Vincristine, an antinoplastic drug, is used for solid tumors and hematologic malignancies. Its use as a chemotherapeutic drug is limited as a result of the development of a dose-related polyneuropathy characterized by painful parethesias and dysesthesias. No effective treatment of chemotherapy-induced neuropathy is currently available. Drugs typically used for neuropathic pain, including anticonvulsants (gabapentin) and antidepressants (amitryptiline), are of limited efficacy for chemotherapy-evoked neuropathy. There is a need for new therapeutic drugs for the prevention and treatment of this pain state.1,2
One potential target for new analgesics effective for neuropathy is the neuronal nicotinic acetylcholine receptor (nAChR).3–6 A variety of nAChR subtypes, the homomeric and heteromeric combinations of α2-α10 and β2-β4 subunits, have been identified so far, but their specific role in analgesia has not been fully elucidated. Significant effort has focused on the α4β2 nAChR found in the central nervous system (CNS) and thought to be involved in analgesia via activation of multiple descending inhibitory pathways originating in the brainstem. The efficacy of several agents acting as agonists at the α4β2 nAChR subtype (nicotine, epibatidine, ABT-894) has been established in preclinical models of pain: nociception, inflammatory pain, neuropathy resulting from nerve trauma or diabetes.7,8 A novel drug from this class, ABT-594, also alleviated allodynia resulting from chemotherapy in rats.9 The α4β2 nAChR is widespread in the CNS, and thus centrally mediated side effects make these agents less desirable analgesic drugs.
A unique approach to avoid side effects related to drug activity at CNS nicotinic receptors has been to focus on the heteromer assembled from 2 different α subunits, α9 and α10. Gene transcripts for α9α10 have been identified in diverse yet limited numbers of tissues but importantly not in the brain; thus, it is unlikely that this subunit may be significantly involved in mediation of CNS-related side effects.10 The α-conotoxins, venom peptides that have been the source of selective antagonists for α9α10 nAChR subtype, demonstrated an interesting pharmacological profile, including effectiveness in rodent models of neuropathic and inflammatory pain; a likely peripheral mechanism of action (no effect after intrathecal administration); ability to accelerate functional recovery from nerve injury; no significant side effects; ability to reduce migration of macrophages, lymphocytes, and ACh-producing cells into the neural and perineural areas of nerve injury.11–13 Modification of the nicotine molecule by quaternization of the pyridine-N atom with a lipophilic substituent (to afford N-substituted analogs) yielded a family of novel nonpeptide small molecule bis-, tris-, tetrakis-azaaromatic quaternary ammonium analogs.14–16 A number of these analogs also showed high affinity and selectivity for α9α10 nAChR expressed in Xenopus oocytes, although they have significantly less potency on other subtypes.17,18 These analogs demonstrated effectiveness against persistent inflammatory pain and posttraumatic neuropathy at doses well below those that produced motor dysfunction in rats.17,19 However, they were not investigated in chemotherapy-evoked pain. To test this possibility the most potent and α9α10-selective agent from the bis-group, ZZ1-61C, was characterized in a rat model of vincristine-induced neuropathy.
Male Sprague-Dawley rats (250 g; Harlan, Indianapolis, IN) were housed individually in transparent cages with a sawdust-covered floor in a humidity- and temperature-controlled Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility (a 12-hour alternative light/dark cycle) with free access to standard laboratory chow and tap water. Body weights (BW) were determined on the day of experimentation. At the end of the experiment, rats were euthanized with medical grade pentobarbital, 150 mg/kg (Vortech, Deaborn, MI) given by the intraperitoneal (IP) route. Rats were allowed to habituate to the housing facility for at least 1 week. Next, they were familiarized (3 times) with the experimental environment/apparatus before the experiments began. In addition, rats were accustomed to the experimental room for at least 30 minutes before testing. All experiments were conducted during the light phase of the cycle (0800–1700). The experiments were performed in accordance with a protocol approved by the University of Kentucky Institutional Animal Care and Use Committee, the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals (1996) prepared by the National Academy of Sciences' Institute for Laboratory Animal Research.
Vincristine sulfate (Tocris Bioscience, Ellisville, MO) was administered IP 100 μg/kg/day (diluted to 1 mL in 0.9% saline) in two 5-day cycles with 2 days break between cycles (10 injections, 1 mg/kg cumulative dose). ZZ1-61c, N,N′-[(1,1′-biphenyl)-4,4′-di-(1-propyn-3-yl)]- bis-(3,4-dimethylpyridinum) dibromide, was synthesized as previously described.18 ZZ1-61c was dissolved in saline and administered 100 μg/kg/day IP either along with or after completion of vincristine treatment.
The experimental design used 1 group of rats per treatment condition with each rat in each group tested using all pain assays. Each treatment was given in two 5-day cycles with 2 days break between cycles (days 1 to 5 and 8 to 12). Group 1 (n = 6 rats) was treated with vincristine, 100 μg/kg/day IP alone to validate a rodent model of chemotherapy-induced neuropathic pain. Group 2 (n = 6 rats) was treated with vincristine, 100 μg/kg/day IP, and ZZ1-61c, 100 μg/kg/day IP to determine the prophylactic effects of ZZ1-61c on inhibition of vincristine-induced neuropathic pain. Control rats were injected with ZZ1-61c alone, 100 μg/kg/day IP (Group 4, n = 6 rats), or saline vehicle, 1 mL/kg/day IP (Group 4, n = 6 rats). Groups 1 to 4 were not drawn from a single pool of subjects. In addition, to assess the treatment effects of ZZ1-61c on vincristine-induced neuropathic pain, another group of rats (n = 12) was exposed to vincristine, 100 μg/kg/day IP, on days 1 to 5 and 8 to 12. Once neuropathic pain was evident (day 15 postvincristine initiation) the rats were randomly divided into 2 groups and then treated with either ZZ1-61c, 100 μg/kg/day IP (group 5A, n = 6 rats), or saline, 1 mL/kg/day (group 5B, n = 6 rats) starting on day 15 and ending on day 33.
Each rat was tested before initiation of the treatment (day 1, baseline, assessed twice, 15 minutes apart) and then every other day (starting at day 3 after the first injection) across the time of study (33 days). Control rats (ZZ1-61c, saline) were also assessed every other day (days 1 to 15). Responsiveness was measured before each injection (except rats in groups 5A and 5B; testing before and 15 minutes after administration of ZZ1-61c or saline). Two well-established tests based on the use of mechanical stimuli were used. First, rats were placed on the elevated wire mesh floor and then von Frey monofilaments (VFH) with bending forces of 4, 8, and 15 g (Stoelting, Wood Dale, IL) were applied in ascending order to the mid-plantar side of the hindpaw (5 times/paw) and held for approximately 5 seconds.20 The number of withdrawals was recorded for each paw, averaged, and expressed as an overall percentage response (paw withdrawal, %PW) in each rat. Next, rats were tested by the paw pressure technique21 using the Basile Analgesimeter (UGO, Milan, Italy). Briefly, increasing pressure was applied to the dorsal side of the paw with vocalization used as the end point (32 g/s; cutoff 300 g). Each paw was tested 3 times; responses were averaged and presented as a vocalization threshold (VT, g). The observer was blinded to treatment.
The effect of ZZ1-61c on motor coordination was determined using the rotarod performance test. Naïve rats were trained to run on the Rat Rota Rod (Ugo Basile, Comeno, Italy) at a constant speed (10 rev/minutes) for 180 seconds (cut off). Thereafter, they were injected with ZZ1-61c 100 μg/kg/day IP for 3 subsequent days. Latency to fall off the drum was recorded before (baseline) and at 15, 30, 45, and 60 minutes after injection.
The effect of ZZ1-61c on hindlimb weakness was assessed in the grip strength test using a Digital Grip Strength Meter (Columbus Instruments, Columbus, OH). The rat grasped the bar with both forepaws and then was steadily pulled backwards by the tail until its grip was broken. Each rat was tested before (baseline) and 15, 30, 45, and 60 minutes (3 measurements per time) after repeated administration of ZZ1-61c 100 μg/kg/day IP on days 1 to 3.
All data are expressed as mean ± SEM for n rats. The time courses (%PW or VT) were generated for each treatment. The areas under the curves (AUC0-t) were calculated by the trapezoidal rule. One-way ANOVA, post hoc Dunnett's method, t-paired test, t-test, and Wilcoxon's signed rank test were used (SigmaPlot for Windows version 11.0, Systat Software Inc.).
The daily injections of vincristine (100 μg/kg/day IP, days 1 to 5 and 8 to 12) resulted in a decrease in thresholds (compared to day 1 pretreatment baseline). This was indicated by exaggerated paw withdrawal responsiveness to monofilament fibers (4, 8, and 15 g) and vocal responses in the paw pressure test (F14,89 = 28.4, 208.6, 22.8, 59.5, respectively; P < 0.001, 1-way ANOVA with Dunnett's test). Significant pain sensitivity was first noted after the fifth vincristine injection (first cycle), it peaked after the 10th injection (second cycle) and gradually declined when dosing ceased (Fig. 1, A–D; group 1).
Responsiveness to VFH and paw pressure was still elevated (relative to day 1 pretreatment baseline) in the presence of ZZ1-61c (coadministration of drugs; 100 μg/kg/day IP of each; days 1 to 5 and 8 to 12) (F14,89 = 4.9, 5.2, 3.2, 13.1 for 4, 8, 15 g VFH and VT, respectively; P < 0.001, one-way ANOVA with Dunnett's test) (Fig. 1, A–D; group 2). Nevertheless, pain hypersensitivity was markedly less (lower %PW, higher VT) for a vincristine and ZZ1-61c combination therapy versus vincristine. The AUC1 to 15 day values were significantly different (t-test) for vincristine plus ZZ1-61c versus vincristine alone: 421.7 ± 42.2 versus 628.2 ± 37.5 (4 g VFH, P = 0.004); 600.8 ± 32.4 versus 871.6 ± 29.8 (8 g VFH, P < 0.001); 875 ± 44.8 versus 1122.2 ± 20.9 (15 g VFH, P < 0.001) and 3356.6 ± 43.8 versus 1653.4 ± 9.8 (VT, P < 0.001). Similar differences (t-test) were observed when the recovery period also was included (AUC1 to 33 day): 910.8 ± 54.6 versus 1174.8 ± 12.7 (P < 0.001); 1422.3 ± 79.0 versus 1812.0 ± 42.2 (P = 0.001); 1998.3 ± 113.1 versus 2411.2 ± 23.9 (P = 0.005); and 4472.0 ± 42.2 versus 4261.9 ± 13.7 (P < 0.001) for 4 g, 8 g, and 15 g VFH and VT, respectively. No significant effects (relative to baseline) were seen after administration of ZZ1-61c or saline in control rats (Fig. 1, A–D; group 3 and 4, respectively).
Pain sensitivity was significantly enhanced after vincristine treatment (measured on day 15 after the initiation of vincristine) as compared to naïve values (day 1, previncristine) (P < 0.001, paired t-test) regardless of noxious stimuli used (Fig. 2, A–D). These rats were subsequently treated with ZZ1-61c or saline during the postvincristine recovery period (commencing on day 15 and continued through day 33 of the study). Overall, thresholds were higher (decreased %PW, increased VT) in rats treated with ZZ1-61c compared to rats which were injected with saline (Fig. 2, A–D; group 5A and 5b, respectively). The AUC17 to 33 day for ZZ1-61c versus saline (t-test) were 390.0 ± 63.4 versus 407.5 ± 29.4 (4 g VFH, nonsignificant); 656.7 ± 63.4 versus 755.8 ± 21.4 (8 g VFH, P = 0.075); 947.5 ± 43.2 versus 1049.2 ± 7.9 (15 g VFH, P < 0.05); and 2212.4 ± 11.7 versus 2506.8 ± 30.0 (VT, P < 0.001). It is notable that the immediate effect of ZZ1-61c (15-minute postinjection versus preinjection pain threshold) was of statistical significance only after the first injection (day 15) but not with the subsequent dosing (Fig. 3, A–D).
The next experiment was conducted to determine the side effects of ZZ1-61c. No changes in motor coordination (rotarod performance test) or muscle strength (grip strength test) were seen during repeated administration of ZZ1-61c, 100 μg/kg/day IP, in naïve rats (Fig. 4A and B).
Vincristine caused a moderate BW loss in rats. This effect was inhibited by coadministration of ZZ1-61c. At maximum (day 12), BW (% of initial value) = 95.5% ± 0.5 versuss 86.7% ± 1.1 (P < 0.01, t-test) for vicristine plus ZZ1-61c versus vincristine alone. At the end of a study, BW gain also was greater in rats previously exposed to combination therapy than vincristine alone (day 33): BW = 128.5% ± 1.5 versus 115.3% ± 2.5 (P < 0.001, t-test). Administration of ZZ1-61c after completion of vincristine treatment had no effect on BW gain (day 33): BW = 116.0% ± 1.7. Naïve rats repeatedly dosed with ZZ1-61c gained weight normally (day 15): BW = 104.1 ± 0.47% (ZZ1-61c) versus 105.0 ± 0.62% (saline). No mortality or apparent morbidity occurred when vincristine was given alone or in combination with ZZ1-61c (eg, no obvious changes noted when rats were observed in their home cages for motor activity, guarding, gait, and abnormally formatted stool).
The ability of a novel nonpeptide small molecule α9α10 nAChR antagonist, bis-quaternary ammonium salt ZZ1-61c, to prevent and/or correct vincristine-evoked neuropathic pain was characterized in the present work. Partial but significant inhibition/reversal of pain hypersensitivity was demonstrated at a dose that did not result in overt side effects. ZZ1-61c's potential as a new drug for chemotherapy-induced neuropathy may be suggested.
Painful neuropathy was repeatedly demonstrated after repeated bolus injections or continuous IV infusion of vincristine in rodents.22–24 The present data confirmed previous findings25 that intermittent systemic exposure to vincristine (100 μg/kg/day IP on a 5-day–on, 2-day–off, 5-day–on schedule) was sufficient enough to reduce nociceptive thresholds in rats. This effect was demonstrated using VFH of different bending forces or an increasing pressure to the paw. Pain sensitization could not be attributed to repeated testing because it was not seen in control rats. As previously reported, naïve rats rarely withdraw paws from the 4-g filament (normally innocuous stimuli), whereas only 10%–20% of rats withdraw from the 15-g force (noxious stimuli) so enhanced responses to these stimuli are thought to be indicative of mechano-allodynia and mechano-hyperalgesia, respectively.20,26 Naïve baseline responsiveness was noticeably higher, in our study. This was a consistent observation.27 The reason for these differences is not clear. Increased sensitivity to VFH (withdrawal threshold) was evident starting with the fifth dose of vincristine and lasted for a period of ∼20 days. The present data also showed marked changes in responsiveness to the paw pressure test. Nociceptive thresholds (a vocal response) also were reduced at day 5 of vincristine dosing but remained lowered for the whole study (33 days). The discrepancy in time courses of hypersensitivity to pressure may likely be explained by facilitation of inputs from different fibers (progressive versus sharp mechanical stimulation).28
The present data clearly showed efficacy of ZZ1-61c. First, it inhibited the development of neuropathy resulting from vincristine (alleviation of pain sensitivity during chemotherapy). Second, it reversed established neuropathy (restoration of pain sensitivity after chemotherapy). Third, the acute effect of ZZ1-61c (pre- versus postinjection responses) was weak and transient, thereby suggesting the persistent effect rather than the immediate effect. Fourth, ZZ1-61c was not an active analgesic agent on its own (naïve rats). Fifth, no motor dysfunction or muscle weakness was seen at the tested dose of ZZ1-61c. Vincristine (100 μg/kg) was ineffective in the rotarod29 and grip strength30 tests; however, potential interaction between these drugs cannot be excluded. In our study, responsiveness was not confounded by motor impairment because an increase in pain thresholds was seen regardless of whether ZZ1-61c was given along with or after discontinuation of vincristine. In agreement with the earlier data,30,31 vincristine caused a moderate BW loss in rats. Notably, this effect also was hampered by coadministration of ZZ1-61c. The present findings may suggest that ZZ1-61c has disease modification/prevention properties, ie, ability to preempt and/or correct neuropathy caused by vincristine. Whether ZZ1-61c interferes with chemotherapeutic activity of vincristine needs to be further investigated.
Because this work was merely of an exploratory nature, its limitations must be considered. First, a dose of ZZ1-61c was based on maximum efficacy in a formalin model of persistent inflammatory pain.18 Caution must be taken when comparisons are made between different pain models; a wide range of doses ought to be implemented in a further study. Second, because ZZ1-61c acts as antagonist at nAChRs17,18 and has no efficacy on its own desensitization of receptors is less likely to occur. Even so, it is of interest to determine if efficacy remains consistent with repeated dosing (a cumulative dose paradigm). Third, no positive control was used. A number of drugs from different classes were characterized earlier in a similar paradigm.32 For example, the nAChR agonist, ABT-594,9 and selective T-type calcium channel blocker, ethosuximide,20 reversed vincristine-induced neuropathy in a dose-dependent manner. The positive effects were noted with anticonvulsant,33,34 neurosteroids,35 cannabinoids,36 and drugs that enhance mitochondrial function.26 Mixed results were demonstrated with common analgesic drugs such as opioids and nonsteroidal antiinflammatory drugs.33,37–39 Fourth, strikingly, the pain-enhancing effect of vincristine was comparable in magnitude to that seen after chronic constriction nerve injury (reduction VT ∼80 g, the paw pressure test).19,27,40 Even though behavioral outcomes appear similar, the mechanisms are thought to be somewhat distinct in these pain models (ie, axonal degeneration in peripheral nerves accompanies painful neuropathy secondary to nerve trauma but not chemotherapy).22–24 From a pain treatment perspective, drugs that modify cellular calcium levels were effective against pain sensitization caused by toxic but not traumatic nerve insult.41 On the contrary, effectiveness of drugs acting as antagonists at the N-Methyl-d-Aspartate receptors was well-established in the later model yet was not seen in a former model of neuropathy.20 In this context, it is remarkable that analogs from a family of tetrakis-, tris-, and bis-quaternary ammonium salts (lead compounds highly selective for α9α10 nAChR) alleviated signs of pain hypersensitivity that was derived from neurotoxicity or nerve trauma; in that, partial reversal of pain hypersensitivity in vincristine-treated rats (present study) was comparable to what was previously seen in rats with chronic nerve injury.19,42 They also blocked, in a dose-related manner, formalin-evoked flinching (a late phase, which is thought to involve central sensitization) in a rat model of persistent inflammatory pain. None of them was active in a rat model of acute nociception (tail-flick test) suggesting a limited role of α9α10 in this type of pain.17,19,42
Activation of central α4β2 (and to lesser extent α7) subunits nAChR have been associated with analgesic effects of nicotinic agonists.3–6 Nevertheless, peptides α-conotoxins (Vc1, RgIA) that act as antagonists at α9α10 nAChR also show analgesic activities in rodent models of neuropathy (chronic constriction nerve injury, spinal nerve ligation, diabetes) and inflammation (complete Freud's adjuvant). Although the mechanism of action is unknown, it has been thought that blockade (antagonism of α9α10 nAChR) may reduce the number of macrophages and T-cells at the site of injury (both α9 and α10 are expressed in a variety of immune cells), which in turn contributes to the cumulative analgesic and restorative effects of α-conotoxins.10,11 The antagonism of α9α10 nAChRs in sensory nerves (expression of α9 and α10 in sciatic nerve is unknown),11,13 interaction with α3(α5)β2 and α3(α5)β4 nAChR43 and action via G-protein-coupled γ-aminobutyric acidB receptors44,45 have also been proposed as mechanisms of action. Novel nonpeptide small molecule quaternary ammonium analogs also potently blocked α9α10 and discriminated between α9α10 and α7 nAChR (IC50 similar to those for α-conotoxins). In this regard, bis-analog ZZ1-61c (used in our study) showed 75-fold greater selectivity for the α9α10 subtype over the α7 subtype (IC50 = 16 versus 1190 nM) and even lower antagonist activities at other nAChR subtypes (eg, IC50 = 4000 nM for the ganglion type α3β4). Lead agents from tris- (GZ5-56A) or tetrakis- (ZZ1-04G) group also demonstrated preference as antagonists for the α9α10 versus the α7 (50- and 10-fold, respectively) or the α3β4 (26- and 1220-fold, respectively) subtype.17,18,42 Discrimination between α9α10 and α7 nAChR is important, whereas antagonism of the former receptor is desirable, antagonism of the latter receptor would not be. The antiinflammatory effectiveness (ie, suppression of release of proinflammatory cytokines from macrophages) appears to be achieved with both α9α10-selective antagonists and α7-selective agonists at nicotinic receptors. The expression of α9α10 appears to overlap with that of α7 nAChR (eg, dorsal root ganglion neurons, lymphocytes); however, the α9α10 subunit nAChR has restricted distribution in tissues whereas the α7 nAChR is abundant in the CNS. The mechanism of action of quaternary ammonium analogs is expected to be predominantly peripheral rather than central (no α9α10 receptors in brain). In addition, the quaternary structure of these compounds makes them less likely to cross the blood–brain barrier and enter the CNS. Although highly speculative, it is likely that antagonism of α9α10 nAChR and reduction of inflammation at the site of injury (decreased accumulation of microphage and T-cells) may underlie efficacy of these agents in rat models of pain with inflammatory components such as the formalin test and chronic constriction nerve injury; however, other mechanism(s) cannot be excluded. The mechanism of action against vincristine-evoked neuropathy seems even more complex, ie, mononuclear inflammatory cells were not evident in neuronal specimens in this type of pain condition. Notably, there is a line of evidence that chemotherapy-induced neuropathy may likely result from inflammatory mediators involving proinflammatory cytokines produced by nonneuronal cells in peripheral nerves (Schwann cells).23 Therefore, more studies are needed to determine whether antagonism of α9α10 nAChR has immune consequences that may play a role in the prophylactic and symptomatic effects against vincristine-evoked pain.
In summary, efficacy of α9α10 nAChR antagonist, a nonpeptide bis-quaternary ammonium analog ZZ1-61c, was shown in a rat model of chemotherapy-evoked neuropathy.
The present preliminary findings may represent a new lead in the search for a novel class of peripherally acting nicotinic antagonists. Such a strategy may provide effective treatments, which circumvent toxicity of centrally acting nicotinic receptor agonists.
Name: Elzbieta P. Wala, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Elzbieta P. Wala has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Peter A. Crooks, PhD.
Contribution: This author helped design the study, conduct the study, and write the manuscript.
Attestation: Peter A. Crooks has seen the original study data and approved the final manuscript.
Name: J. Michael McIntosh, MD.
Contribution: This author helped design the study and write the manuscript.
Attestation: J. Michael McIntosh has seen the original study data and approved the final manuscript.
Name: Joseph R. Holtman, Jr., MD, PhD.
Contribution: This author helped design the study and write the manuscript.
Attestation: Joseph R. Holtman, Jr. has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Steven L. Shafer, MD.
Technical help of Mitchell Elliott and Jonathan Walter (University of Kentucky) is greatly appreciated.
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