A painful peripheral neuropathy in the rat produced by the chemotherapeutic drug, paclitaxel

Pain 94 (2001) 293–304 www.elsevier.com/locate/pain

A painful peripheral neuropathy in the rat produced by the chemotherapeutic drug, paclitaxel Rosemary C. Polomano a,*, Andrew J. Mannes b, Uraina S. Clark c, Gary J. Bennett c,1 a

Departments of Anesthesiology and Neuroscience & Anatomy, Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA b Department of Anesthesiology, University of Pennsylvania, Philadelphia, PA 19104, USA c Department of Neurology, MCP Hahnemann University, Philadelphia, PA 19102, USA Received 2 October 2000; received in revised form 18 May 2001; accepted 12 June 2001

Abstract Paclitaxel, an effective anti-neoplastic agent in the treatment of solid tumors, produces a dose-limiting painful peripheral neuropathy in a clinically significant number of cancer patients. Prior work has demonstrated paclitaxel-induced neurodegeneration and sensory loss in laboratory rodents. We describe here an experimental paclitaxel-induced painful peripheral neuropathy. Adult male rats were given four intraperitoneal injections on alternate days of vehicle or 0.5, 1.0, or 2.0 mg/kg of paclitaxel (Taxolw). Behavioral tests for pain using mechanical and thermal stimuli applied to the tail and hind paws, and tests for motor performance, were taken before, during and after dosing for 22–35 days. All three doses of paclitaxel caused heat-hyperalgesia, mechano-allodynia, mechano-hyperalgesia, and cold-allodynia, but had no effect on motor performance. Neuropathic pain began within days and lasted for several weeks. We did not detect any dose–response relationship. Tests at the distal, mid, and proximal tail failed to show evidence of a length-dependent neuropathy. Vehicle control injections had no effect on any measure. No significant systemic toxicities were noted in the paclitaxel-treated animals. Light-microscopic inspection of the sciatic nerve (mid-thigh level), L4–L5 dorsal root ganglia, and dorsal and ventral roots, and the gray and white matter of the L4–L5 spinal cord, showed no structural abnormalities. Electron microscopic examination of the sciatic nerve (mid-thigh level) and the L4–L5 dorsal root ganglia and dorsal horns demonstrated no degeneration of myelinated and unmyelinated axons in the sciatic nerve and roots, but revealed endoneurial edema. This model may be useful in understanding a significant source of pain in cancer patients, and in finding ways to avoid the neurotoxicity that limits paclitaxel therapy. q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Allodynia; Chemotherapy neurotoxicity; Hyperalgesia; Neuropathy; Paclitaxel; Painful neuropathy; Taxanes; Taxol

1. Introduction Paclitaxel (Taxolw, Bristol Myers-Squibb; Princeton, NJ, USA) is one of the most effective and commonly used antineoplastic drugs for the treatment of solid tumors. It has two serious side effects, myelosuppression and peripheral neurotoxicity. Granulocyte colony-stimulating factor effectively counteracts the neutropenia in most patients. Unfortunately, there are no acceptable therapies to prevent or minimize the nerve damage, making neurotoxicity a significant doselimiting side effect (Rowinsky et al., 1993a,b; Wasserheit et al., 1996; Gordon et al., 1997). * Corresponding author: Tel.: 11-717-531-4085; fax: 11-717-531-5434. E-mail address: [email protected] (R.C. Polomano). 1 Present address: Anesthesia Research Unit, McGill University, 1202 McIntyre Bldg, 3655 Promenade Sir William Osler, Montreal, QC, Canada, H3G 1Y6.

Clinically, paclitaxel-induced neurotoxicity typically presents as a sensory neuropathy, with the most common complaints being numbness, tingling and burning pain. Sensory symptoms usually start symmetrically in the feet, but sometimes appear simultaneously in both hands and feet. Most cases resolve within months after paclitaxel treatment is discontinued, but the sensory abnormalities and pain sometimes become a chronic problem (Rowinsky et al., 1993b; Chaudhry et al., 1994; Cavaletti et al., 1995a; Forsyth et al., 1997; Gordon et al., 1997; van den Bent et al., 1997). The incidence of peripheral neuropathy with paclitaxel monotherapy at doses of 250 mg/m 2 and greater ranges from 22 to 100%. The occurrence and severity of the neuropathy is dependent on single dose intensity, duration of infusion, cumulative dose, prior or concurrent treatment with cisplatin, and co-existing conditions such as diabetes and alcohol abuse (Wiernik et al., 1987a,b; Lipton et al.,

0304-3959/01/$20.00 q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(01)00363-3

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1989; Brown et al., 1991; Rowinsky et al., 1993b; Chaudhry et. al., 1994; Cavaletti et al., 1995a; Connelly et al., 1996; McGuire et al., 1996; Rowinsky and Donehower, 1995; Wasserheit et al., 1996; Gordon et al., 1997; Forsyth et al., 1997; van den Bent et al., 1997; Helsing et al., 1999; Akerley, 2000; Langer et al., 2000). A clinically significant number of patients with paclitaxel-induced neuropathy experience neuropathic pain (Kaplan et al., 1993; Rowinsky et al., 1993b; Chaudhry et al., 1994; van Gerven et al., 1994; Hilkens and van de Bent, 1997; Pace et al., 1997; Helsing et al., 1999). For example, of the 37 advanced breast cancer patients treated with doses of 200–250 mg/m 2, 81% complained of dysesthesiae (numbness and tingling) and 22% also experienced neuropathic pain, which was classified as severe in 11% (Forsyth et al., 1997). In another series of 60 patients with various types of cancer, Lipton et al. (1989) reported eight patients (13%) who received 250 mg/m 2 or more who experienced lancinating pain, ‘hyperpathia’, and/or ‘burning dysesthesiae’. Wiernik et al. (1987a) reported severe neuropathic pain in five of the five patients receiving 275 mg/m 2 or more. Laboratory investigations of this clinically significant problem have been limited by the absence of a suitable animal model. We report here our success in producing a paclitaxel-induced painful peripheral neuropathy in the rat. Early work on this model appears in abstract form (Polomano et al., 1998). 2. Methods This investigation was conducted at MCP Hahnemann University in accordance with a protocol that was approved by our Institutional Animal Care and Use Committees and in compliance with Federal law, the regulations of the National Institutes of Health, and the guidelines of the International Association for the Study of Pain (Zimmermann, 1983). 2.1. Animals and drug treatment Adult male Sprague–Dawley rats (Harlan Sprague– Dawley Inc., Indianapolis, IN, USA) were housed with sawdust bedding under a natural light:dark cycle with unrestricted access to food and water. In the rat, paclitaxel’s major route of elimination is via unaltered drug and metabolites in the feces and about 10% of unaltered drug may be present in the urine for up to 24 h after injection (Monsarrat et al., 1990). Therefore, soiled bedding was treated as potentially toxic waste and handled and disposed of appropriately. After habituation to the test environments and baseline measurements of pain sensitivity (see below), four groups of rats were injected intraperitoneally (i.p.) on four alternate days (days 1, 3, 5, and 7) with vehicle or paclitaxel (0.5, 1.0, or 2.0 mg/kg); using an injection volume of 1 ml/kg. Cumu-

lative paclitaxel doses were thus 2.0, 4.0, or 8.0 mg/kg. The vehicle was a mixture of 10% saline and Cremophor EL w, a derivative of castor oil and ethylene oxide that is used clinically for paclitaxel injections (Sigma Chemicals, St. Louis, MO, USA). Previous work suggested that 8 mg/kg approximates the rat’s i.p. threshold dose for systemic toxicity (Cavaletti et al., 1995b). Tests for altered pain sensitivity began on day 3 and continued to day 35. When an injection was to be given on the same day as behavioral testing, all rats were injected after the measurements were taken. All behavioral assays were conducted by an observer who was blinded to drug condition. 2.2. Behavioral assays The plantar surface of the hind paws (sciatic nerve territory) was tested for heat-hyperalgesia, mechano-allodynia, and cold-allodynia. Measurements of mechano-allodynia preceded cold-allodynia in order to avoid cold-induced reductions in mechanical thresholds (Kauppila, 2000). The tail was tested for heat-hyperalgesia, mechano-hyperalgesia, and cold-allodynia. Mechano-allodynia was not assessed in the tail because the tail’s skin is scaly and sparsely covered with short, stiff bristles, which precludes the use of von Frey hairs. 2.2.1. Hind paw heat-hyperalgesia Heat-hyperalgesia of the hind paw was tested using methods described by Hargreaves et al. (1988) and Bennett and Hargreaves (1990)). Briefly, the rat was placed within a plastic compartment atop a glass floor; a light source beneath the floor was aimed at the skin of the fat part of the heel. The nocifensive withdrawal reflex interrupts the light reflected from the heel onto a photocell and automatically turns off the light and a timer. The intensity of the light was adjusted at the start of the experiment such that average baseline latencies were about 7 s and a cut-off latency of 15 s was imposed. Latency to withdrawal defines the heat-pain threshold. Three latencies were obtained alternately from each hind paw 5-min apart and the first measurement per side was discarded (as in the tail-flick test, the first response is often anomalously long). We observed no consistent left vs. right differences, and so each rat’s score is the average of the four latencies obtained bilaterally. 2.2.2. Hind paw mechano-allodynia Mechano-allodynia of the hind paw was assessed with the von Frey hair test (Tal and Bennett, 1994; Xiao and Bennett, 1994; Johansson and Bennett, 1997). Hairs (nylon monofilaments; North Coast Medical Inc., San Jose, CA, USA) from the standard Semmes–Weinstein series (Semmes et al., 1960) were ranked (omitting the three stiffest hairs) from smallest (rank #1 ¼ 0.005 g of force) to the largest (rank #17 ¼ 75 g of force), with rank #17 assigned if there was no response to any hair. Testing began with the monofilament ranked #10 (2.06 g of pressure). With the rat stand-

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ing on a wire mesh floor and confined beneath an inverted plastic cage, the monofilament was pressed against the plantar surface of the hind paw until it bent. This was repeated three times and if there was no withdrawal response to any of the three applications, then the next higher stimulus was tried. If there was a response, then the next lower stimulus was tried. Testing continued in the descending or ascending order until the rank value of the lowest stimulus evoking at least one withdrawal response was determined for each paw, which were averaged and designated as the threshold for the testing session. 2.2.3. Hind paw cold-allodynia Cold-allodynia of the hind paw was assessed using the acetone drop method described by Choi et al. (1994). With the rats standing on the wire mesh floor and confined beneath an inverted plastic cage, a drop of acetone was placed on the skin of the heel. Stimulation was repeated for three trials on each side with an inter-trial interval of 5 min. Normal rats either ignore the stimulus or occasionally respond with a very small and brief withdrawal. Neuropathic rats respond frequently with a withdrawal that is clearly exaggerated in amplitude and duration. Responses to cold were expressed as a frequency of response (percent), calculated by taking the number of trials accompanied by brisk paw withdrawal £ 100/the total number of trials (Choi et al., 1994). Three baseline sessions were conducted on separate days and the last two were averaged to yield one baseline measure. 2.2.4. Tail heat-hyperalgesia Heat-hyperalgesia of the tail was assessed as described above for the hind paws, except that the rat was confined in a clear Plexiglas cylinder from which its tail protruded. A cutoff latency of 20 s was established. The heat was directed to the distal tail (22 mm from the tip), three trials were done at intervals of 5 min, and one score was assigned for each session by averaging the last two trials. A value for the baseline measurement was obtained by averaging the results from two sessions conducted on different days prior to dosing. 2.2.5. Tail mechano-hyperalgesia Mechano-hyperalgesia of the tail was measured using a modified Ugo-Basile Analgesymeter (Biological Research Apparatus, Varese, Italy). The device delivers gradually increasing pressure to the tail, which is placed upon a pedestal and opposed by a stylus. The pressure needed to evoke a withdrawal reflex defined the pain threshold. We replaced the standard round-tipped stylus with a bar-shaped stylus with a slightly rounded edge, approximately 1 mm wide, which was placed at a right angle to the tail. With the rat confined within a Plexiglas cylinder, three sites (22, 45, and 90 mm from the tip of the tail) were tested consecutively, starting at the distal site and moving proximally. Three trials were done at each of the three sites, with an inter-trial inter-

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val of 5 min. A separate score for each site was obtained by averaging the thresholds found in the last two trials. A value for the baseline measurement was obtained for each of the three sites by averaging the results obtained from two sessions conducted on different days prior to dosing. Clinically, paclitaxel-induced neuropathy has features resembling both a symmetrical, length-dependent neuropathy of the dying-back type, and of a neuronopathy (Lipton et al., 1989; Rowinsky et al., 1993b; Chaudhry et al., 1994; Cavaletti et al., 1995a; Forsyth et al., 1997). We attempted to examine this issue by measuring mechanically evoked pain thresholds at three locations on the tail: distal, mid, and proximal (22, 45, and 90 mm from the tip, respectively). If the effect were a dying-back neuropathy, then one would expect to see symptoms (hypo- or hyper-responsiveness) first at the distal tail, then at mid tail, and subsequently at the proximal tail. 2.2.6. Tail cold-allodynia Cold-allodynia of the tail was measured by immersing the tip of the tail in ice water (48C) and timing the latency to a withdrawal reflex (Na et al., 1994). A cut-off latency of 15 s was imposed. Normal rats rarely respond within this period, while neuropathic rats respond frequently. 2.2.7. Motor coordination Motor coordination was evaluated with an accelerating rota-rod device (diameter of rotating rod, 7.0 cm) (modified Rollodrum, New Brunswick Scientific Co., Edison, NJ, USA). Accelerating rota-rod testing, originally described by Jones and Roberts (1968), has been commonly used to assess motor performance associated with chemotherapy neurotoxicity (Aley et al., 1996; Cliffer et al., 1998; Tredici et al., 1998). We tested 24 additional rats that received either the Cremophor vehicle or 1 or 2 mg/kg of paclitaxel (n ¼ 8/group) over 22 days. Our initial setting was 6 rpm and the rotation was accelerated by 2.5 rpm every 10 s. The performance on the rod was measured in seconds from placement of the rat on the stationary rod to the time that it fell off following acceleration of the rod. Three baseline sessions were performed on separate days prior to treatment; each included two trials separated by about 30 s. Testing was repeated periodically during the dosing regimen and thereafter until day 22. The scores for each session were the average of two trials. 2.3. Histology For light microscopy, three rats that had received the highest cumulative dose (8 mg/kg) and one vehicleinjected control were sacrificed on day 14 (1 week after the last injection, and a time when pain hyper-sensitivity is clearly developed, see below). When deeply anesthetized following a sodium pentobarbital overdose (100 mg/kg i.p.), the rats were perfused transcardially with 150 ml of saline followed by 300 ml of freshly prepared 4% parafor-

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maldehyde. The lumbosacral vertebral column and a segment of the sciatic nerve from mid-thigh were harvested and post-fixed in the same solution. The vertebral column was dissected to obtain the L4 and L5 segments (which receive the bulk of the sciatic innervation of the hind paw), and the corresponding dorsal root ganglia, and dorsal and ventral roots. The tissue was embedded in paraffin, sectioned at 4 mm, and stained with hematoxylin and eosin, or Luxol Fast Blue. For electron microscopy, five rats that had received the highest cumulative dose (8 mg/kg) and three vehicleinjected controls were sacrificed between days 19 and 22. Following deep anesthesia with sodium pentobarbital (100 mg/kg; i.p.), rats were perfused transcardially with 150 ml of heparinized (1.5 U/ml) saline with lidocaine (0.015 mg/ ml) followed by 300 ml of freshly prepared Karnovsky solution of a 1:7 dilution of full strength Karnovsky fixative (4% paraformaldehyde and 5% gluteraldehyde) in 0.1 M sodium cacodylate buffer containing 4% polyvinylpyrrolidone (PVP-40T). A segment of the sciatic nerve from midthigh, L4 and L5 dorsal root ganglia (DRG), and the L4/ L5 spinal dorsal horns were harvested and post-fixed for 24 h in full strength Karnovsky fixative. Following fixation, tissues were rinsed in 0.1 M sodium cacodylate to postfixation with 1% osmium tetroxide in 0.1 M sodium cacodylate for 2 h. Tissues were dehydrated by moving them sequentially through 30, 50, 70, and 95% ethyl alcohol. Infiltration with serial dilutions of propylene oxide and EMbed 812 (2:1, 1:1, 1.2) or a single 1:1 dilution of propylene oxide followed by Embed 812. Thin sections were stained with uranyl acetate and lead citrate and examined in a Philips 400 electron microscope.

3. Results 3.1. Animals’ general health Body weight averaged 300.7 g (^SD 32.6, N ¼ 40) at the start of the experiment. Animals in all groups continued to gain weight normally, and there was no significant difference in the average amount of weight gain (g) between groups during 28 days of testing (one-way ANOVA, Fð3; 33Þ ¼ 0:962, P ¼ 0:422, post hoc analysis LSD) (Fig. 1). All rats survived until the end of the study with the exception of one rat from the 1.0-mg/kg group, which was sacrificed on day 11 when it was obviously moribund (its data are omitted from the behavioral results given below). At necroscopy, a distended, hemorrhagic lower bowel was noted, possibly from a misplaced i.p. injection complicated by thrombocytopenia (a side-effect of paclitaxel). A rat in the Cremophor control group was also eliminated from the analysis after completing the study when it was found to have pneumonia (confirmed at necroscopy). For the remaining animals, general appearance was normal, except for mild alopecia in some of the rats receiving 2.0 mg/kg. Concern about the possible deleterious effects of i.p. injections of both paclitaxel and the Cremophor vehicle prompted us to perform a pilot evaluation prior to the study in which eight rats (two each receiving Cremophor vehicle, or 0.5, 1.0, or 2.0 mg/kg of paclitaxel) were dosed as described above and sacrificed on day 9 (2 days after the last dose). At necroscopy, only one of the rats (0.5 mg/kg dosing group) showed any sign of intra-abdominal pathol-

2.4. Statistical analysis Continuous data (e.g. tests for hind paw and tail heathyperalgesia and tail mechano-hyperalgesia) were analyzed using repeated measures analysis of variance (ANOVA) and one-way ANOVA followed by post hoc analyses with least significant difference (LSD) test. Non-parametric statistics (Kruskal–Wallis and Mann–Whitney tests) were applied to the results yielding rank or ordinal level data (tests for hind paw mechano- and cold-allodynia). For cold-allodynia of the tail, a large percentage of animals reached the cut-off latency (15 s), and therefore the data were not normally distributed. These data were analyzed by logistic regression analyses using generalized estimating equations (GEE) which accounts for correlations between serial measurements in the same rat (Liang and Zeger, 1993). The level of significance for all statistical tests was set at P , 0:05. Bonferonni adjustments in alpha values were made when multiple comparisons of the data were conducted. All statistical tests were performed using SPSS 9.0 for Windows (SPSS, Inc., Chicago, IL, USA) and SAS 8.0 (SAS Inc., Cary, NC, USA).

Fig. 1. Mean increase in body weight (g) for the vehicle control group and the three paclitaxel dosing groups are plotted for all rats during the experiment. Arrows beneath the abscissa indicate days of treatment (Tx). Overall weight gain (mean ^ SEM) for each group up to day 28 was 61.1 g ^ 4.8 for the vehicle control group (n ¼ 9), and 61.4 g ^ 4.3 (n ¼ 8), 74.4 g ^ 8.3 (n ¼ 13), and 62.3 g ^ 8.1 (n ¼ 9) for the 0.5, 1.0, and 2.0 mg/ kg paclitaxel dosing groups, respectively. No statistically significant difference in the average amount of weight gain between groups was observed (one-way ANOVA).

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ogy: four non-hemorrhagic lesions on the outer surface of the small intestine and approximately 3 ml of ascites fluid were present. The origin of this pathology is obscure. In summary, adverse effects were infrequent, there were no major toxicities noted in the highest paclitaxel dosing group (2.0 mg/kg) and there was no effect on weight gain. We conclude that with i.p. injections, the Cremophor vehicle, paclitaxel doses, and dosing schedule used in this study have little or no significant effect on the animals’ general health. 3.2. Behavioral assays All three doses of paclitaxel produced significant thermal and mechanical hyperalgesia and allodynia in both the hind paws and tail. We observed no evidence of a dose–response relationship. In the hind paw tests, there were no consistent left–right differences, suggesting that paclitaxel induced a symmetrical neuropathy. 3.2.1. Heat-hyperalgesia in the hind paw At baseline (Fig. 2), there were no significant betweengroup differences in mean scores for heat-evoked response latencies (s). Paclitaxel treatment produced significant heathyperalgesia (repeated measures ANOVA, Fð3; 24Þ ¼ 6:60; P , 0:01). Post hoc analyses of overall group means showed that the 0.5 (P , 0.05) and 1.0 mg/kg (P , 0:001) dosing groups, but not the 2.0 mg/kg dosing group, had significantly greater heat-hyperalgesia than the vehicle control group. Fig. 2 shows statistically significant differ-

Fig. 2. The effects of paclitaxel on heat-hyperalgesia of the hind paw. The results for the three paclitaxel dosing groups are represented as the group’s mean ^ SEM for each day. Arrows beneath the abscissa indicate days of treatment (Tx). Overall group means from baseline (B) to day 28: vehicle control (n ¼ 7) 7.9 s ^ 0.3; 0.5 mg/kg (n ¼ 8) 6.7 s ^ 0.3, 1.0 mg/kg (n ¼ 5) 5.7 s ^ 0.2; and 2.0 mg/kg (n ¼ 8) 7.3 s ^ 0.4. Statistically significant differences (one-way ANOVA and post hoc comparisons with LSD) between each paclitaxel treatment group and the vehicle control group on testing days are shown (*, P , 0:05; **, P , 0:01).

Fig. 3. The effects of paclitaxel on mechano-allodynia in the hind paws using von Frey hairs. Results are presented as mean threshold stimulus rank. Arrows beneath the abscissa indicate days of treatment (Tx) and baseline data (B) are plotted. Statistically significant comparisons for each of the three paclitaxel dosing groups vs. the vehicle control group (Mann–Whitney test) are shown (*, P , 0:05; **, P , 0:01).

ences (one-way ANOVAs and post hoc analyses) between the vehicle control group and dosing groups by testing day. The heat-hyperalgesia lasted until at least day 28. For the 0.5 and 1.0 mg/kg groups, the greatest reduction (relative to the control group) in the response latencies was about 20– 40%. For all the paclitaxel dosing groups, the changes are comparable to the threshold reductions seen in models of post-traumatic nerve injury such as the chronic constriction injury (CCI) model (Bennett and Xie, 1988) and neuritisevoked neuropathic pain (Eliav et al., 1999), but distinctly smaller than the threshold reduction seen in some models of inflammatory hyperalgesia (Hargreaves et al., 1988). 3.2.2. Mechano-allodynia in the hind paw At baseline (Fig. 3), there were no significant betweengroup differences (Kruskal–Wallis test) in the responses to von Frey hair stimulation. The baseline thresholds to evoke a withdrawal response (expressed as the mean ^ SEM of the stimulus ranks) were as follows: vehicle control ðn ¼ 7Þ 16:2 ^ 0:6; 0:5 mg=kg ðn ¼ 8Þ 16:0 ^ 0:5; 1:0 mg=kg ðn ¼ 5Þ 15:0 ^ 0:9; and 2:0 mg=kg ðn ¼ 8Þ 15:6 ^ 0:7: This corresponds to baseline thresholds of about 15–38 g of force. The thresholds of the Cremophor vehicle control group varied little during the entire experiment. Paclitaxel produced significant mechano-allodynia (Kruskal–Wallis test, Chi-square ¼ 12.3, d.f. 3, P , 0:01) which appeared as early as day 5 for the 1.0-mg/kg dosing group (Mann– Whitney test, P , 0:01) but not until day 16 for the other two dosage groups (0.5 mg/kg, P , 0:01; 2 mg/kg, P , 0:01). The decrease in threshold for the period of 5– 28 days was equivalent to a decrease in rank of 3–5, corresponding to post-treatment thresholds ranging from 5.7 to

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15 g of force; a decrease of 47–80% relative to the control. The severity of the mechano-allodynia produced by paclitaxel is approximately the same as that seen in other models of painful peripheral neuropathy (Tal and Bennett, 1994; Eliav et al., 1999). 3.2.3. Cold-allodynia in the hind paw At baseline (Fig. 4), the cold stimulus evoked a response in only 5–15% of the trials. There were no significant between-group differences in baseline responding. Animals in the vehicle control group (n ¼ 7) continued to respond to the cold stimulus at a low rate for the entire 28 days of observation. However, by day 10, a significant difference (Kruskal–Wallis test, Chi-square ¼ 8.56, d.f. 3, P , 0:05) was noted in the rate of responding in the paclitaxel treated rats. Mann–Whitney pair-wise comparisons for each paclitaxel group relative to the control group revealed significant increases (0.5 mg/kg, n ¼ 8, P , 0:01; 1 mg/kg, n ¼ 8, P , 0:01, and 2.0 mg/kg, n ¼ 8, P , 0:05) in the rate of responding. This continued such that the cold stimulus evoked a response in 40–75% of the trials in the paclitaxel treated rats for at least 28 days (Fig. 4). Paclitaxel also produced a change in the topography of the response to the cold stimulus. Prior to treatment, the response consisted of a very brief, small magnitude flick of the paw. After paclitaxel treatment, the response was usually much larger and accompanied by intense licking of the paw and ankle. 3.2.4. Heat-hyperalgesia in the tail At baseline (Fig. 5), there were no significant betweengroup differences in mean heat-evoked response latencies.

Fig. 4. The effects of paclitaxel on cold-allodynia in the hind paws. Results are expressed as the mean ^ SEM response frequency (percent) for the vehicle control and 0.5, 1, and 2.0 mg/kg paclitaxel dosing groups. Arrows beneath the abscissa indicate days of treatment (Tx), and baseline data (B) are plotted. Statistically significant comparisons for each of the three paclitaxel dosing groups vs. the vehicle control group (Mann–Whitney test) are shown (*, P , 0:05; **, P , 0:01).

Fig. 5. The effects of paclitaxel on heat-hyperalgesia in the tail. Results for the three paclitaxel dosing groups are represented as the group’s mean ^ SEM for each day. Arrows beneath the abscissa indicate days of treatment (Tx). Overall group means from baseline (B) to day 28: vehicle control (n ¼ 7) 13.0 s ^ 0.5; 0.5 mg/kg (n ¼ 8) 11.2 s ^ 0.5; 1.0 mg/kg (n ¼ 5) 10.4 s ^ 0.6; and 2.0 mg/kg (n ¼ 8) 11.3 s ^ 0.5. Statistically significant differences (one-way ANOVA and post hoc comparisons with LSD) between each paclitaxel treatment group and the vehicle control group on testing days are shown (*, P , 0:05; **P , 0:01).

Paclitaxel treatment produced significant heat-hyperalgesia (repeated measures ANOVA, Fð3; 24Þ ¼ 4:08, P , 0:05) (baseline to day 28). Post hoc analysis (LSD) on overall group means showed that the 0.5 mg/kg (P , 0:05), the 1 mg/kg (P , 0:01), and 2.0 mg/kg (P , 0:05) groups were significantly different from the vehicle control group, but not from each other. For the 2.0 mg/kg group, heat-hyperalgesia lasted until at least day 33. All paclitaxel dosing groups experienced the greatest reduction (relative to the control group) in the response latency ranging from 15 to 20%. 3.2.5. Mechano-hyperalgesia in the tail At baseline, the average mean thresholds (g of pressure) for the three test sites (22, 45, and 90 mm from the tip of the tail) for the vehicle control group and 0.5 and 2 mg/kg paclitaxel dosing groups are shown in Fig. 6. The 1 mg/kg dose did not produce any significant mechano-hyperalgesia in the tail relative to the control at any site or any day, except for day 19 at the 22-mm location (P , 0:05), therefore, data from this group are not shown. The average baseline threshold at the 45-mm site for the 0.5-mg/kg group was significantly lower than the control group, but this difference was not apparent on day 3, which was prior to the onset of the treatment effect. Paclitaxel at both doses produced significant (repeated measures ANOVA) mechano-hyperalgesia at the 22 mmðFð2; 20Þ ¼ 5:24; P , 0:05) and 45 mmðFð2; 20Þ ¼ 6:71; P , 0:01) test sites, but not at the 90-mm site ðFð2; 20Þ ¼ 2:41; P ¼ 0:115). The onset of drug effect was evident by differences from the vehicle control group by day

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P , 0:05). Peak symptom severity appeared throughout days 14–24. A trend toward recovery was noted by day 33 (Fig. 6). Neither dose produced any clear evidence of a length-dependent process.

3.2.6. Cold-allodynia in the tail Data were analyzed by logistic regression using GEE to account for correlations between serial measurements in the same rat (Liang and Zeger, 1993). A model with the lowest and highest paclitaxel doses (0.5 and 2 mg/kg) compared to the vehicle control was used to determine the percent probability of a tail withdrawal on or before 10 s. No interpretable model was possible when the data from the 1 mg/kg group were included which may indicate that the pattern of responses were not consistent; thus the results from this group are not shown. At baseline (Fig. 7), the cold stimulus rarely evoked a withdrawal response (5.2% of trials). The vehicle control group continued in this way for the duration of the experiment. Both doses of paclitaxel (0.5 and 2.0 mg/ kg) evoked cold-allodynia. The percent probability of a tailflick response within 5, 10, and 15 s was modeled as an ordinal response depending on dose and day of the study. The second and third trials for each rat were entered into the percent probability model over the entire testing period of 35 days. Significant differences between both paclitaxel treated rats and vehicle controls were evident by day 14 and continue throughout the testing period. Rats treated with the highest dose, 2 mg/kg, demonstrated significant cold-allodynia (tail-flick at 10 s or before) sooner than the 0.5 mg/kg dosing group. The percent probability for a tail-flick for the 2

Fig. 6. The effects of paclitaxel on mechano-hyperalgesia in the tail. Data from three test sites (22, 45, and 90 mm from the tip of the tail) are plotted relative to the vehicle control group for the 0.5 and 2.0 mg/kg dosing groups. Arrows beneath the abscissa indicate days of treatment (Tx). Overall group means ^ SEM from third baseline measurement (B3) to day 33 at the 22, 45, and 90-mm sites: vehicle control (n ¼ 7) 387.7 g ^ 12.7, 433.7 g ^ 14.2, and 490.9 g ^ 16.8; 0.5 mg/kg (n ¼ 8) 337.9 g ^ 10.2, 374.4 g ^ 13.5, and 441.4 g ^ 21.1; and 2.0 mg/kg (n ¼ 8) 341.8 g ^ 12.6, 366.5 g ^ 18.2, and 448.2 g ^ 16.3. Statistically significant differences (one-way ANOVA and post hoc comparisons) between each paclitaxel treatment group and the vehicle control group on testing days are shown (*, P , 0:05; **, P , 0:01).

9 at the 45-mm site of the tail (0.5 mg/kg, P , 0:05; 2 mg/kg, P , 0:05) and day 14 at the 22-mm site (0.5 mg/kg,

Fig. 7. The effects of paclitaxel on cold-allodynia of the tail. The percent probabilities of a tail-flick on or before 10 s are represented for the vehicle control group (n ¼ 7) and the 0.5 (n ¼ 8) and 2 mg/kg (n ¼ 8) paclitaxeltreated groups. Arrows beneath the abscissa indicate days of treatment (Tx), and baseline data (B1–B3) are plotted. Statistically significant differences by contrast estimate results using GEE between each paclitaxel treatment group and the vehicle control group on testing days are shown with probability Chi-square values (*, P , 0:05; **, P , 0:01).

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mg/kg dosing group was significantly different than control on day 4 (6.6%, P , 0:01, n ¼ 8) and day 8 (15.1%, P , 0:01) and worsened throughout the testing period with the most severe cold-allodynia on day 35 (approximately 23.3%, P , 0:01). The 0.5-mg/kg dosing group experienced significant cold-allodynia by day 14 (8.8%, P , 0:01) and this was most pronounced on day 21 (22.6%, P , 0:05). Symptom severity lessened by day 28, but remained significantly different from the controls (12.3%, P , 0:01).

was no degeneration in the dorsal columns or in Lissauer’s tract. Cell bodies in the DRG and in the ventrolateral motor neuron pools were normal. Electron-microscopic examination confirmed that there was no degeneration of myelinated or unmyelinated axons in the sciatic nerve and no degeneration in the dorsal root ganglia. However, there was endoneurial edema apparent in the sciatic nerves. 4. Discussion

3.2.7. Motor coordination Rota-rod performance was equivalent for the vehicle control group (n ¼ 8) and 1 mg/kg (n ¼ 8) and 2 mg/kg (n ¼ 8) paclitaxel-treated rats for all three pre-dosing baseline sessions (Fig. 8). During and after dosing, the animals in all groups continued to improve their performance with no significant between-group differences (repeated measures ANOVA, Fð2; 21Þ ¼ 0:21; P ¼ 0:82) in rota-rod performance.

We found that 0.5, 1.0, and 2.0 mg/kg paclitaxel given i.p. every other day for a total of four injections (cumulative doses of 2.0, 4.0, and 8.0 mg/kg) to adult rats produced a painful peripheral neuropathy without significant impairment of the animals’ general health, without impairment of motor function and without degeneration of somatosensory or motor axons. 4.1. Behavioral observations

3.2.8. Histology Light-microscopic examination found no evidence of abnormalities in peripheral nerves or dorsal root ganglia in the paclitaxel-treated animals compared to controls at a time when abnormal pain sensations were clearly evident. Transverse sections of the dorsal and ventral roots (L4–L5) were normal. Longitudinal sections showing the axons of the sciatic nerves (mid-thigh level) were also normal. There

Fig. 8. The effects of paclitaxel on motor coordination as assessed by the rota-rod test. Mean ^ SEM for the vehicle control (n ¼ 8) and 1 mg/kg (n ¼ 8) and 2.0 mg/kg (n ¼ 8) paclitaxel-treated rats are represented by the duration(s) on an accelerating rota-rod for three pre-dosing baseline/training sessions (B1–B3), during treatment and post-dosing. Arrows beneath the abscissa indicate days of treatment (Tx). The overall group means for the period encompassing the third baseline trial to day 22 were 78.3 s ^ 3.3 for the vehicle control group, and 79.9 s ^ 4.6 for the 1 mg/kg and 81.9 s ^ 3.5 for the 2 mg/kg paclitaxel dosing groups. There were no statistically significant between-group differences at any time.

The paclitaxel-induced neuropathic pain sensations began within days and lasted for at least several weeks. No dose produced a loss of large or small fiber sensory function. There was no indication of a dose–response relationship in the range that we examined. The inspection of the time of onset of mechano-hyperalgesia at three locations on the tail provided no clear evidence of a length-dependent process. It is possible that even with our lowest dose the evolution of the neuropathy is so rapid that length-dependency is obscured. A similar effect may explain the clinical observation of the apparently simultaneous appearance of symptoms in hands and feet. There was no evidence of any effect on gross motor neuron function: the animals behaved normally and had no deficits in the rota-rod test. We are aware of seven prior reports of experimental peripheral neuropathy due to systemic administration of paclitaxel where the animals’ sensory status was evaluated (Apfel et al., 1991; Cavaletti et al., 1995b, 1997; Campana et al., 1998; Cliffer et al., 1998; Boyle et al., 1999; Authier et al., 2000). All of these studies found decreases in sensory function, but only one of them (Authier et al., 2000) documented any signs of neuropathic pain. These studies used different single dose intensities, different cumulative doses, and different dosing schedules; clinical experience suggests that each of these variables might influence the appearance of neuropathy. With two exceptions (Cavaletti et al., 1995b; Campana et al., 1998), our single dose intensities and cumulative doses were considerably lower than those tested previously (our single doses of 0.5, 1.0, or 2.0 mg/kg vs. 5.0–21.6 mg/kg; and our cumulative doses of 2.0, 4.0, and 8.0 mg/kg vs. 18.0–160 mg/kg). Our largest cumulative dose (8.0 mg/ kg) equals the smallest cumulative dose examined by Cavaletti et al. (1995b), but they did not examine pain responses. The lowest dose used by Campana et al. (1998), 1.2 mg/kg,

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was within our range, but their lowest cumulative dose (18 mg/kg) was greater than our highest (8.0 mg/kg). Therefore, it is possible that neuropathic pain is a low-dose effect of paclitaxel, and that it might be an earlier or more sensitive indicator of the onset of neurotoxicity. Our results are similar in some respects to those reported by Authier et al. (2000) who reported hind paw mechanohyperalgesia (Randall–Selitto test) in rats receiving a single injection of 32 mg/kg paclitaxel or five such injections given at weekly intervals for 5 weeks (total dose ¼ 160 mg/kg). However, unlike our results, their rats did not display mechano- and cold-allodynia, and the hindpaws were hypo-sensitive to noxious heat. The doses used by Authier et al. (2000) did not affect rota-rod performance. The behavioral abnormalities that we observed with paclitaxel are also similar in some respects to those seen by Aley et al. (1996) and Authier et al. (2000) with vincristine, a vinca alkaloid chemotherapeutic agent. In addition to an acute and transient effect on the pain threshold, Aley et al. (1996) showed that chronic treatment with vincristine (100 and 200 mg/kg; intravenous (i.v.); five weekly injections for 2 weeks) produced mechano-hyperalgesia and mechano-allodynia (measured on the hind paws) that began after 1 week of treatment and persisted for about 1 week after the treatment ceased. Chronic vincristine treatment caused only a small and short-lived heat-hyperalgesia (consistent with the study by Rebert et al. (1984) which used higher doses of vincristine), and the highest dose (200 mg/ kg) severely affected rota-rod performance. Authier et al. (1999) gave ten consecutive daily i.v. injections of 50 or 75 mg/kg. The 75 mg/kg was highly toxic, but there was no significant effect on general health with the 50 mg/kg dose. The lower dose yielded mechano-hyperalgesia and mechano-allodynia, but hypo-sensitivity to heat.

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letti et al. (1995b, 1997) and Authier et al. (2000) described modest pathology in the sciatic nerve, and Cliffer et al. (1998) demonstrated significant degeneration in the dorsal (but not ventral) roots. Our results are also consistent with those of Tanner et al. (1998) who did not find any anatomical abnormalities at the light-microscopic level in rats with a painful peripheral neuropathy due to vincristine treatment. However, electron microscopy of the vincristine-treated animals (Tanner et al., 1998; Topp et al., 2000) did find subtle signs of pathology. In the peripheral nerve, unmyelinated and myelinated sensory axons were swollen and the normal linear organization of their microtubules was disrupted. In the dorsal root ganglion, the large cells were swollen and had accumulations of neurofilaments. We note that there is precedence for the appearance of an experimental painful peripheral neuropathy in the absence of structural injury to axons (Eliav et al., 1999). 4.3. Mechanism of action The mechanism whereby paclitaxel evokes neuropathy is

4.2. Anatomical observations Our light-microscopic investigations found no evidence of axonal degeneration in sensory or motor axons at the level of the sciatic nerve, dorsal and ventral roots, or spinal cord dorsal columns or Lissauer’s tract. We also found no anatomical evidence of neuronopathy in dorsal root ganglia cells or spinal motor neuron pools. However, endoneurial edema was identified in peripheral nerves of animals treated with paclitaxel 2 mg/kg (Fig. 9). The pathophysiological significance of this is unknown. Our anatomical data do not discount the possibility of a dying-back process where the pathology is confined to the region near the sensory axons’ receptors, nor does it discount the possibility of subtle alterations that would be evident only by quantitative ultrastructural study (e.g. Tanner et al., 1998; Topp et al., 2000). The absence of structural injury to axons in our animals is consistent with the report of Campana et al. (1998) who found no light-microscopic evidence of abnormal anatomy in the sciatic and sural nerves, even though their animals were hypo-responsive in the tail-flick test. In contrast, Cava-

Fig. 9. Electron photomicrographs of mid-section of the sciatic nerve. (A) Cremophor vehicle control demonstrating normal myelinated and unmyelinated axons and normal perineurium. (B) Rat treated with paclitaxel 2 mg/ kg. Endoneurial edema present, but no degeneration of myelinated and unmyelinated axons and no difference in appearance of myelin sheaths. Magnification: £4125.

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not known. Its ability to kill cancer cells is generally thought to be due to its effects on the mitotic spindle. The mitotic spindle is composed of homopolymers of the protein, betatubulin. Paclitaxel binds to beta-tubulin and stabilizes its polymerization. This is thought to disrupt the dynamic organization of the mitotic spindle, arresting the process of cell division, and engaging the apoptotic pathway (Schiff and Horowitz, 1980; De Brabander et al., 1981; Parness and Horwitz, 1981; Rowinsky et al., 1988). However, recent studies suggest that paclitaxel may destroy cancer cells via other mechanisms (Fan, 1999). It is noteworthy that in the rat the two principle hepatic metabolites of paclitaxel are as effective as the parent compound in stabilizing beta-tubulin polymerization, but nine and 39 times less cytotoxic against leukemia cells (Monsarrat et al., 1990). Paclitaxel is known to reduce mitochondrial respiration (Manzano et al., 1996) and subtle, hereditary defects in mitochondrial function give rise to sensorimotor neuropathies (Schro¨ der, 1993). Moreover, paclitaxel has some of the same effects as immune stimulation with bacterial lipopolysaccharide (LPS); in particular, it evokes the release of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNFa; Ding et al., 1990; Manthey et al., 1992; Salkowski and Vogel, 1992; Burkhart et al., 1994). The LPS-like effect is known to be independent of paclitaxel binding to beta-tubulin; instead, it involves binding with a member of the heat-shock protein-90 family (Byrd et al., 1999). Neuroimmune interactions generally, and an effect of TNFa in particular, have been implicated in the pathogenesis of painful peripheral neuropathies (for review see Eliav et al., 1999). Homopolymerized beta-tubulin also composes the microtubules found in neuronal dendrites and axons. It is natural to suspect that paclitaxel-induced neuropathy is due to betatubulin binding and subsequent disruption of microtubular axoplasmic flow. However, there is no clear evidence to support this hypothesis. Numerous studies show that microtubules are severely affected when paclitaxel is injected directly into a peripheral nerve (Komiya and Tashiro, 1988; Komiya, 1992; Ro¨ ytta¨ and Raine, 1985, 1986; Raine et al., 1987; Nennesmo and Reinholt, 1988; Vuorinen et al., 1988a,b, 1989a,b; Vuorinen and Ro¨ ytta¨ , 1990a,b), but it is difficult to estimate the relevance of these studies. These experiments used relatively high doses of paclitaxel (10–20 ml of 10 mM, or 0.2 ml of 1 mM paclitaxel) in a vehicle that contained dimethylsulfoxide (DMSO; 0.1–0.2 or 10%). The endoneurial and especially the intra-axonal concentrations of paclitaxel after systemic administration must be very much less than that achieved after injection directly into the nerve of a large dose in a DMSO vehicle. However, a recent report shows that in rats systemically administered paclitaxel, in doses that produce significant sensorimotor deficits, has ready access to peripheral nerves, and especially to DRG where it accumulates (Cavaletti et al., 2000). There is no evidence that clinically relevant doses of systemically administered paclitaxel have any effect on

axonal transport. Direct observation of microtubular transport in cultured dorsal root ganglion neurons failed to find any effect with doses of paclitaxel that had a measurable effect on microtubule polymerization (Horie et al., 1987). Glutamate (Boyle et al., 1999) and prosaptide (Campana et al., 1998) protect against experimental paclitaxel-induced neuropathy; neither result is easily explained with reference to impaired axoplasmic transport. Clinical experience and the behavioral and anatomical studies of systemic paclitaxel in laboratory rodents suggest that paclitaxel-induced neuropathy may be a graded phenomenon. It is likely to begin with a painful peripheral neuropathy without gross nerve pathology, progress to a predominately sensory neuropathy with loss of both large and small sensory fiber function and modest nerve pathology, and culminate in a profound loss of sensory and motor function with extensive anatomical disruption in the nerve. Such a progressive process might be due entirely to the same paclitaxel mechanism of action, or different components of the process might be due to distinct mechanisms. Whatever its mechanism of action, it is clear that paclitaxel-induced neuropathy limits therapy and is a major source of pain in cancer patients. Further investigations with the model reported here might be valuable in understanding and solving these problems. Acknowledgements R.C.P. was supported by a Scientific Fellowship Award to the University of Pennsylvania, School of Nursing, from the United States Pharmacopeial Convention, Inc. and a grant from the NINR (KO1 NR 00136-01A1). We acknowledge David T. Mauger, PhD for his statistical advice, Roland Meyers BS for EM photomicrographs and Philip J. Boyer, MD, PhD for EM interpretations. R.C.P. is grateful to Kevin D. Alloway, PhD and Nancy C. Tkacs, PhD, RN for their continued support with this project. References Akerley W. Paclitaxel in advanced non-small cell lung cancer: an alternative to high-dose weekly schedule. Chest 2000;117:152S–155S. Aley KO, Reichling DB, Levine JD. Vincristine hyperalgesia in the rat: a model of painful vincristine neuropathy in humans. Neuroscience 1996;73:259–265. Apfel SC, Lipton RB, Arezzo JC, Kessler JA. Nerve growth factor prevents toxic neuropathy in mice. Ann Neurol 1991;29:87–90. Authier N, Coudore F, Eschalier A, Fialip J. Pain induced behaviour during vincristine-induced neuropathy in rats. NeuroReport 1999;10:965–968. Authier N, Gillet JP, Fialip J, Eschalier A, Coudore F. Description of a short-term taxol-induced nociceptive neuropathy in rats. Brain Res 2000;887:239–249. Bennett GJ, Hargreaves KM. Reply to Hirata and his colleagues. Pain 1990;42:255. Bennett GJ, Xie Y-K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988;33:87– 107.

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