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There have been numerous anecdotal reports of venoms being employed as analgesics in attempts to relieve severe pain
associated with cancer, immune dysfunction and viral infection. Current methods to treat severe pain mainly comprise
opiate-based products that have short-lived activity and the potential to produce dependence. New methods to relieve
chronic pain are of great interest, and venoms are rich in peptides with the potential to modulate chronic pain. This aspect
is highlighted by the recent approval of ziconitide (conotoxin SNX111) for the treatment of chronic pain.
Snake venoms have demonstrated antinociceptive activity, and certain isolated neurotoxins have demonstrated
significant analgesia in animal models. Cobra venoms contain high levels of neurotoxins that target nicotinic acetylcholine
receptors (NAChR). Cobrotoxin (CT)[1], a short-chain postsynaptic
a-neurotoxin isolated usually from Naja naja
atra, is reported to have analgesic
activity[2,3] and is commercially available in China for this purpose. CT is consi-dered to be a
specific ligand for the muscle-based alpha1 subtype of the NAChR, although it produces strong, apparently
centrally-mediated analgesic effects through an opiate-independent
mechanism[3]. The recent demonstration that the analgesics
ketamine and tramadol inhibit nicotinic currents carried by alpha7 receptors expressed in
Xenopus oocytes[4] implicates the
alpha7 receptor subtype. It was concluded that ketamine inhibits the presynaptic nicotinic receptors responsible for
facilitating neurotransmitter release, as well as the direct ligand-gated inward
current[4]. Ketamine was found to inhibit the
nicotine-evoked presynaptic facilitation of glutamate
release[5]. It has been reported that alpha-bungarotoxin can block the
effects of ketamine. Alpha-cobratoxin (CTX), a long-chain neurotoxin and homolog of alpha-bungarotoxin, is a high-affinity
ligand for the alpha7 subtype[6-8], whose localization is reported to be mainly pre-synaptic in the peripheral nervous system,
although with high-affinity sites within the brain but not in the spinal cord. It is also known that alpha7 subtypes can
conduct Ca2+ ions, thereby impacting directly on neurotransmitter
release[9]. However, it is unknown if such long-chain
neurotoxins can reach the CNS and exert antinociceptive activity.
The antinociceptive effects of neurotoxins from snake venoms other than that of
Naja naja atra have been
reported[10]. Crotamine (ip, sc), one of the main components in the venom of
Crotalus durissus terrificus, produced analgesic
effects[11]. Recently, we have demonstrated that crotoxin, another major neurotoxin of
Crotalus durissus terrificus venom, in addition
to its antitumor effects[12,13], has separate antinociceptive effects (HL Zhang
et al, unpublished data) and demonstrates
synergism with acetylsalicylic acid. It has been associated with reduced pain in patients with solid tumors in a phase I clinical
trials[14]. These studies suggest that snake neurotoxins could provide the basis for new treatments to combat pain. The
present study reports that CTX from the Thailand cobra,
Naja kaouthia, provides antinociception in rodent pain models and
that this activity is independent of the opioid pathway.
Materials and methods
Animals Male and female Kunming mice weighing 18-22 g and Sprague-Dawley rats weighing 200-250 g were purchased
from the Center for Medical Experimental Animals, Soochow University, China (grade 2, certification
No 98018). The National Institutes of Health guidelines for the care and use of laboratory animals were followed in all animal procedures.
Drugs and drug administration CTX was supplied by ReceptoPharm (Plantation, Florida, USA). Naloxone hydrochloride,
acetylsalicylic acid and atropine sulfate were purchased from Sigma (St Louis, MO, USA).
Intra-cerebral ventricle injection The injection of CTX was performed as previously
described[15]. Briefly, mice were anesthetized with methoxyflurane and a small incision was made on the skull to access the bregma. Mice were randomly
divided into 2 groups (n=10 in each group): the saline control and 4.5 µg/kg CTX groups. CTX was administered in a volume
of 5 µL normal saline through a puncture point 2 mm lateral to the bregma, using a 10-µL Hamilton syringe with a truncated
27 gauge needle so that it penetrated into the brain 3 mm from the top of the skull. The impact on the pain threshold was
measured using the mouse acetic acid-writhing test 1 h after icv CTX or saline administration. To confirm that the drugs were
administered into the cerebral ventricle, several mice were injected with 5 µL of diluted blue ink, and after the mice were killed,
their brains were examined macroscopically after sectioning. The accuracy of the injection technique was found to be good,
with 95% of injections being correctly located.
Periaqueductal gray cannulation and microinjection
Under chloral hydrate anesthesia, a guide cannula (stainless steel
tube of 0.3 mm OD) was stereotaxically implanted and positioned at 3 mm above the target area in rats. The coordinates were:
AP, 6.3 mm posterior to the bregma; H, 5.8 mm below the dura mater; and L, 0.5 mm lateral to the
midline[16,17]. The cannula was
anchored on the skull with dental acrylic cement. Rats were housed individually in cages with food and water provided
ad libitum and given 1 week to recover from surgery. One microliter of the drug solution (5 µg/kg) was injected over 5 min using
the syringe cannula, which projected 3 mm beyond the tip of the guide cannula. The injection cannula was left in place for
an additional 5 min to minimize the backflow of the drug. The delivery of drug to the periaqueductal gray (PAG) was also
verified by injecting 1 µL ink through the guide cannula upon completing the observation.
Analgesic assessments
Hot-plate assay In the hot-plate test, female mice and rats were placed on a hot plate with the temperature setting
controlled at 55 °C. The latency period required for mice and rats to lick their hind paws was recorded as the pain threshold.
The baseline pain threshold was obtained by averaging the values of 2 measurements before drug or placebo administra-tion.
Mice with 5-20 s or rats with 5-15 s pain thresholds were used in the experiments. Following drug administration, the pain
threshold was determined as described and cut-off times of 60 s in mice and 30 s in rats were used in order to minimize
injurious effects to the animals.
To assess the effects of CTX on pain response in the hot-plate test, female mice were randomly divided into 4 groups
(n=10 in each group): normal saline (NS) was used as a control (group 1), and CTX was administered at doses of 30, 45, 68
µg/kg (groups 2-4). The pain threshold was measured at 1 to 24 h after drug (ip) administration.
Acetic acid writhing assay In the acetic acid writhing test, 10 min after the administration of 0.1 mL/10 g (ip) 1% acetic
acid solution to mice, the number of writhing movements was counted from 10 min to 20 min after acetic acid injection.
The effects of CTX on pain response in the acetic acid writhing test was measured by randomly dividing mice into 4
groups (n=10 in each group), using saline as the control and CTX at 30, 45, and 68 µg/kg as for the hot-plate test. The pain
threshold was measured 3 h after drug administration.
Assessment of effects of atropine and naloxone on CTX-induced
analgesia The influence of atropine on CTX-induced
analgesia in these models was also studied. In each assay, 4 groups of mice
(n=10 in each group) were used: group 1, normal
saline (NS) control; group 2, CTX at 45
mg/kg; group 3, atropine (Atr) at 0.5 mg/kg (in hot-plate test) or 10 mg/kg (in acetic acid writhing test); and group 4, CTX plus
atropine (CTX+Atr) at the doses described for the individual assays. Atropine at a dose of 0.5 mg/kg (im) or 10
mg/kg (ip), or NS was administered 1.5 h after CTX. The pain threshold was determined 1.5 h after atropine administration.
The effects of naloxone on CTX-induced analgesia were assessed in the hot-plate test. Female mice were randomly
divided into 6 groups (n=10 in each group): normal saline (NS) control, 45 µg/kg CTX, 1 or 5 mg/kg naloxone (Nal), and CTX
plus 1 mg/kg or 5 mg/kg naloxone (CTX+Nal 1 mg/kg and CTX+Nal 5 mg/kg, respectively) as per the individual assays.
Naloxone or NS (ip) was administered 2.5 h after CTX. The pain threshold was determined 30 min after naloxone administration.
Study of analgesic combinations The impact of acetylsalicylic acid (ASA) on the analgesia induced by CTX was
investigated using the hot-plate test. In a manner similar to the assays described earlier, female mice were randomly divided
into 4 groups (n=10 in each group): 1, normal saline (NS) control; 2, 45 µg/kg CTX; 3, 300 mg/kg ASA; and 4, CTX+ASA at the
levels described previously. ASA (300
mg/kg, ip) was administered 2 h after cobratoxin (45 µg/kg). The pain threshold was determined 1 h after ASA administra-tion.
Effects of CTX on locomotor function Locomotor activity in mice was measured using an Animex activity Type S meter
(LKB, Farad, Sweden) with the setting at maximum sensitivity. Every movement by the mice was recorded
automatically by the instrument. On the day of the experi-ment, the mice were treated with the highest level of CTX (68
µg/kg) or saline. The locomotor activity of mice was then observed for 15 min at 1, 2, and 3 h after ip injection of CTX or control.
Data analysis Data are expressed as mean±D. Statistical significance of differences was determined by one-way
analysis of variance (ANOVA).
Results
Effects of CTX on pain responses in mice and rats
CTX at 30, 45, or 68 µg/kg (ip) produced a dose-dependent
prolongation of the latency for mice to respond to pain stimulation induced by heat. The analgesic effect of CTX appeared
at 2 h and peaked at 3 h after drug administration
(Figure 1A). The ED50 of the antinociceptive effect of CTX was 57.6 µg/kg
(35.32-93.93, 95% confidence limit) in the hot-plate test. Similarly, CTX produced a dose-dependent inhibition of the writhing response
to acetic acid administration in mice (Figure 1B). The
ED50 of the antinociceptive effect of CTX was 54.04 µg/kg (41.04-71.16,
95% confidence limit) for the acetic acid writhing test. The efficacy of CTX in analgesia was comparable to that of CT
(ED50 was 63.10 µg/kg as assessed in the hot-plate model, or 55.86 µg/kg as assessed in the acetic acid writhing model in our parallel
studies; data not shown). The analgesic effects disappeared 5 h after CTX (data not shown).
Analgesic actions of centrally administered CTX
In mice, intra-cerebral ventricular (icv) administration of CTX (4.5
µg/kg), 1/12th of the systemic dose of CTX, significantly reduced the writhing response induced by acetic acid
(P<
0.05), indicating that icv injection of CTX was effective
(Figure 2A). In the rat hot-plate test, administration of CTX (4.5
µg/kg) to PAG, 1/12th of a mouse systemic dose of CTX, did not produce a significant analgesic action (Figure 2B).
Influence of the antagonists atropine and naloxone
In the hot-plate and acetic acid writhing test in mice, 0.5 mg/kg (im)
or 10 mg/kg (ip) atropine alone had no significant effect on the pain threshold. In the hot-plate test, both CTX and CTX
combined with a small dose of atropine (0.5 mg/kg) produced significant analgesia
(P<0.05). There was no significant difference between the 2 groups
(Figure 3A). How-ever, in the acetic acid writhing test, CTX produced marked analgesia,
whereas CTX combined with a larger dose of atropine (10 mg/kg) did not produce significant analgesic action
(Figure 3B), indicating that a large dose of atropine could antagonize the effects mediated by CTX.
Naloxone at doses of 1 and 5 mg/kg (ip) had no significant influence on the pain threshold in the hot-plate assay. Both
CTX (45 µg/kg) and CTX combined with naloxone produced similar analgesic effects
(P<0.05). There was no significant difference between these groups (Figure 4).
CTX combined with ASA In the hot-plate test, CTX (45 µg/kg), ASA (300 mg/kg) and CTX combined with ASA produced
marked analgesia. There was no significant difference among these 3 groups (Figure 5), providing evidence that no
antagonism or synergism between the 2 products exists.
Locomotor effects of CTX To rule out the possibility that the effects of CTX on pain response were caused by an
impairment of motor activity, mice pretreated with the highest dose of CTX (68 µg/kg, ip) were evaluated for spontaneous
mobility 1, 2, and 3 h after drug administration with an Animex apparatus. The spontaneous mobility of mice did not change
with treatment at the highest dose of CTX (68 µg/kg) used in the study as compared with saline-treated mice (Figure 6).
Discussion
It is known that a-neurotoxins such as CTX and CT have neurotoxic effects because they block nicotinic receptors at the
neuromuscular junction[18], more specifically at the diaphragm. Despite this fact, cobra neurotoxins have been employed as
analgesics for decades[2], and early studies in animal
models confirmed that CT from Naja naja
atra had antinociceptive
properties[3]. In our present study CTX, a long-chain postsynaptic
a-neurotoxin from Naja kaouthia, produced potent
antinociceptive effects in both the hot-plate test and the acetic acid writhing test in mice when administered via icv and ip
routes. The analgesic effects of CTX appeared 2 h following administration and reached a peak 3 h later. The
ED50 of the antinociceptive effects of CTX was 57.60 µg/kg in the hot-plate test and 54.04 mg/kg in the acetic acid writhing test in mice.
The observations made in the present study for CTX are very similar to those reported for CT, with the exception that atropine
can inhibit the activity of CT at lower doses than observed for CTX. The present study showed that a large dose of atropine
(10 mg/kg, ip) could antagonize the analgesic effects of CTX in the acetic acid writhing test in mice, suggesting that the
analgesia induced by this toxin may be mediated by the central cholinergic system. Our study showed that naloxone had no
effect on the analgesia induced by CTX, implying that the central endogenous opioid peptidergic system is not involved.
This property is similar to that described for
CT[3]. These results suggest that CTX, despite its higher toxicity/receptor
affinity, is as effective as CT in the induction of analgesia and that the effect is not associated with the neurotoxic properties
of the proteins. Studies using crotoxin have suggested a potent synergism with ASA (unpublished data), so CTX was
assessed in combination with ASA, but no meaningful synergistic effects were noted.
Systemic and icv administration was effective for both neurotoxins, suggesting that the site of analgesic action of the
neurotoxins may be, at least partially, in the central nervous system. The PAG matter has long been suggested to play a
critical role in central pain inhibitory systems and morphine-mediated
analgesia[19,20]. However, the present results
demonstrated that microinjection of CTX at the same dose as that of icv (4.5 µg/kg) into the PAG did not result in significant
analgesia, suggesting that the PAG is unlikely to be an active site. Constant administration of small doses of CT has been
reported to increase the Leu-enkephalin content in the hypothalamus, striatum and midbrain, and increase the
Met-enkephalin content in the hypothalamus and midbrain, especially the
thalamencephalon[21]. In general, the onset of activity averaged
3 h after administration, whether administered by icv or ip, and in the case of CT persisted for more than 6 h. This activity is
not consistent with the known pharmacokinetics of these neurotoxins, and such neurotoxins show little accumulation in the
central nervous system[22]. It is therefore not possible to rule out the possibility that CTX also exerts its antinociceptive
effects peripherally or, as proposed by Chen and
Robinson[3], that these neurotoxins act through a second messenger
system. These postulations could serve to explain the time course of the measured responses.
It has been demonstrated that activation of the central
cholinergic system produces antinociception in
animals[15-20]. Activation of cholinergic pathways by nicotine and nicotinic agonists has been shown to elicit antinociceptive
effects in a variety of species and pain tests. During the 1990s, the discovery of the antinociceptive properties of the potent
nAChR agonist epibatidine in rodents sparked interest in the analgesic potential of this class of
compounds[23]. The identification of considerable nAChR diversity suggested that the toxicities and therapeutic actions of the compound might
be mediated by distinct receptor subtypes and, accordingly, epibatidine and its derivatives were used to identify nAChR with
mainly alpha4 receptors, although
receptors with alpha3 were also sensitive to these com-pounds. The involvement of alpha7 nicotinic receptors in nicotinic
analgesia has been assessed through spinal (it) and icv administration in mice. Dose-dependent antinocicep-tive effects
were seen with the alpha7 agonist choline after spinal and supraspinal injection using the tail-flick
test[24]. Furthermore, alpha7 antagonists MLA and alpha-bungaro-toxin significantly blocked the effects of choline. These studies suggested that
activation of alpha7 receptors in the central nervous system elicits antinociceptive effects in an acute thermal pain model.
Nicotine’s analgesic effects are now believed to arise as a result of the desensitized state of the receptor following its
activation[24]. In contrast to the roles of agonists discussed earlier, our own studies and those of others suggest that
nicotinic antagonists may also have a role in pain relief. CTX and its homologue, alphabungaro-toxin, preferentially target the
alpha7 and alpha1 nAchR in nerve and muscle tissue, respectively, and function by preventing the activation of such
acetylcholine receptors in pre- and post-synaptic membranes. The lack of effect of atropine on the analgesic action of CTX
in the present study could due to the low dose of atropine used. However, in a mouse hot-plate pain model, atropine at a dose
larger than 0.5 mg/mg elevated the pain threshold. We thus tested a high dose of atropine (10 mg/kg) in the acetic acid pain
model. We found that 10 mg/kg atropine had no effect on acetic acid-induced pain, but antagonized the analgesic action of
CTX.
Cobra neurotoxins, mainly CT, have been employed clinically for the relief of pain. Derivatives of CTX are also being
investigated in clinical
applications[25]. The ease of administration provides such toxins with some advantages over
conotoxin-based analgesics, which are administered
intra-thecally[26]. Additionally, research is emerging that cobro-toxin can substitute
for morphine and suppress the effects of morphine
withdrawal[21]. The results of the present study suggest that CTX has
analgesic effects and that the site of analgesic action may be the central nervous system, but the PAG is unlikely to be
involved in the mechanism of action. The central cholinergic system appears to be involved in the antinociceptive action of
CTX, although details of the mechanism remain unclear, and further studies on the relationship between central and
peripheral cholinergic activity for the analgesic effect of CTX are necessary. These data may provide support for the development
of a new analgesic drug based on CTX.
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