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NEUROPHARMACOLOGY
Research and Development Department, Chiesi Farmaceutici S.p.A., Parma, Italy (G.V., M.B., F.B., P.T.B., M.M., C.P., I.R.); Cerep, Biology Department, Rueil-Malmaison Cedex, France (P.C.C.); and Department of Clinical and Experimental Medicine, Section of Pharmacology, and Neuroscience Center, University of Ferrara, Ferrara, Italy (M.S., M.B.)
Received February 4, 2003; accepted May 2, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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). However, systematic reviews reveal that only 30
to 50% of patients suffering from neuropathic pain achieve clinically
significant (>50%) pain relief with any available single therapy
(Sindrup and Jensen, 1999).
Furthermore, side effects of these therapies often limit their usefulness.
Several neurotransmitters/neuromodulators have now been identified as being
involved to varying degrees in the pain processing pathways. Among them, the
amino acid glutamate, the dominant excitatory neurotransmitter of the
mammalian central nervous system, seems to be a key effector in diseases
reflecting long-term plastic changes in the central nervous system, such as
chronic pain and pain-related neurotoxicity
(Bennett, 2000). Glutamate is
found in nerve terminals on spinal nociceptive neurons
(Broman and Adahl, 1994) and is
released in the spinal cord after stimulation of peripheral nociceptors
(Ueda et al., 1994). Glutamate
acts on a variety of ligand-gated ion channels or G protein-coupled
metabotropic receptors. The glutamate-gated ion channels are classified in
three major subclasses: 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid, kainate, and N–methyl-D-aspartate (NMDA)
receptors. Recent preclinical studies suggest that hyperalgesia and allodynia
after peripheral tissue or nerve injury may result from both an increase in
transduction sensitivity of primary afferent receptors at the site of injury
and an increase in the excitability of spinal cord neurons mediated by NMDA
receptors (Dickenson, 1997).
In fact, NMDA antagonists have been reported to block formalin-induced pain
behavior (Chaplan et al.,
1997), hyperalgesia induced by chronic constriction (CCI) of the
rat sciatic nerve (Quartaroli et al.,
1999), and peripheral inflammation
(Ren et al., 1992).
A number of human studies have evaluated the efficacy of clinically
available NMDA receptor antagonists in chronic pain. This class of compounds
has been shown to modulate pain and hyperalgesia, but the efficacy has been
hampered by dose-limiting side effects
(Fisher et al., 2000). As a
consequence, the challenge has been to develop new "therapeutically
safe" NMDA antagonists, specifically compounds able to prevent the NMDA
receptor activation in pathological pain states without affecting the
physiological activation of the receptor. Indeed, there is now considerable
evidence that new moderate-affinity NMDA channel blockers,
glycineB, and NR2B-selective antagonists are endowed with
antinociceptive activity in animal models at doses devoid of obvious side
effects (Parsons, 2001).
N-(2-Indanyl)-glycinamide hydrochloride (CHF3381) is a novel
low-affinity, noncompetitive NMDA antagonist. Binding studies have shown that
CHF3381 inhibits [3H]1-[1-(2-thienyl)ciclohexyl] piperidine binding
to brain membranes with micromolar affinity (IC50 = 8.8 µM), in
the absence of significant binding to other NMDA receptor complex subsites
and/or to other glutamate receptor subtypes
(Villetti et al., 2001).
CHF3381 is endowed with neuroprotective activity in vitro
(Gandolfi et al., 2001) and in
vivo (Zucchini et al., 2002)
and with anticonvulsant activity in models of generalized and partial seizures
(Villetti et al., 2001). These
effects were observed at doses devoid of negative side effects on motor
coordination or behavior (Villetti et al.,
2001). In view of the above-mentioned considerations, the
capability of CHF3381 to affect the NMDA receptor complex is of interest in
the context of nociception.
The present study, therefore, was designed to characterize the
antihyperalgesic profile of CHF3381. The in vitro CHF3381 effect on wind-up,
which is considered to be an electrophysiological phenomenon mechanistically
similar to spinal central sensitization
(Barbieri and Nistri, 2001),
was investigated. CHF3381 activity was evaluated in in vivo models of
persistent inflammatory pain and against a variety of behavioral signs in two
models of neuropathic pain and compared with those of memantine and
gabapentin, compounds reported to be active in pain animal models and in
clinical trials (Parsons,
2001; Backonja,
2002). The possible development of tolerance to the
antihyperalgesic activity of CHF3381 was also addressed.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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).
Surgery
The CCI was carried out as described previously
(Bennett and Xie, 1988).
Briefly, rats (125–150 g at time of surgery) were anesthetized with
sodium pentobarbital (50 mg/kg i.p., supplemented if required). The common
right sciatic nerve was exposed at mid-thigh level, proximally to the sciatic
trifurcation, and four ligatures (3-0 silk) with about 1-mm spacing were
loosely tied around the nerve. All operations were completed by closing the
muscles in layers and applying two wound clips to close the skin incision.
Rats were then treated with enrofloxacin (10 mg/kg s.c.) to minimize the
incidence of subsequent infection. All behavioral tests were conducted on
animals at least 1 week after surgery. Neuropathic rats were able to drink and
eat unaided.
Electrophysiology
Wind-up. Wind-up experiments were performed as described previously
(Barbieri and Nistri, 2001).
Briefly, spinal cord preparations (comprising a region from mid-thoracic level
to conus medullaris) were isolated from 5- to 10-day-old Wistar rats (Harlan
Italy, San Pietro al Natisone, Udine, Italy) under ether terminal anesthesia.
The spinal cord was fixed to the bottom of the recording chamber and
superfused (12 ml/min) with Krebs' solution of the following composition: 113
mM NaCl, 4.5 mM KCl, 1 mM MgCl2 · 7H2O, 2 mM
CaCl2, 1 mM NaH2PO4, 25 mM NaHCO3,
and 11 mM glucose, gassed with 95% O2, 5% CO2; pH 7.38
at room temperature (22°C). DC ventral root (VR) recordings (usually from
L5 VRS) were obtained with glass suction microelectrodes
filled with Krebs' solution. Mono- and polysynaptic potentials were amplified
and digitally stored on computer hard disk. Homolateral dorsal roots (DRs)
were electrically stimulated via miniature bipolar suction electrodes.
Stimulus intensity (1–20-V range; 1-ms duration) was adjusted to be at
least 2 times above threshold defined as the minimum intensity to elicit a
detectable polysynaptic response from the homolateral VR. The standard DR
train protocol consisted of 16 train pulses (1 Hz; 5-min intertrial) that
generated an incrementing depolarization (cumulative depolarization). As
parameters of sensitization, we considered the amplitude of the cumulative
depolarization and its rate of rise. The former was measured as difference
between the baseline potential and the potential 1 s after the last stimulus;
the rate of rise (expressed as percentage of control) was calculated as the
increment of baseline depolarization per second, starting after the second
response to a train of 16 pulses. All agents were bath-applied via the
superfusing solution at the concentration mentioned in the text.
Behavioral Tests
Carrageenan-Induced Thermal Hyperalgesia in the Rat. Thermal
hyperalgesia was assessed using the rat plantar test (Ugo Basile, Comerio,
Italy), according to a method modified by Hargreaves et al.
(1988). Briefly, rats were
habituated to the apparatus that consisted of three individual Perspex boxes
on a glass table. A mobile radiant heat source was located under the table and
focused onto the desired paw. Paw withdrawal latencies (PWLs) were recorded
three times for both hind paws of each animal, the mean of which represented
baseline for left and right hind paws. The apparatus was calibrated to give a
PWL of approximately 10 s. To prevent tissue damage of the plantar zone, a
20-s cut-off was observed. After baseline testing was conducted, animals
received an intraplantar injection of carrageenan (100 µl of a 20-mg/ml
solution) into the right hind paw. PWLs were reassessed according to the same
protocol as after 2.5 h after carrageenan, to ascertain that hyperalgesia had
developed. Test compounds or the corresponding vehicle was administered 3 h
after carrageenan, and PWLs were taken again at 3.5, 4, and 5 h after
carrageenan.
Carrageenan-Induced Mechanical Hyperalgesia in the Rat. Mechanical
nociceptive thresholds were measured in the rat paw pressure test
(Randall and Selitto, 1957) by
an analgesimeter (Ugo Basile). The day before testing, rats received three
training sessions. Pressure was gradually applied to the right hind paw, and
paw withdrawal thresholds (PWTs) were assessed as the pressure (grams)
required to elicit paw withdrawal. A cut-off point of 250 g was used to
prevent any tissue damage to the paw. On the test day, two to three baseline
measurements were taken before animals were administered carrageenan (100
µl of a 20-mg/ml solution) into the right hind paw. PWTs were determined
again 2.5 h after carrageenan to establish that mechanical hyperalgesia had
developed. Test compounds were then administered 3-h postcarrageenan, and PWTs
thresholds were taken again at 3.5, 4, and 5 h postcarrageenan.
Mouse Paw Formalin Test. The mouse paw formalin test was a slight
modification of the method originally described by Wheeler-Aceto and Cowan
(1991). The day before
formalin injection, mice were placed individually into clear plastic cylinders
for 30 min to allow adaptation to the new environment. The day of testing, 20
µlof 1% formalin was injected into the plantar surface of the left hind paw
and the animals were again placed into the cylinders on a clear plastic table
for behavioral observation. A camera was placed under the table, and the
behavioral experimental sessions were recorded. The amount of time in seconds
that the animals spent licking and flinching the injected paw for the first 5
min (early phase), and then from 10 to 40 min (late phase) after formalin
injection, was used as measurement of pain intensity. CHF3381 was administered
i.p. 15 min before formalin; memantine and gabapentin were administered 1 h
before formalin i.p. or s.c., respectively.
Tolerance Studies. The mouse paw formalin test was used to ascertain
whether, after chronic treatment, tolerance develops to the antihyperalgesic
activity of CHF3381 and morphine, as reported previously for the latter
compound (Quartaroli et al.,
1999). Furthermore, in a second study, CHF3381 was administered to
animals chronically treated with morphine to establish whether
morphine-induced tolerance cross-generalized with CHF3381. Evaluation of
compound effects was carried out as described previously for testing after
acute treatment. In the first study, mice were divided randomly into five
groups (n = 12) and treated once daily for 8 days as follows: three
groups with saline i.p., one group with CHF 3381 60 mg/kg i.p., and one group
with morphine 20 mg/kg i.p. On day 9, these groups were treated in the
following manner: one saline-pretreated group was treated with saline i.p.;
two saline-pretreated groups were treated either with CHF3381 30 mg/kg i.p. or
with morphine 6 mg/kg i.p.; the group pretreated with CHF3381 60 mg/kg was
treated with CHF3381 30 mg/kg i.p., and the group pretreated with morphine 20
mg/kg was treated with morphine 6 mg/kg i.p. CHF3381 and morphine were
administered 15 and 30 min before formalin injection, respectively. In the
second study, two groups of animals (n = 12) were treated once daily
for 8 days with morphine 20 mg/kg i.p. Three other groups of animals
(n = 12) received chronic dosing of saline i.p. On day 9, animals
treated with chronic morphine received either morphine (6 mg/kg i.p., 30 min
before formalin) or CHF3381 (60 mg/kg i.p., 15 min before formalin,
respectively), whereas three saline-treated groups received either a similar
administration of saline, morphine (6 mg/kg i.p.), or CHF3381 (60 mg/kg
i.p.).
Cold Allodynia in Neuropathic Rats. For the measurement of cold
allodynia, each rat was placed upon a metal floor chilled by an underlying
water bath (5 ± 1°C) for a maximum of 20 s, as described previously
(Bennett and Xie, 1988).
Neuropathic animals responded to the contact with the cold surface by lifting
the paw on the ligated side off the floor. The cold stimulus did not elicit
any pain-related paw withdrawal in the sham-operated group (data not shown).
For each set of experiments, animals were prescreened twice with 20-min
interval between tests, to select animals displaying clear signs of allodynia,
i.e., animals with a paw withdrawal latency on the ligated side of <10 s in
both trials. Animals were then stratified into groups based on their mean
withdrawal latency, so that the mean baseline did not differ between groups.
The latency to paw withdrawal was then determined at 1, 2, and 4 h
post-treatment.
Mechanical Allodynia in Neuropathic Rats. Rats were placed on a
metal mesh table and adapted to the new environment. The mechanical stimulus
was delivered to the plantar surface of both hind paws from below the floor of
the test chamber by an automated testing device (dynamic plantar
aesthesiometer; Ugo Basile), as described previously
(Gibbs et al., 2001). A steel
rod (diameter of 0.5 mm) was pushed against the hind paw with ascending force.
The force went from 0 to 50 g over a 20-s period. When the animal withdrew its
hind paw, the mechanical stimulus was automatically stopped, and the force at
which the animal withdrew its paw was recorded to the nearest 0.1 g.
Withdrawal responses were taken from four consecutive trials with at least 10
s between trials and averaged to select for animals displaying a clear
reduction of the threshold (>40%) of the ligated paw in comparison with the
contralateral paw. Animals were then stratified into groups based on their
mean withdrawal threshold, so that the mean baseline did not differ between
groups. The latency to paw withdrawal was then determined at 1, 2, and 4 h
post-treatment.
Mechanical Hyperalgesia in Rat Diabetic Neuropathy. On day 0,
diabetes was induced by the i.p. injection of STZ (75 mg/kg). Control animals
received a similar administration of saline. On day 23, to confirm the
presence of diabetes, glycemia was measured using blood glucose strips and the
glycemia reader Reflolux SF type 970819 (Roche Diagnostics, Mannheim,
Germany). On day 24, from 4:00 PM up to pain measurement, diabetic rats were
submitted to a controlled diet (30% of the normal daily diet), with water
available ad libitum. On day 25, distilled water, CHF3381 (25–100
mg/kg), and gabapentin (100 mg/kg) were administered by oral route and
memantine (15 mg/kg) by intraperitoneal route to diabetic rats, 60 min before
pain measurement. Control animals received a similar administration of
distilled water. The nociceptive threshold was evaluated in all groups using a
mechanical nociceptive stimulation (paw pressure test;
Randall and Selitto, 1957) by
an analgesimeter (Ugo Basile). The pain threshold was measured in both hind
paws.
CHF3381 Plasma Levels in Streptozotocin-Treated Rats. At the end of pain measurement, animals were sacrificed (under ether anesthesia) by bleeding from the abdominal aorta to determine plasma levels of CHF3381 after drug administration at different doses. Plasma was separated from blood and CHF3381 was quantitated by HPLC after liquid-liquid extraction. Briefly, 1.0 ml of plasma was added to internal standard and 0.5 ml of 0.5 M phosphate buffer, pH 11.7. Then, the samples were extracted with 6 ml of a diethyl ether/butanol mixture (80:20) containing 0.3% tetra-n-octylammonium bromide. To the organic phase, 0.2 ml of 0.1 N HCl was added and 30 µl of the acidic phase was injected into the HPLC system. CHF3381 and internal standard were separated using reversed phase chromatography, with retention time of 13.3 and 18.5 min, respectively. The mobile phase contained 15% methanol and 85% 0.5 M phosphate buffer, pH 2.7, and was pumped at a flow rate of 0.5 ml/min. The stationary phase was a C18 column (X-Terra MS, 150 x 4.6 mm, 3.5 µm; Waters, Milford, MA). Analytes were detected by using a fluorescence HPLC detector (model 474; Waters) set at 266 nm (excitation) and 286 nm (emission). The method was validated and was found to be linear in the 2- to 2000-ng/ml concentration range. Plasma samples were adequately diluted with blank rat plasma if the concentration was greater than 2000 ng/ml. The lower limit of quantitation was 2 ng/ml.
Drugs and Chemicals Used for Testing. CHF3381 was synthesized at the Chiesi Farmaceutici Chemical Synthesis Department (Parma, Italy). Memantine and morphine hydrochloride were obtained from Sigma-Aldrich (Milan, Italy). Gabapentin was extracted from Neurontin tablets (Parke-Davis, Milan, Italy). The other chemicals were purchased from Sigma-Aldrich. Drugs were diluted in distilled water for oral administration and in 0.9% saline for parenteral administration. The volume of administration was 10 ml/kg for mice and ranged from 2 to 5 ml/kg in rats. For comparison with compound-treated groups, a control group received in all cases the relevant vehicle. The volume of administration was identical for vehicle- and compound-treated animals.
The selection of CHF3381 doses to be tested in behavioral tests was based
on the work of Villetti et al.
(2001). The antinociceptive
activity of CHF3381 has been investigated at doses that had been previously
shown not to impair motor performance in the mouse and rat rotarod task and
not to disrupt learning and memory in the rat Morris water maze task
(Villetti et al., 2001).
Statistics. Data are expressed as mean ± S.E.M. In the diabetic neuropathy study, nociceptive thresholds were calculated as mean values of the nociceptive thresholds obtained from both hind paws. Only for mechanical allodynia in the CCI model data were normalized for each animal as maximum possible effect (MPE). This value was calculated as follows: MPE = (PDR – IBR)/(CBR – IBR), where PDR is the postdrug response of the ipsilateral paw, IBR is the ipsilateral paw baseline response, and CBR is the contralateral paw baseline response. Accordingly, the individual values reported or depicted are the MPE ± S.E.M. Data obtained for the carrageenan and formalin tests, tolerance studies, and neuropathic pain models were subjected to a one-way ANOVA followed, when significant, by post hoc Dunnett's t test. When comparing the means of only two groups, Student's t test was used. Data obtained for the wind-up, were analyzed using Mann-Whitney U test. All comparisons were analyzed separately for each time point. For all tests, a p value lower than 0.05 (p < 0.05) was considered significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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), morphine produced a much more profound effect than
CHF3381 and memantine, being capable of almost completely abolishing wind-up
at the concentration tested. In contrast, gabapentin was devoid of any effect
at a concentration of 300 µM (Fig.
1C) and up to 1 mM (data not shown). The effect of CHF3381 was
concentration-dependent and more potent on the cumulative depolarization
amplitude than on the rate of rise development
(Fig. 2A), as indicated by the
fact that, at a concentration of 300 µM, the CHF3381 effect is nearly
maximal for the former parameter and barely detectable for the latter. At
higher concentrations (1 mM), however, CHF3381 was found to dramatically
reduce the rate of rise as well (Fig.
2A), i.e., to essentially abolish the wind-up phenomenon. For
comparison, the effect of 300 µM memantine on both wind-up parameters is
also plotted in Fig. 2A. These
effects nearly exactly match those described for CHF3381. The time course of
onset of 300 µM CHF3381 effects on the cumulative depolarization has been
studied in detail (Fig. 2B).
Nearly maximal effects were obtained within 15 to 20 min of perfusion, a short
time interval compared with memantine, as indicated by a significantly greater
effect at the 15-min time point (Fig.
2B).
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Carrageenan-Induced Thermal Hyperalgesia in the Rat. Animals showed baseline PWLs of 10 to 12 s. At 2.5 h after carrageenan injection, the PWLs of the carrageenan-injected paw were significantly reduced, averaging approximately 5 s in all experiments. This hyperalgesic state was maintained in vehicle-treated animals at all time points tested. The p.o. administration of CHF3381 (10–100 mg/kg) 3 h after carrageenan dose dependently antagonized the maintenance of thermal hyperalgesia, with a minimum significantly effective dose (MED) of 30 mg/kg (Fig. 3A). At the highest dose tested, CHF3381 produced a significant and sustained (>2 h) elevation in the PWLs of the inflamed paw. There were no significant differences in the mean change in thermal threshold of the noninflamed contralateral hind paw in groups treated with any oral dose of CHF3381 (data not shown). The s.c. administration of gabapentin (10–100 mg/kg) was devoid of antihyperalgesic effects (Fig. 3B). After i.p. administration, 10 mg/kg memantine partially reversed the maintenance of thermal hyperalgesia at 4 h after carrageenan, whereas at the 15-mg/kg dose the effect was significant and sustained up to 5 h after carrageenan (Fig. 3C).
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Carrageenan-Induced Mechanical Hyperalgesia in the Rat. On the test day, animals showed baseline withdrawal thresholds of about 150 to 160 g. At 2.5 h after carrageenan injection, the ipsilateral paw exhibited marked mechanical hyperalgesia, averaging approximately 90 g in all experiments. This hyperalgesic state was maintained in vehicle-treated animals at all of the tested time points. The p.o. administration of CHF3381 (10–200 mg/kg) at 3 h after carrageenan produced a significant and dose-dependent reversal of mechanical hyperalgesia with a MED of 30 mg/kg (Fig. 4A). The highest dose of CHF3381 produced a complete reversal of the inflammatory-induced mechanical hyperalgesia, and this effect was sustained up to 5 h after carrageenan. The s.c. administration of gabapentin (10–100 mg/kg) dose dependently antagonized the maintenance of mechanical hyperalgesia (Fig. 4B), with a MED of 10 mg/kg. The highest dose of gabapentin produced a complete reversal of the inflammatory-induced mechanical hyperalgesia. Memantine after i.p. administration partially relieved mechanical hyperalgesia at 3.5 h after carrageenan injection (MED = 15 mg/kg); however, the antihyperalgesic effect did not persist at the subsequent time points (Fig. 4C).
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Mouse Paw Formalin Test. In the vehicle-treated groups, s.c. injection of 1% formalin into the left hind paw of mice induced a biphasic licking/flinching nociceptive response with an early and a late phase. CHF3381 i.p. administration resulted in a dose-dependent and significant reversal of the licking/flinching response in both phases of the formalin test (Table 1). In the late phase, all four doses of CHF3381 produced a reduction of the nociceptive response, with a MED of 30 mg/kg. The s.c. injection of gabapentin resulted in a significant and dose-dependent suppression of the late phase behavior with a MED of 30 mg/kg (Table 1) and a maximal 71% reduction observed at 300 mg/kg. Gabapentin yielded only a modest effect on the early phase behavior at the highest dose (300 mg/kg) tested (p < 0.05). However, at this dose some mild motor weakness was observed. The i.p. administration of memantine significantly attenuated nociceptive responding in both phases of the formalin test (Table 1), even if this compound seemed to be somewhat more potent in blocking the late phase of formalin pain. Indeed, the largest reduction in the formalin-induced pain behavior was observed at the 20-mg/kg dose (53%) in the late phase.
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Tolerance Studies. Morphine (6 mg/kg i.p.) significantly attenuated basal nociceptive response in both phases of formalin test in chronic vehicle-treated animals. However, the same dose of morphine administered at day 9 in animals chronically treated with 20 mg/kg i.p. morphine failed to show such effect, indicating development of tolerance (Fig. 5A). In contrast, 30 mg/kg i.p. CHF3381 showed a comparable activity in mice given chronic treatment of either 60 mg/kg i.p. CHF3381 or vehicle, indicating lack of tolerance development (Fig. 5A). Moreover, 60 mg/kg i.p. CHF3381 still demonstrated antihyperalgesic activity in mice chronically treated with morphine, indicating that no cross-tolerance exists with morphine (Fig. 5B).
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Cold Allodynia in Neuropathic Rats. At baseline, animals with CCI to the sciatic nerve displayed cold allodynia by lifting the ligated hind paw off the floor with mean baseline withdrawal latencies ranging from 3.74 ± 0.29 to 5.77 ± 0.45 s. CHF3381 (10–200 mg/kg p.o.) blocked cold allodynia with a MED of 100 mg/kg. This action was dose-dependent and maximum at 2 h after administration (5.19 ± 0.53, 7.96 ± 1.56, 9.71 ± 1.59, and 11.08 ± 1.90 s for 10, 30, 100, and 200 mg/kg, respectively, versus 4.00 ± 1.19 for the vehicle-treated group; Fig. 6A). The effect disappeared by 4 h. Gabapentin (10–100 mg/kg s.c.) blocked cold allodynia at the highest dose tested (Fig. 6B). This effect remained significant for up to 4 h (3.32 ± 0.81, 6.49 ± 1.65, and 10.69 ± 2.18 for 10, 30, and 100 mg/kg, respectively, versus 4.55 ± 1.84 for the vehicle-treated group). Memantine (5–15 mg/kg i.p.) failed to have a significant effect on the maintenance of cold allodynia (Fig. 6C). At the highest dose of memantine tested (15 mg/kg i.p.), the latency to paw withdrawal at 4 h postdose was 9.22 ± 2.33 s (versus 5.52 ± 1.38 s for the vehicle; p > 0.05).
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Mechanical Allodynia in Neuropathic Rats. At baseline, animals with CCI to the sciatic nerve displayed allodynia in response to the mechanical stimulus with a mean withdrawal latency of the ligated paw of 22.7 ± 0.2 g; the mean withdrawal latency of the contralateral paw was 43.7 ± 0.3 g. CHF3381 (10–200 mg/kg p.o.) produced a dose-dependent antiallodynic effect, with a MED of 100 mg/kg (Fig. 7A). This effect remained significant for up to 4 h and was greatest at the 200-mg/kg dose, reaching an almost complete reversal of mechanical allodynia at 120 min after the administration (MPE = 0.76 ± 0.11). Gabapentin (10–100 mg/kg s.c.) failed to produce a significant effect on the maintenance of CCI-induced mechanical allodynia (Fig. 7B). A partial effect was observed at the 100-mg/kg dose 2 and 4 h after drug administration (MPE = 0.41 ± 0.14 and 0.41 ± 0.08, respectively). Memantine (10–15 mg/kg i.p.) was devoid of significant effects at the doses tested, maximal activity being observed at the 10-mg/kg dose 2 h after the administration (MPE = 0.36 ± 0.06; Fig. 7C).
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Mechanical Hyperalgesia in Rat Diabetic Neuropathy. A marked and significant decrease in the nociceptive threshold was evidenced in the vehicle-treated diabetic rats (136.7 ± 18.0 g) compared with the nondiabetic group (314.2 ± 11.6 g) (Fig. 8). CHF3381 orally administered at 25 mg/kg did not significantly modify the nociceptive threshold, even though the percentage of increase reached 83% (250.0 ± 17.0 g). At 50 and 100 mg/kg, however, CHF3381 significantly increased the nociceptive threshold in diabetic rats by 134% (319.2 ± 48.5 g) and 110% (287.5 ± 20.3 g), respectively (Fig. 8). Memantine (15 mg/kg i.p.) increased in a significant manner the nociceptive threshold in diabetic rats compared with the vehicle-treated diabetic group (108%; 284.2 ± 22.0 g) (Fig. 8). Conversely, gabapentin did not significantly modify the nociceptive threshold, although the percentage of variation reached 68% (229.2 ± 32.3 g) (Fig. 8).
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CHF3381 Plasma Levels in Streptozotocin-Treated Rats. CHF3381 plasma
levels (expressed as means ± standard deviation) of CHF3381 measured 1
h after drug administration (at the end of pain measurement) of 25, 50, and
100 mg/kg were 469 ± 280, 1206 ± 588, and 2980 ± 1753
ng/ml, respectively. A dose-proportional concentration level was therefore
found. At 50 mg/kg, levels of CHF 3381, after 1-h drug administration, were
similar to those found in normal rats
(Villetti et al., 2001),
suggesting that the absorption and distribution kinetics were not modified by
3.5 weeks after the induction of diabetes by STZ. Because the nociceptive
threshold increased from 25 to 50 mg/kg CHF3381, but not further at 100 mg/kg,
it may be assumed that an intermediate value between 469 and 1206 ng/ml
circulating drug level is needed to obtain a significant pharmacological
effect 25 days after the induction of diabetes.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). This is consistent with the observation that CHF3381
suppressed spinal nociceptive transmission in vitro with a similar profile and
magnitude of activity to the well established noncompetitive NMDA antagonist
memantine. Furthermore, CHF3381 showed an antihyperalgesic activity in
experimental pain models without affecting the physiological nociceptive
threshold, in line with previous reports for NMDA receptor antagonists
(Ren et al., 1992;
Quartaroli et al., 1999).
The first part of this study investigated the CHF3381 effect on the wind-up
phenomenon, an increased excitability of dorsal horn neurons induced by
repetitive C-fiber stimulation. As observed with memantine, the most
pronounced CHF3381 action was on the amplitude of cumulative depolarization.
Cumulative depolarization is strongly dependent on activation of
high-threshold afferents and NMDA receptors
(Thompson et al., 1992).
Therefore, the actions of CHF3381 and memantine on cumulative depolarization
are consistent with their ability to modulate the NMDA receptor ion channel.
However, high CHF3381 and memantine concentrations (300 µM) were necessary
to potently inhibit cumulative depolarization. Indeed, the potency of NMDA
receptor antagonists is often apparently much lower in in vitro slice
preparations used for electrophysiological recordings than in isolated neurons
or in membranes (Asghar et al.,
2000). This likely reflects the slow penetration of lipophilic
substances into the spinal cord compared with cultured neurons. It should be
also pointed out that other neuromediators, besides glutamate, are critically
involved in the wind-up phenomenon
(Sivilotti et al., 1995).
Gabapentin did not affect the wind-up response, confirming previous reports
(Patel et al., 2001).
CHF3381 potently and dose dependently inhibited carrageenan-induced thermal
and mechanical hyperalgesia, in agreement with studies indicating a
contribution of the NMDA receptor in this model
(Ren et al., 1992;
Taniguchi et al., 1997).
CHF3381 did not modify the physiological responses of the uninjured paw to
thermal and mechanical stimuli at the doses tested. A significant
antinociceptive activity was evident at doses about 10-fold below those known
to disrupt rat motor performance in the rotarod test
(Villetti et al., 2001). In
contrast, memantine only partially reversed thermal hyperalgesia; the
inhibition of mechanical hyperalgesia was transient and evident only at the
highest dose tested. Gabapentin reduced mechanical hyperalgesia in a
dose-dependent manner, but did not affect the maintenance of thermal
hyperalgesia. Gabapentin was previously shown to be equally effective at
blocking carrageenan-induced mechanical and thermal hyperalgesia
(Field et al., 1997). This
study and the present one are similar with respect to doses administered,
route of administration, carrageenan concentration, and subjects. Therefore,
the reason for this unexpected finding is not clear. Possibilities may include
distinct mechanisms underlying inflammation-induced mechanical and thermal
hyperalgesia, differing painful intensities evoked by mechanical and thermal
tests used as experienced by the inflamed paw, or a combination of these
possibilities.
In the mouse paw formalin test, noncompetitive NMDA antagonists have been
reported to preferentially alleviate pain behavior during the late phase,
confirming an important role for spinal NMDA receptor in contributing to
central sensitization after tissue injury
(Chaplan et al., 1997).
Similarly, CHF3381 abolished the late phase response (up to 99%) and, to a
lesser extent, inhibited the early phase (up to 65%). A similar pattern of
activity was observed for memantine, even if this compound suppressed both the
early (up to 44%) and the late phase (up to 53%) of formalin-induced responses
only partially. Therefore, as for the carrageenan model, these results suggest
that CHF3381 inhibits spinal NMDA receptors in pain models of peripheral
inflammation. However, the relative contribution of spinal NMDA receptor
inhibition to CHF3381 antinociceptive effect must be further verified by
comparing the present results with those for CHF3381 administered by the
intrathecal route. Gabapentin activity was in line with published data
(Field et al., 1997).
The results reported here suggest that, unlike morphine, CHF3381 does not
induce tolerance to its antihyperalgesic effect after chronic administration
in the formalin test. Eight days of 20 mg/kg morphine administration produced
significant tolerance in mice treated at day 9 with 6 mg/kg morphine. In
contrast, chronic treatment with 60 mg/kg CHF3381 did not modify the day 9
antihyperalgesic activity of 30 mg/kg CHF3381. We can exclude that the lack of
tolerance development after CHF3381 treatment can be attributed to the use of
a low dose, because CHF3381 was chronically administered at the dose maximally
active in the late phase of formalin-induced pain. The present study further
demonstrates that in the doses administered, morphine tolerance dose not
cross-generalize to CHF3381. Whether CHF3381 can block or delay the
development of opiate tolerance, as reported for other NMDA antagonists
(Quartaroli et al., 2001),
remains to be determined.
Recent studies suggest that both hyperalgesia and allodynia in peripheral
nerve-injured rats are sensitive to NMDA receptor antagonism
(Chaplan et al., 1997;
Quartaroli et al., 1999;
Yashpal et al., 2001). In
agreement with these studies, CHF3381 showed the ability to partially relieve
cold and mechanical allodynia in the CCI model. These effects were
dose-dependent and, for mechanical allodynia, represented a 70% recovery to
physiological level of response at 100 mg/kg. No significant effect was
observed on contralateral withdrawal thresholds. Interestingly, the
administration of doses 3-fold higher than those endowed with antihyperalgesic
activity in inflammatory pain models was necessary to relieve allodynia in
peripheral nerve-injured rats. Although the reason for the different potency
of CHF3381 in inflammatory and neuropathic pain models remains unclear, these
results might be explained by different levels of central sensitization in
these two conditions and/or a different involvement of the NMDA receptor in
the maintenance of allodynia and hyperalgesia in various pain states. In our
experimental conditions, we failed to demonstrate an antiallodynic efficacy of
memantine. This was somewhat surprising given that this compound has been
reported to attenuate mechanical allodynia in peripheral nerve-injured rats
(Carlton and Hargett, 1995;
Chaplan et al., 1997). As for
cold allodynia, we are not aware of previous behavioral assessment of
memantine effect on this parameter in the CCI model. However, memantine was
ineffective in reducing cold allodynia in clinical studies
(Eisenberg et al., 1998). It
seems plausible that CHF3381 and memantine block the NMDA receptor via the
same mechanism. It is unlikely that large differences in pharma-cokinetics and
tissue distribution may explain the different activity of these compounds in
the CCI model and also in carrageenan-induced inflammation. It is possible
that this different profile of activity arises as a result of the maximum dose
of drug, which could be administered without overt side effects. Indeed, the
occurrence of side effects can be expected after acute administration of
memantine at doses 
20 mg/kg i.p.
(Parsons et al., 1999).
Additional activities besides the NMDA receptor antagonism may also account
for the superior activity profile of CHF3381. CHF3381 behaves as a
nonselective monoamine oxidase (MAO) inhibitor (IC50 = 7.2 µM
for MAOA; IC50 = 60.3 µM for MAOB;
Villetti et al., 2002). Such
an action would activate inhibitory monoaminergic descending processes
originating at supraspinal levels (Millan,
2002). Thus, this could result in an interaction with NMDA
receptor antagonism for the inhibition of nociceptive transmission within the
spinal cord. Indeed, MAO inhibitors have been recently reported to possess
antinociceptive activity in experimental models of neuropathic pain
(Apaydin et al., 2001).
However, the functional significance of CHF3381 MAO inhibition in experimental
pain models remains to be addressed. Contrasting results were found after
gabapentin treatment. The gabapentin positive effect on cold allodynia was
consistent with other reports (Hunter et
al., 1997). The magnitude of this effect was comparable with that
observed after CHF3381 treatment. However, we could not detect a pronounced
antinociceptive effect against mechanical allodynia. To verify whether the
inefficacy of gabapentin was due to inadequate dosing, we treated neuropathic
rats with 300 mg/kg gabapentin and observed a complete reversal of mechanical
allodynia, as reported previously (Hunter
et al., 1997).
Finally, we examined the antinociceptive effect of CHF3381 in rats with
STZ-induced diabetic neuropathy. We confirmed that STZ-treated rats show
mechanical hyperalgesia (Courteix et al.,
1993). The administration of CHF3381 and memantine restored the
nociceptive threshold in diabetic rats. Our results are supported by recent
data indicating that magnesium and MK-801 reverse mechanical hyperalgesia in
diabetic rats (Begon et al.,
2000). Together, these data suggest an important role for NMDA
receptor antagonism in mediating mechanical hyperalgesia in diabetic
peripheral neuropathy. A single administration of gabapentin weakly improved
mechanical hyperalgesia. This observation is consistent with published data,
showing that gabapentin has to be administered over an extended period to
attenuate mechanical hyperalgesia in neuropathic rats
(Patel et al., 2001).
Interestingly, CHF3381 plasma levels achieved in diabetic rats 1 h after the
administration of the 50-mg/kg p.o. dose, which yielded a maximal
antinociceptive effect, were comparable with those previously observed in
normal rats (Villetti et al.,
2001), suggesting that the absorption and distribution kinetics of
CHF3381 were not modified 3.5 weeks after the induction of diabetes with
STZ.
Several clinical investigations have evaluated the activity of NMDA
receptor antagonists in chronic pain patients
(Fisher et al., 2000).
Although results from these studies have been encouraging, unwanted side
effects have hampered a wide use of this class of compounds. In this study, we
provide evidence that the low-affinity, noncompetitive NMDA antagonist CHF3381
inhibits spinal nociceptive transmission in vitro and is endowed with
antinociceptive activity in a number of animal models of inflammatory and
neuropathic pain at doses devoid of obvious side effects
(Villetti et al., 2001). In
light of the current therapeutic need for neuropathic pain treatment and of
the proven analgesic efficacy of NMDA antagonists in patients, it could be
worth assessing the therapeutic potential of CHF3381 for the treatment of
neuropathic pain in double blind placebo-controlled clinical studies.
|
Acknowledgements |
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|
Footnotes |
|---|
ABBREVIATIONS: NMDA, N-methyl-D-aspartate; CCI, chronic constriction injury; STZ, streptozotocin; VR, ventral root; DR, dorsal root; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; HPLC, high-performance liquid chromatography; MPE, maximal possible effect; ANOVA, analysis of variance; MED, minimum significantly effective dose; MAO, monoamine oxidase; MK-801, (–)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine maleate.
Address correspondence to: Dr. Gino Villetti, R & D Department, Chiesi Farmaceutici S.p.A., Via Palermo 26/A-43100, Parma, Italy. E-mail: g.villetti{at}chiesigroup.com
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