather than use knockout mice to evaluate candidate genes, tests can be done in vitro, he said. “I would love to take this into pain models, into whatever we can model in cell culture to help us untangle all these data we have,” he said. Other exciting possibilities include the ability to "knock in" genes carrying mutations or combinations of common SNPs.
Michael Costigan, Children’s Hospital Boston, US, has pioneered the approach of using differential gene expression in rodent models to identify pathways and genes involved in neuropathic pain (Costigan et al., 2002). One pathway is immune activation, and Costigan and colleagues showed that T cell infiltration into the central nervous system (CNS) plays an important role in producing hypersensitivity after nerve injury in rats (Costigan et al., 2009). Since then, they have found that reconstituting immune-deficient animals with T cells, but not B cells, can produce hypersensitivity.
In support of the importance of T cells in neuropathic pain, Costigan mentioned unpublished GWAS data on 180 patients enrolled in a prospective study of pain after surgery for lumbar disc sciatic pain. The second strongest association seen in the study, he said, was with an unnamed gene that controls T cell activity. Other genes identified in the study included known pain genes GCH1, KCNS1, and Nav1.7.
To produce pain in rats, T cells need to cross the blood-brain barrier, and Costigan proposed that preventing T cells from entering the CNS might be a new therapy for neuropathic pain. Although there is as yet no direct evidence for T cell infiltration associated with neuropathic pain in people, Costigan said it is intriguing that one of the symptoms of the autoimmune disease multiple sclerosis (MS) is trigeminal neuralgia (a neuropathic chronic pain syndrome). New data about how the nervous system regulates immune cell access through the blood-brain barrier in animal models of multiple sclerosis (see Arima et al., 2012, and associated comment by Costigan) raise the intriguing possibility that treatments for multiple sclerosis aimed at keeping T cells out of the nervous system might also have utility in chronic neuropathic pain, he said.
Into the clinic
A sure sign of enthusiasm for the meeting was how many participants stayed on through the last session, turning out for the final talks on new clinical approaches to pain. David Fink, University of Michigan, Ann Arbor, US, spoke about targeted gene delivery for treating localized pain. He has been developing herpes simplex virus (HSV)-based vectors that can retrogradely carry therapeutic genes from sensory nerve endings in the skin into the dorsal horn of the spinal cord. In published Phase 1 trial data, an HSV-proenkephalin vector appeared safe and even showed some evidence of efficacy in cancer patients (Fink et al., 2011). Enrollment in a Phase 2 randomized, placebo-controlled trial is nearly complete, he said, and results should be available later in 2012.
Genes that are close to clinical testing include GAD67, which encodes glutamic acid decarboxylase, a GABA-synthesizing enzyme. In rats, HSV vector-based transfer of GAD67 to dorsal root ganglion neurons results in the continuous release of GABA and reductions in hyperalgesia and allodynia in a model of painful diabetic neuropathy (Chattopadhyay et al., 2011). Fink also reported he recently received a grant to produce a neurotropin-3 vector as a potential therapy for chemotherapy-induced peripheral neuropathy.
Simon Tate, Convergence Pharmaceuticals, Cambridge, UK, presented data on a selective voltage-gated sodium channel (Nav1.7) blocker that recently began clinical trials. He showed unpublished data supporting the drug-like properties of the compound (orally dosed, achieves adequate blood levels, and well tolerated with few CNS side effects), and a unique activity profile of state-dependent channel inhibition that is relatively selective for Nav1.7 compared to other voltage-gated sodium channels. The clinical plan for CNV1014802 includes two proof-of-concept studies with novel designs. “If Nav1.7 blockers have a chance in the clinic, this molecule will tell us,” Tate said.
After Phase 1 testing in healthy volunteers, CNV1014802 moved to Phase 2 in July 2011, in a crossover design in lumbosacral radiculopathy. Results are expected in the second half of 2012. A second Phase 2 trial is planned in trigeminal neuralgia. In that condition, the pain is so intense and carbamazepine offers an effective (but poorly tolerated) therapy, so patients will not be randomized immediately to placebo or active treatment. The trial will have an initial three week open-label phase, and then responders will be randomized to four weeks of placebo or drug in a double blind design. The primary outcome of the study will be the number of failures on CNV1014802 compared to the number of failures on placebo during the double blind treatment period. Results of that trial are due in early 2013.
It didn’t take human genetic data to interest researchers in Nav1.7, but the discovery in 2004-2006 of mutations in the gene for the channel in the painful conditions erythermalgia and paroxysmal extreme pain disorder (Yang et al., 2004; Cummins et al., 2004; Fertleman et al., 2006), and in congenital insensitivity to pain (first reported in Cox et al., 2006), helped to validate the target. “We were already working on the channels, but if we hadn’t been, we would have started,” Tate said.
With a look forward to these exciting clinical prospects, the meeting ended, leaving the attendees with much to ponder. If you missed the meeting, but want to learn more about pain genetics, never fear. The Pain Genetics Special Interest Group is sponsoring a satellite symposium this summer at the IASP World Pain Congress in Milan, Italy. Click on the PDF file below for more information.
