DISCUSSION and CONCLUSION
GlyT2 inhibition holds promise as a new analgesic target, however severe side effects of compounds such as ORG25543 have tempered development. Here, we have shown that a reversible GlyT2 inhibitor is analgesic while eluding serious side effects. Detailed in vivo characterisation of ol-D-lys in mice showed it was an effective analgesic for neuropathic pain, with minimal side effects after systemic administration. We conducted a side-by-side comparison of ol-D-lys and ORG25543 – currently the only blood-brain barrier permeable, GlyT2-selective inhibitor commercially available. While useful as a research tool in vitro , the high toxicity of ORG25543 precludes its use in reliably characterising the in vivoeffects of inhibiting GlyT2. By virtue of its larger therapeutic window, we were able to test ol-D-lys in multiple pain and side-effect models and show that inhibition of GlyT2 appears to be a safe and beneficial approach to treating chronic neuropathic pain, but not chronic inflammatory pain or acute anti-nociception.
Ol-D-lys produced near complete reversal of allodynia in the mouse CCI neuropathic pain model. A dose-dependent relationship was seen between 10 and 30 mg kg-1, while higher doses of 50 and 100 mg kg-1 were less effective. This may be due to the high lipophilicity and poor aqueous solubility of ol-D-lys leading to precipitate formation at higher doses, and/or the high degree of binding in the CNS, likely due to sequestration to lipid-rich brain tissue after crossing the blood-brain barrier (Mostyn et al., 2019). The analgesic effects of 30 mg kg-1 ORG25543 were modest but sustained for the 6 h duration of testing. At 50 mg kg-1, ORG25543 caused mice to reduce responding to the stimulus at every timepoint tested, however this is confounded by side effects, discussed below. Neither inflammatory pain nor acute thermal pain were alleviated by ol-D-lys or ORG25543. We propose that this is due to the underlying glycinergic changes that occur in chronic pain states, wherein loss of inhibitory glycinergic tone over time leads to increased nociceptive signalling. These changes are the target of GlyT2 inhibitors and may be absent in the shorter timescale CFA inflammatory model and acute pain model used here.
At the highest dose, ol-D-lys caused mild side effects on only one measure, while ORG25543 caused severe side effects as well as lethal toxicity. Ol-D-lys produced no convulsions at any dose. Mild to moderate clinical behaviours were scored for some mice on the numerical scale, but only at 100 mg kg-1; well above the peak analgesic dose. These behaviours included decreased activity, hunched posture, and mild pain behaviours of abdominal constriction, which suggest that a depot of the drug may have formed at the site of injection. Our prediction of precipitate formation at high doses supports this idea of a lipid depot forming and may account for the relatively low and sustained analgesic activity at 100 mg kg-1 (Zuidema et al., 1994). In contrast, ORG25543 produced severe side effects, convulsions and toxicity at 50 mg kg-1, with one mouse dying 1 h post-injection. Of those animals that were administered the 50 mg kg-1dose, most reached the maximum (ceiling) score for the numerical rating scale, and one animal reached the ceiling for the convulsions score.
Ol-D-lys did not impede motor coordination in the rotarod test, with a significant effect only in the positive direction. The nature of such tests that involve subtle behavioural and motivational factors complicates interpretation of this result. Non-motor factors such as arousal and executive function (e.g. volition and motivation to stay on the rotarod; Yogev-Seligmann et al., 2008) may account for the behaviours, rather than drug-induced motor enhancementper se . With no other observable changes in this group at the time of testing, further experiments are required to determine the true cause of the enhanced rotarod performance. ORG25543 caused profound and immediate aberration of motor coordination in 2 out of 3 mice tested at 50 mg kg-1. However, due to the low numbers tested, this effect could not be statistically evaluated. The peak effects of ORG25543 on the numerical side-effect scale and convulsions score align temporally with the decrease in responses to the von Frey stimulus, thus, confounding the interpretation of the analgesic effects of ORG25543 at this dose. The von Frey test is highly sensitive to state of arousal, alertness and attention (Callahan et al., 2008). Furthermore, the von Frey test relies on discrete motor behaviours as endpoints for scoring pain thresholds, while ORG25543 caused motor incoordination. Thus, the reduced responding to von Frey stimuli likely reflects side effects such as convulsions preventing or distracting the mice from responding, not just analgesic effects of ORG25543.
Respiratory depression is a major side effect of opioids and has been shown to occur following administration of GlyT2 inhibitors (Hermanns et al., 2008). Here, we compared ol-D-lys with morphine, the current putative gold-standard analgesic drug for many types of pain. We observed no respiratory depression caused by the peak analgesic dose of ol-D-lys, 30 mg kg-1, compared with vehicle and an analgesic dose of morphine. A comparative increase in respiratory frequency, but not minute volume, at 30 min post-injection was observed. ORG25543 was not tested here due to ethical considerations, however these results support the proposal that reversible and selective GlyT2 inhibitors will not substantially affect respiration.
In this study, as we have previously shown in rats (Mostyn et al., 2019), we have observed severe side effects at minimally analgesic doses following i.p. delivery of ORG25543. Previous reports found complete reversal of allodynia at 100-fold lower doses of ORG25543 than in our study, and vastly differing pharmacodynamics (Morita et al., 2008; Motoyama et al., 2014). These past studies used a variety of pain models including a similar neuropathic pain model and the same von Frey test as used here. However, those studies found that the analgesic effects peaked from 3-24 h and lasted several days after a single bolus intravenous (i.v.) injection, with no side effects at analgesic doses. The lack of side effects may be due to the lower doses used compared to our study, although, Motoyama et al (2014) tested a single 1 mg kg-1 dose, orally (p.o.) administered, without reporting any side effects. There are two key points of difference between these reports and our findings; the route of administration (i.v. or p.o., compared with i.p.) and the vehicle (aCSF or saline, compared with 1% DMSO, 10% solutol in saline). While the route of administration may partially account for the differences in effective dose range, it is unlikely to account for this drastic pharmacodynamic difference. ORG25543 has good plasma and hepatic metabolic stability (Caulfield et al., 2001), thus hepatic metabolism upon i.p. administration is unlikely to account for the effects lasting for hours versus days. The vehicle used here was based on the requirements of dissolving the lipophilic ol-D-lys and may have affected the pharmacology of ORG25543. Indeed, an independent study (Mingorance-Le Meur et al. 2013) also used DMSO (5%) in their vehicle and found that higher doses of ORG25543, 20 mg kg-1, were required to reach peak analgesic effect and observed convulsions and lethality at analgesic doses. Our findings indicate ORG25543 to have a narrow therapeutic window with severe on-target side effects at analgesic doses.
The utility of GlyT2 inhibitors as analgesics is being increasingly supported by pharmacological and genetic studies. There is evidence that irreversible blockers of GlyT2 (e.g. ORG25543; Mingorance-Le Meur et al., 2013) will lead to side effects such as convulsions and high toxicity, while reversible compounds (e.g. ol-D-lys) will have a larger therapeutic window. Additionally, the efficacy of compounds may influence side-effect liability, with full inhibitors causing more side effects than partial inhibitors. The difference is attributed to the role of GlyT2 in clearing synaptic glycine and repackaging it into presynaptic vesicles (Jursky and Nelson, 1995); complete depletion of vesicular glycine stores following complete, irreversible GlyT2 inhibition abolishes glycinergic signalling. However, partial or reversible inhibition of the transporter preserves vesicle repackaging while slowing down synaptic clearance and prolonging glycine receptor activation. This concept is mirrored in mouse genetic studies where global deletion of GlyT2 (compare to full/irreversible inhibitor) causes spasticity, tremor, inability to right, and death at post-natal week 2 (Gomeza et al., 2003), whereas partial knockdown of GlyT2 (compare to partial/reversible inhibitor) imparts analgesia with no apparent side effects (Morita et al., 2008; Motoyama et al., 2014).
The potential of targeting GlyT2 clinically is currently being established with the multi-target drug opiranserin (VVZ-149, Vivizon). Opiranserin is a novel analgesic drug, acting as antagonist at both GlyT2 and serotonin receptor 2A (5HT2A; Pang et al., 2012). Opiranserin has successfully completed recruitment for its first phase 3 clinical trial, with recruitment for a second trial underway (https://clinicaltrials.gov/ct2/results?term=vvz-149). All trials for opiranserin have been conducted/planned in post-operative pain. Our findings suggest that the best indication for a single-target GlyT2 inhibitor is in chronic pain states, where spinal glycinergic changes have become aberrant, and may be corrected with reversible GlyT2 inhibitors. The present results also build evidence that reversible GlyT2 inhibitors such as ol-D-lys elude side effects observed with irreversible GlyT2 inhibition.
References
Aroeira, R.I., Sebastião, A.M., and Valente, C.A. (2014). GlyT1 and GlyT2 in brain astrocytes: Expression, distribution and function. Brain Struct. Funct. 219 : 817–830.
Benbouzid, M., Pallage, V., Rajalu, M., Waltisperger, E., Doridot, S., Poisbeau, P., et al. (2008). Sciatic nerve cuffing in mice: A model of sustained neuropathic pain. Eur. J. Pain 12 : 591–599.
Betz, H., Gomeza, J., Armsen, W., Scholze, P., and Eulenburg, V. (2006). Glycine transporters: Essential regulators of synaptic transmission. Biochem. Soc. Trans. 34 : 55–58.
Borges, K., Gearing, M., McDermott, D.L., Smith, A.B., Almonte, A.G., Wainer, B.H., et al. (2003). Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model. Exp. Neurol.182 : 21–34.
Breivik, H., Collett, B., Ventafridda, V., Cohen, R., and Gallacher, D. (2006). Survey of chronic pain in Europe: Prevalence, impact on daily life, and treatment. Eur. J. Pain 10 : 287–333.
Callahan, B.L., Gil, A.S.C., Levesque, A., and Mogil, J.S. (2008). Modulation of Mechanical and Thermal Nociceptive Sensitivity in the Laboratory Mouse by Behavioral State. J. Pain 9 : 174–184.
Caulfield, W.L., Collie, I.T., Dickins, R.S., Epemolu, O., McGuire, R., Hill, D.R., et al. (2001). The first potent and selective inhibitors of the glycine transporter type 2 [1]. J. Med. Chem. 44 : 2679–2682.
Cheng, W., Yin, Q., Cheng, M.Y., Chen, H.S., Wang, S., Feng, T., et al. (2009). Intracerebroventricular or intrathecal injection of glycine produces analgesia in thermal nociception and chemical nociception via glycine receptors. Eur. J. Pharmacol. 614 : 44–49.
Cioffi, C.L. (2018). Modulation of glycine-mediated spinal neurotransmission for the treatment of chronic pain. J. Med. Chem.61 : 2652–2679.
Curtis, M.J., Alexander, S., Cirino, G., Docherty, J.R., George, C.H., Giembycz, M.A., et al. (2018). Experimental design and analysis and their reporting II: updated and simplified guidance for authors and peer reviewers. Br. J. Pharmacol. 175 : 987–993.
Dohi, T., Morita, K., Kitayama, T., Motoyama, N., and Morioka, N. (2009). Glycine transporter inhibitors as a novel drug discovery strategy for neuropathic pain. Pharmacol. Ther. 123 : 54–79.
Gomeza, J., Ohno, K., Hülsmann, S., Armsen, W., Eulenburg, V., Richter, D.W., et al. (2003). Deletion of the mouse glycine transporter 2 results in a hyperekplexia phenotype and postnatal lethality. Neuron 40 :.
Harvey, R.J., and Yee, B.K. (2013). Glycine transporters as novel therapeutic targets in schizophrenia, alcohol dependence and pain. Nat. Rev. Drug Discov. 12 : 866–885.
Hehn, C.A. von, Baron, R., and Woolf, C.J. (2012). Deconstructing the Neuropathic Pain Phenotype to Reveal Neural Mechanisms. Neuron73 : 638–652.
Hermanns, H., Muth-Selbach, U., Williams, R., Krug, S., Lipfert, P., Werdehausen, R., et al. (2008). Differential effects of spinally applied glycine transporter inhibitors on nociception in a rat model of neuropathic pain. Neurosci. Lett. 445 : 214–219.
Imlach, W.L., Bhola, R.F., Mohammadi, S.A., and Christie, M.J. (2016). Glycinergic dysfunction in a subpopulation of dorsal horn interneurons in a rat model of neuropathic pain. Sci. Rep. 6 : 1–14.
Jursky, F., and Nelson, N. (1995). Localization of Glycine Neurotransmitter Transporter (GLYT2) Reveals Correlation with the Distribution of Glycine Receptor. J. Neurochem. 64 : 1026–1033.
Kochanek, K.D., Murphy, S.L., Xu, J., and Arias, E. (2019). Deaths: Final data for 2017. Natl. Vital Stat. Reports 68 : 1–18.
Mingorance-Le Meur, A., Ghisdal, P., Mullier, B., Ron, P. De, Downey, P., Perren, C. Van Der, et al. (2013). Reversible inhibition of the glycine transporter GlyT2 circumvents acute toxicity while preserving efficacy in the treatment of pain. Br. J. Pharmacol. 170 : 1053–1063.
Mohammadi, S., and Christie, M.J. (2014). Α9-Nicotinic Acetylcholine Receptors Contribute To the Maintenance of Chronic Mechanical Hyperalgesia, But Not Thermal or Mechanical Allodynia. Mol. Pain10 : 1–9.
Morita, K., Motoyama, N., Kitayama, T., Morioka, N., Kifune, K., and Dohi, T. (2008). Spinal antiallodynia action of glycine transporter inhibitors in neuropathic pain models in mice. J. Pharmacol. Exp. Ther.326 : 633–645.
Mostyn, S.N., Rawling, T., Mohammadi, S., Shimmon, S., Frangos, Z.J., Sarker, S., et al. (2019). Development of an N-Acyl Amino Acid That Selectively Inhibits the Glycine Transporter 2 to Produce Analgesia in a Rat Model of Chronic Pain. J. Med. Chem. 62 : 2466–2484.
Motoyama, N., Morita, K., Shiraishi, S., Kitayama, T., Kanematsu, T., Uezono, Y., et al. (2014). Relief of cancer pain by glycine transporter inhibitors. Anesth. Analg. 119 : 988–995.
Pang, M.H., Kim, Y., Jung, K.W., Cho, S., and Lee, D.H. (2012). Foundation review: A series of case studies: Practical methodology for identifying antinociceptive multi-target drugs. Drug Discov. Today17 : 425–434.
Pitzer, C., Kuner, R., and Tappe-Theodor, A. (2016). Voluntary and evoked behavioral correlates in neuropathic pain states under different social housing conditions. Mol. Pain 12 : 1–12.
Raiteri, L., Stigliani, S., Usai, C., Diaspro, A., Paluzzi, S., Milanese, M., et al. (2008). Functional expression of release-regulating glycine transporters GLYT1 on GABAergic neurons and GLYT2 on astrocytes in mouse spinal cord. Neurochem. Int. 52 : 103–112.
Schlösser, L., Barthel, F., Brandenburger, T., Neumann, E., Bauer, I., Eulenburg, V., et al. (2015). Glycine transporter GlyT1, but not GlyT2, is expressed in rat dorsal root ganglion-Possible implications for neuropathic pain. Neurosci. Lett. 600 : 213–219.
Scholl, L., Seth, P., Kariisa, M., Wilson, N., and Baldwin, G. (2018). Drug and Opioid-Involved Overdose Deaths — United States, 2013–2017. MMWR. Morb. Mortal. Wkly. Rep. 67 : 2013–2017.
Seth, P., Rudd, R.A., Noonan, R.K., and Haegerich, T.M. (2018). Quantifying the epidemic of prescription opioid overdose deaths. Am. J. Public Health 108 : 500–502.
Takahashi, Y., Hara, K., Haranishi, Y., Terada, T., Obara, G., and Sata, T. (2015). Antinociceptive effect of intracerebroventricular administration of glycine transporter-2 inhibitor ALX1393 in rat models of inflammatory and neuropathic pain. Pharmacol. Biochem. Behav.130 : 46–52.
Tanabe, M., Takasu, K., Yamaguchi, S., Kodama, D., and Ono, H. (2008). Glycine transporter inhibitors as a potential therapeutic strategy for chronic pain with memory impairment. Anesthesiology 108 : 929–937.
Tung, V.W.K., Burton, T.J., Quail, S.L., Mathews, M.A., and Camp, A.J. (2016). Motor performance is impaired following vestibular stimulation in ageing mice. Front. Aging Neurosci. 8 : 1–10.
Vandenberg, R.J., Ryan, R.M., Carland, J.E., Imlach, W.L., and Christie, M.J. (2014). Glycine transport inhibitors for the treatment of pain. Trends Pharmacol. Sci. 35 : 423–430.
Yogev-Seligmann, G., Hausdorff, J.M., and Giladi, N. (2008). The role of executive function and attention in gait. Mov. Disord. 23 :.
Zafra, F., Aragón, C., Olivares, L., Danbolt, N.C., Giménez, C., and Storm-Mathisen, J. (1995). Glycine Cells Are Differentially Expressed CNS in the spinal. J. Neurosci. 15 : 3952–3969.
Zeilhofer, H.U., Studler, B., Arabadzisz, D., Schweizer, C., Ahmadi, S., Layh, B., et al. (2005). Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J. Comp. Neurol. 482 : 123–141.
Zuidema, J., Kadir, F., Titulaer, H.A.C., and Oussoren, C. (1994). Release and absorption rates of intramuscularly and subcutaneously injected pharmaceuticals (II). Int. J. Pharm. 105 : 189–207.