Differential Morphological and Biochemical Recovery from Chemotherapy-Induced Peripheral Neuropathy Following Paclitaxel, Ixabepilone, or Eribulin Treatment in Mouse Sciatic Nerves.

The reversibility of chemotherapy-induced peripheral neuropathy (CIPN), a disabling and potentially permanent side effect of microtubule-targeting agents (MTAs), is becoming an increasingly important issue as treatment outcomes improve. The molecular mechanisms regulating the variability in time to onset, severity, and time to recovery from CIPN between the common MTAs paclitaxel and eribulin are unknown. Previously (Benbow et al. in Neurotox Res 29:299-313, 2016), we found that after 2 weeks of a maximum tolerated dose (MTD) in mice, paclitaxel treatment resulted in severe reductions in axon area density, higher frequency of myelin abnormalities, and increased numbers of Schwann cell nuclei in sciatic nerves. Biochemically, eribulin induced greater microtubule-stabilizing effects than paclitaxel. Here, we extended these comparative MTD studies to assess the recovery from these short-term effects of paclitaxel, eribulin, and a third MTA, ixabepilone, over the course of 6 months. Paclitaxel induced a persistent reduction in axon area density over the entire 6-month recovery period, unlike ixabepilone- or eribulin-treated animals. The abundance of myelin abnormalities rapidly declined after cessation of all drugs but recovered most slowly after paclitaxel treatment. Paclitaxel- and ixabepilone- but not eribulin-treated animals exhibited increased Schwann cell numbers during the recovery period. Tubulin composition and biochemistry rapidly returned from MTD-induced levels of α-tubulin, acetylated α-tubulin, and end-binding protein 1 to control levels following cessation of drug treatment. Taken together, sciatic nerve axons recovered more rapidly from morphological effects in eribulin- and ixabepilone-treated animals than in paclitaxel-treated animals and drug-induced increases in protein expression levels following paclitaxel and eribulin treatment were relatively transient.

Still NAAG'ing After All These Years: The Continuing Pursuit of GCPII Inhibitors.

Nearly two decades ago, Joe Coyle published a single-authored review with the provocative title, The Nagging Question of the Function of N-Acetylaspartylglutamate (Coyle, 1997). In this review, Coyle documented NAAG's localization to subpopulations of glutamatergic, cholinergic, GABAergic, and noradrenergic neurons, Ca(2+)-dependent release, mGlu3 receptor agonist and NMDA receptor antagonist activity, and cleavage by the glial enzyme glutamate carboxypeptidase II (GCPII). However, at the time of his review, NAAG's physiological function as a neurotransmitter remained elusive. Ironically his review was published months following the discovery of the first potent and selective GCPII inhibitor, 2-(phosphonomethyl)pentanedioc acid (2-PMPA) (Jackson et al., 1996). Over the ensuing decades, over a dozen independent laboratories used 2-PMPA and other GCPII inhibitors to elucidate two distinct neurotransmitter functions for NAAG. Under basal conditions, when GCPII activity is relatively low, intact NAAG dampens synaptic activity via presynaptic mGlu3 receptor activation and NMDA receptor blockade. However, under stimulated conditions, NAAG release and GCPII activity are enhanced resulting in excess glutamate generation, activating NMDA and other glutamate receptors, often pathologically. Diverse classes of GCPII inhibitors have been synthesized and shown to increase NAAG, decrease glutamate, and provide robust efficacy in many disease models wherein abnormal glutamatergic transmission is presumed pathogenic. In addition, over the past 20 years, basic questions regarding NAAG's synthesis, packaging into vesicles, and receptor selectivity profile have been eloquently elucidated. The purpose of this chapter is to summarize these advances and the promise of regulating NAAG metabolism through GCPII inhibition as a therapeutic strategy.

The role of glutamate signaling in pain processes and its regulation by GCP II inhibition.

Glutamate is the predominant excitatory neurotransmitter used by primary afferent synapses and neurons in the spinal cord dorsal horn. Glutamate and glutamate receptors are also located in areas of the brain, spinal cord and periphery that are involved in pain sensation and transmission. Not surprisingly, glutamate receptors have been an attractive target for new pain therapies. However, their widespread distribution and array of function has often resulted in drugs targeting these sites having undesirable side-effects. This chapter will review, in general terms, the current knowledge of glutamate and its effects at various glutamate receptors with regards to nociception. In addition, we will briefly review the glutamatergic drugs currently in use as treatments for pain, as well as known novel candidates in various stages of clinical trial. Lastly, we will summarize the data supporting a novel target for pain intervention by way of GCPII inhibition, which appears devoid of the side-effects associated with direct glutamate receptor antagonists and thus holds major promise for future therapy. GCPII (glutamate carboxypeptidase II) cleaves the prevalent neuropeptide NAAG into NAA and glutamate and there is widespread evidence and belief that targeting the glutamate derived from this enzymatic action may be a promising therapeutic route.

The preventive and therapeutic effects of GCPII (NAALADase) inhibition on painful and sensory diabetic neuropathy.

Excitotoxic glutamate release occurs in several neurological disorders. One source is derived from the hydrolysis of the neuropeptide N-acetyl aspartyl glutamate (NAAG) by glutamate carboxypeptidase II (GCPII, also known as NAALADase). Drugs that attenuate glutamate transmission have been shown to relieve neuropathic pain, however side effects have limited their clinical use. It appears that GCPII is exclusively recruited to provide a glutamate source in hyperglutamatergic, excitotoxic conditions and therefore would be devoid of such side effects. Here we report on the therapeutic effects of an orally bio-available GCP II inhibitor on established painful and sensory neuropathy in the spontaneously diabetic BB/Wor rat. It significantly improved hyperalgesia, nerve conduction velocity and underlying myelinated fiber atrophy. The data suggest that GCP II inhibition may provide a meaningful and effective approach to the treatment of painful diabetic neuropathy.

GCPII (NAALADase) inhibition prevents long-term diabetic neuropathy in type 1 diabetic BB/Wor rats.

AIMS/HYPOTHESIS: Hyperglutamatergic activity induced by ischemia is believed to underlie neuronal damage in a variety of neurological disorders, including neuropathic pain. Since ischemia is believed to be a prominent mechanism involved in diabetic polyneuropathy (DPN), we investigated the effect of the glutamate carboxypeptidase II (GCPII, EC #3.4-17.21; previously termed NAALADase), an enzyme responsible for the hydrolysis of the neuropeptide NAAG to NAA and glutamate, on the development of DPN in type 1 diabetic BB/Wor rats. METHODS: Diabetic animals were treated with 10 mg/kg/day i.p. of the selective GCPII inhibitor GPI-5232 from onset of diabetes for 6 months. Hyperalgesia to thermal stimulation and nerve conduction velocity (NCV) were measured monthly. The effect on structural DPN was assessed by scoring of single, teased myelinated fibers, myelinated fiber morphometry and ultrastructural examination of C-fibers at 6 months. RESULTS: GCPII inhibition showed significant but partial effects on hyperalgesia (p<0.001), nerve conduction slowing (p<0.01) axonal and nodal structural changes (p<0.001), small myelinated fiber atrophy, and degenerative changes of C-fibers. CONCLUSIONS: GCPII inhibition has beneficial effects on hyperalgesia, nerve function, and structural degenerative changes in DPN, which are likely mediated by inhibition of ischemia-induced glutamate release.

Selective inhibition of NAALADase, which converts NAAG to glutamate, reduces ischemic brain injury.

We describe here a new strategy for the treatment of stroke, through the inhibition of NAALADase (N-acetylated-alpha-linked-acidic dipeptidase), an enzyme responsible for the hydrolysis of the neuropeptide NAAG (N-acetyl-aspartyl-glutamate) to N-acetyl-aspartate and glutamate. We demonstrate that the newly described NAALADase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) robustly protects against ischemic injury in a neuronal culture model of stroke and in rats after transient middle cerebral artery occlusion. Consistent with inhibition of NAALADase, we show that 2-PMPA increases NAAG and attenuates the ischemia-induced rise in glutamate. Both effects could contribute to neuroprotection. These data indicate that NAALADase inhibition may have use in neurological disorders in which excessive excitatory amino acid transmission is pathogenic.

The role of striatal dopaminergic mechanisms in rotational behavior induced by phencyclidine and phencyclidine-like drugs.

Phencyclidine (PCP) and phencyclidine-like drugs (TCP, dexoxadrol, MK-801, and SKF 10,047) were evaluated for their ability to induce rotational behavior in rats with unilateral 6-OHDA lesions of the medial forebrain bundle and for their ability to alter striatal dopamine (DA) overflow with microdialysis procedures. All of the compounds tested produced rotational behavior ipsilateral to the lesion, suggesting that they were enhancing extracellular dopamine in the intact striatum. The microdialysis studies, however, did not support this contention. There appeared to be a complete dissociation between the ability of the five compounds to produce ipsilateral rotations and their ability to enhance extracellular dopamine levels in the striatum. PCP was the only compound able to elicit significant increases in striatal dopamine overflow following i.p. injections and also produce dramatic rotational behavior. MK-801 was the most potent compound in enhancing rotational output while it had no effect at all on striatal dopamine overflow. Dexoxadrol also produced significant rotational output without having any effect on extracellular levels of dopamine following i.p. injections. TCP and SKF 10,047, at doses which produced significant rotational behavior, only elevated dopamine 16% and 12%, respectively, at peak effect. It is most parsimonious to conclude that the effects of PCP-like drugs on nigro-striatal function are mediated through their ability to act as indirect NMDA receptor antagonists and not through their ability to alter striatal dopamine activity.

Evidence that m-chlorophenylpiperazine-induced hyperthermia in rats is mediated by stimulation of 5-HT2C receptors.

Intraperitoneal administration of m-chlorophenylpiperazine (m-CPP) to Wistar rats produced hyperthermia with a peak effect at 30 min. Pretreatment with low doses of metergoline (5-HT1/5-HT2 antagonist), mesulergine and mianserin (5-HT2C/5-HT2A antagonists) blocked m-CPP-induced hyperthermia. Pretreatment with propranolol (beta-adrenergic receptor antagonist that also has binding affinity for 5-HT1A, 5-HT1B and 5-HT2B sites), yohimbine (alpha 2-noradrenergic antagonist that also has binding affinity for 5-HT2B sites), MDL-72222 or ondansetron (5-HT3 antagonists) did not attenuate m-CPP-induced hyperthermia. Only high doses of ketanserin, LY-53857 and ritanserin (5-HT2A/5-HT2C antagonists) as well as spiperone (5-HT1A/5-HT2A/D2 antagonist) attenuated m-CPP-induced hyperthermia. Daily administration of m-CPP produced complete tolerance to its hyperthermic effect by day 5. However, there was no cross-tolerance to 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, a 5-HT2A agonist that also has high affinity for 5-HT2C receptors)-induced hyperthermia. m-CPP-induced increases in temperature were found to be significantly less in the Fawn-Hooded (FH) rat strain as compared to the Wistar rat strain; in prior studies, FH rats have been found to be subsensitive to other 5-HT2C-mediated pharmacologic responses. Altogether, these findings suggest that m-CPP-induced hyperthermia in rats is mediated by selective stimulation of 5-HT2C receptors.

Evidence that 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced hyperthermia in rats is mediated by stimulation of 5-HT2A receptors.

The effects of various 5-HT receptor subtype-selective antagonists were studied on phenylisopropylamine hallucinogen 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced hyperthermia in Wistar rats, in an attempt to characterize the 5-HT receptor subtype mediating DOI-induced hyperthermia. Intraperitoneal administration of DOI to rats produced hyperthermia with a peak effect at 60 min. Pretreatment with propranolol (beta-adrenoceptor antagonist that also has binding affinity for 5-HT1A, 5-HT1B and 5-HT2C sites), MDL-72222 or ondansetron (5-HT3 antagonists) did not attenuate DOI-induced hyperthermia. In contrast, pretreatment with metergoline (5-HT1/5-HT2 antagonist), ketanserin, LY53857, mesulergine, mianserin and ritanserin (5-HT2C/5-HT2A antagonists), as well as spiperone (5-HT1A/5-HT2A/D2 antagonist), significantly attenuated DOI-induced hyperthermia. Furthermore, daily administration of DOI (2.5 mg/kg per day) for 17 days did not produce either tolerance to its hyperthermic effect or modify m-CPP-induced hyperthermia in rats. These findings suggest that DOI-induced hyperthermia in rats is mediated by stimulation of 5-HT2A receptors.

Functional and biochemical evidence for altered serotonergic function in the fawn-hooded rat strain.

Administration of various doses of DOI (a 5-HT2A/5-HT2C agonist) produced hyperthermia that was significantly less in the FH rat strain relative to the Wistar rat strain. Similarly, administration of various doses of ipsapirone (a 5-HT1A agonist) produced hypothermia that was significantly less in the FH rat strain relative to the Wistar rat strain. Furthermore, m-CPP (a 5-HT agonist)-induced increases in growth hormone levels were also significantly less in the FH rat strain relative to the Wistar rat strain. There was no significant difference in the levels of either 5-HT or 5-HIAA between the two rat strains in the frontal cortex, hippocampus, hypothalamus, and striatum. In the brain stem, however, both 5-HT and 5-HIAA levels were significantly lower in the FH rat strain relative to the Wistar rat strain. On the other hand, 5-HT turnover rate was significantly higher in the hypothalamus and striatum and significantly lower in the hippocampus in the FH rat strain relative to the Wistar rat strain. These findings provide further evidence for altered serotonergic function in the FH rat strain and, in addition, suggest that the FH rat strain may prove to be a useful genetic model for some neuropsychiatric disorders with possible abnormalities in serotonergic function such as depression, obsessive-compulsive disorder, and the eating disorders.

Evidence that 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane-induced hypophagia and hyperthermia in rats is mediated by serotonin-2A receptors.

The administration of various doses of the phenylisopropylamine hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) to rats produced dose-related decreases in 1-hr food intake in a food-restricted paradigm and in locomotor activity. DOM also produced dose-related increases in temperature. Pretreatment with propranolol [a beta adrenoceptor antagonist that also has high binding affinity for serotonin (5-HT) 5-HT1A, 5-HT1B and 5-HT2C sites], bemesetron or ondansetron (5-HT3 antagonists) did not attenuate either DOM-induced hypophagia or hyperthermia. In contrast, pretreatment with metergoline (a 5-HT1/5-HT2 antagonist) and ritanserin (a 5-HT2A/5-HT2C antagonist) significantly attenuated both DOM-induced hypophagia and hyperthermia. However, pretreatment with mesulergine (a 5-HT2C/5-HT2A antagonist) significantly attenuated DOM-induced hyperthermia but not hypophagia. On the other hand, spiperone (5-HT1A/5-HT2A/D2 antagonist) pretreatment significantly attenuated DOM-induced hyperthermia but accentuated DOM-induced hypophagia. Daily administration of DOM (1.0 mg kg-1 day-1) produced complete tolerance to its hypophagic effect by day 4 but did not produce cross-tolerance to m-chlorophenylpiperazine-induced hypophagia. In contrast, daily administration of DOM for 7 days did not produce either tolerance to its hyperthermic effect or modify m-chlorophenylpiperazine-induced hyperthermia in rats. These findings suggest that DOM-induced hypophagia and hyperthermia in rats are mediated by stimulation of 5-HT2a receptors.

Recent advances in pharmacological research on alcohol. Possible relations with cocaine.

Alcohol dependence is a major public health problem. Studies have shown that a person dependent on alcohol often coabuses other substances, such as cocaine. Cocaine is a powerful stimulant whereas ethanol is generally considered to be a depressant, with some stimulating properties. The subjective effects of these two substances in a dependent individual may often appear to be more similar than they are different. Animals also self-administer both substances. Basically, although both substances have anesthetic properties and both act to functionally increase catecholaminergic function, especially that of dopamine, there are some differences in their actions. Both alcohol and cocaine have various effects on several neurotransmitters and systems, which ultimately interact to produce the feeling of well-being avidly sought by many individuals today. This drive often eventually produces a dependence which has associated social and medical consequences. It seems likely that the neurochemical changes that ensue following abuse of these substances underlie the phenomena of dependence, tolerance, and subsequent withdrawal. The apparent similarities and differences between these two substances will be reviewed in this chapter.

Differential effects of antidepressant treatments on fenfluramine-induced increases in plasma prolactin and corticosterone in rats.

Intravenous administration of 5-HT releasing agent, fenfluramine, to rats produced increases in plasma prolactin and corticosterone concentrations. Short-term or long-term treatment with either clorgyline or imipramine did not affect baseline levels of prolactin or corticosterone. On the other hand, short-term but not long-term lithium treatment significantly increased baseline levels of corticosterone but not of prolactin. Short-term treatment with lithium but not clorgyline or imipramine potentiated fenfluramine-induced increases in plasma prolactin but not corticosterone. On the other hand, long-term treatment with clorgyline but not imipramine or lithium attenuated fenfluramine's effect on plasma prolactin but not on corticosterone. These findings demonstrate differential effects of antidepressant treatments on fenfluramine-induced increases in plasma prolactin and corticosterone in rats and are consistent with several other clinical and animal studies demonstrating dissimilar actions of different antidepressant treatments on two different 5-HT-mediated neuroendocrine functions.

Focal application of alcohols elevates extracellular dopamine in rat brain: a microdialysis study.

Dopaminergic systems are thought to play a major role in the stimulant and reinforcing properties of drugs of abuse, including ethanol. The present study describes the effects of local perfusion with ethanol (and other alcohols) on extracellular dopamine in the striatum and nucleus accumbens. Following the establishment of basal dopamine levels (2-3 h), various concentrations of ethanol in artificial CSF (0.01-10% v/v) were slowly perfused through a microdialysis probe. Each dose of ethanol was found to increase dopamine concentrations in both the striatum and nucleus accumbens. This increase was dose-related in the striatum. The exclusion of calcium and inclusion of 12.5 mM magnesium in the perfusion medium prevented, or greatly attenuated the ethanol-induced dopamine (DA) release. Thus, the release of DA by ethanol is exocytotic in nature and involves calcium-dependent processes. The other alcohols tested, namely methanol and butanol, demonstrated a structure-activity relationship together with ethanol, in their ability to increase extracellular DA. The relative potency was butanol greater than ethanol greater than methanol. The diffusion of ethanol into the brain tissue was investigated following perfusion through the probe. Relatively low concentrations of ethanol were found in striatal tissue during perfusion and they declined rapidly with time, following the removal of ethanol from the perfusate. The concentrations of ethanol achieved in brain tissue following focal application through the microdialysis probe were relevant to human intoxication.

Is NAN-190 an effective antagonist of the hypothermia and hyperglycemia induced by the 5-HT1A receptor agonist, 8-OH-DPAT?

The putative 5-HT1A receptor antagonist properties of 1-(2-methoxyphenyl)-4-[4-(2-phtalimmido)butyl] piperazine (NAN-190) were studied in mice. The responses studied were hypothermia- and hyperglycemia-induced by the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). NAN-190 (0.3-3 mg/kg) did not antagonize either response, but rather appeared to be additive with the effect produced by 8-OH-DPAT (0.25 mg/kg) alone, at least with respect to temperature. NAN-190, given alone in similar doses, caused hypothermia and hyperglycemia. These results suggest that NAN-190 has similar properties to 8-OH-DPAT with regard to temperature and glucose effects. Therefore, it does not appear to be a effective antagonist for all 5-HT1A-mediated responses.

Hyperglycemic properties of serotonin receptor antagonists.

Several serotonin (5-HT) receptor antagonists with varying specificities for the 5-HT receptor types, were studied with regard to their effects on blood glucose levels in mice. The non-selective antagonists, metergoline and methysergide, proved to be hyperglycemic at doses commonly used to antagonize 5-HT receptors. In contrast, ritanserin (a 5-HT2 and 5-HT1c antagonist) and MDL 72222 (a 5-HT3 antagonist) were effective only at doses which surpassed the dose range considered to be selective for their respective receptors. The results suggest that 5-HT systems play a role in maintaining glucose homeostasis and that 5-HT1 receptors may be particularly important in this function. Furthermore, the inherent hyperglycemic properties of non-selective serotonin antagonists described here, are pertinent to studies using these agents to investigate glucose metabolism.

Modulation of the hypothermic and hyperglycaemic effects of 8-OH-DPAT by alpha 2-adrenoceptor antagonists.

1. The effects of pretreatment with two novel and relatively specific alpha 2-adrenoceptor antagonists on the hypothermic and hyperglycaemic responses induced by the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) were investigated in mice. The alpha 2-adrenoceptor antagonists used were, atipamezole, which occupies both central and peripheral receptors, and L 659,066, which poorly penetrates the blood brain barrier. 2. Atipamezole (1 and 3 mg kg-1) alone had no effect on body temperature but significantly attenuated the 8-OH-DPAT-induced hypothermic response. The hyperglycaemic effect of 8-OH-DPAT was also attenuated by pretreatment with atipamezole; however, 3 mg kg-1 atipamezole did cause some hypoglycaemia when administered alone. 3. Pretreatment with L 659,066 (3-30 mg kg-1) failed to alter the hypothermic effects of 8-OH-DPAT. All doses of L 659,066 tested attenuated 8-OH-DPAT-induced hyperglycaemia, but the highest dose (30 mg kg-1) produced hypoglycaemia when administered alone. 4. The results suggest that the attenuation of 8-OH-DPAT-induced hypothermia by alpha 2-adrenoceptor antagonist may be centrally mediated whereas the blockade of 8-OH-DPAT-induced hyperglycaemia may involve peripheral mechanisms.

Differential effect of lithium treatment on fenfluramine-induced decreases in food intake and locomotor activity in rats.

Administration of fenfluramine to rats decreased 1-h food intake and locomotor activity. Short-term (2-6 days) but not long-term (21-25 days) lithium treatment potentiated fenfluramine-induced suppression of food intake. However, neither short-term nor long-term lithium treatment had any significant effect on fenfluramine-induced suppression of locomotor activity. These findings demonstrate a differential effect of lithium treatment on fenfluramine-induced suppression of food intake and locomotor activity. Furthermore, these findings are consistent with results from several other animal and clinical studies demonstrating a differential effect of lithium treatment on two different serotonin-mediated functions.

Antagonism of 5-HT3 receptors attenuates the effects of ethanol on extracellular dopamine.

The effect of a 5-HT3 antagonist, ICS 205-930, on ethanol-induced changes in extracellular dopamine, was investigated with in vivo microdialysis. Pretreatment of rats with ICS 205-930 effectively attenuated the subsequent increases in dopamine, in both the nucleus accumbens and corpus striatum. This suggests that 5-HT may be involved in the effects of ethanol on dopaminergic systems.