Pain Killers from Snails? What next?
From the neuroethological perspective, pain exists to protect an individual from an undesirable situation that might result in an adverse bioeconomical outcome such as death or serious injury. However in the case of chronic pain, which often exists as the bi-product of diseases such as severe diabetes, infection, nerve damage or permanent cellular damage, there seems to be little physiological benefit (Reichling et al. 2011). Patients who suffer from chronic pain thus are often faced with a lifetime of intense debilitating painful sensation which add unnecessary stress to their body systems and which in turn prevent them from functioning in their society with any level of acceptable normalcy (Baron et al. 2010). Due to the intensity of the discomfort and the large population of patients who suffer from chronic pain, a large portion of the efforts of those in the medical and scientific community as well as financial investment have gone into specifically understanding and discovering methods for the treatment and relief of this type of pain.
Pain nocicieption like many other sensations is subject to interpretation and evaluation by a given individual’s central nervous system. As a result, the amount of pain any two patients might experience under a set level of stimulation could greatly differ (Baron et al. 2010). This makes the quantification of pain intensity very difficult and presents a major hurdle in the treatment and clinical testing of potential analgesic drugs, namely with respect to their level of efficacy (Zamponi et al. 2009). It is generally accepted however, that on a cellular level pain, as a neurological mechanism, functions the same in every individual. This provides medical researchers with standards for drug action target sites namely in the peripheral nervous system, which in turn allows them to develop novel analgesic drugs that can help to treat chronic pain symptoms (Lewis et al. 2003).
Within the umbrella category of chronic pain there exists many subsets of specific pain categories—typically defined by the originating site of stimulation and their target action location. These could include “referred pain” resulting from internal body system damage such as in the case of severe arthritis or gastroesophogeal reflux disease, or pain resulting from permanent tissue trauma such as in the case of a burn victim. Chronic pain that results from a lesion or disease to the body’s peripheral or central nervous system is thus defined as neuropathic pain. Sciata is an example of a neuropathic pain disease resulting in a chronic sensation/perception of pain from the sciatic nerve of an affected individual without any obvious external noxious stimuli.
In order to accurately diagnose neuropathic pain, rather than nociceptive pain, the specific somatosensory abnormalities that present themselves must be examined (Baron et al. 2010). In patients who suffer from neuropathic pain, hypersensitivity to an affected area coupled with an adjacent lack of sensation is typically observed (Zamponi et al. 2009). The hypersensitive symptom often is presented in the patient as paraesthesia (a tingling sensation) or spontaneous shooting pain (such as that found in an electric shock). Patients with neuropathic pain experience hypersensitivity to mechanical and thermal stimuli as well as pressure or even temperature (Baron et al. 2010). Often times allyodynia (a pain response to a non nociceptive stimulus) or even hyperalgesia (an increased response to a normally nocicieptive stimulus) is also observed (Zamponi et al. 2009). To the medical examiner, the diagnosis of neuropathic pain in a presenting patient is critically dependent on these given modalities. To a medical researcher or pharmacologist, however, the underlying physiological mechanisms of nociception on a neurophysiologic level and the potential changes to those systems in patients suffering from neuropathic pain is key to designing therapies to combat and relieve suffering patients (Lewis et al. 2003).
From a pharmacological perspective, the current therapeutic approach to developing neuropathic pain treatments is done in a stepwise fashion in order to discern which drug or even drug combination provides the best relief for a patient with the least number of side effects (Hama et al.2009). Various types of drugs such as antidepressants which inhibit reuptake of norepinephrin and serotonin, calcium channel ligands,opioid based analgesics such as morphine and even topical based cocaine derivatives such as lidocaine have shown to exhibit some success in the treatment of neuropathic pain. With the exception of the antidepressants, the analgesics all tend to act by altering the permeability of neuronal receptor sites to its given ion species. However the majority of these treatment mechanisms tend to also act with relatively low specificity, showing action on receptors found in other body systems such as the gastrointestinal tract and lymphatic system (Baron et al. 2010). As a result of this general mode of action, the number of side effects presented with each of these drugs as treatment, tends to be detrimental or undesirable to patients, especially due to the typically older age of patients who are generally suffering from multiple issues (Zamponi et al. 2009).
Natural products have been a source for drug templates and treatments for hundreds of years and over 50% of current market drugs are derived or modeled from natural sources rather than theoretically developed synthetic candidates (Clark 1996). Naturally-derived compounds show a high occurrence in animal, plant and microbial systems as secondary metabolites (compounds made by the organism which are not essential to normal biochemical functioning but instead provide the organism with some sort of biological adaptation, thus allowing it greater bio-economical success.) (Clark 1996.) Several advantages of studying a naturally derived secondary metabolite candidate compound, rather than a theoretically derived synthetic molecule, include reduced efforts to test mode of action, kinetics, reproducibility, binding specificity, as well as potency and stability. These advantages are mostly due to the fact that these compounds evolved, function and exist in biological models (Lewis et al. 2003).
Venoms, secondary metabolites typically used by organisms for the capture and incapacitation of prey, arguably provide pharmacologists with an ideal source of drug candidates due to their unique methods of action, highly diverse pharmacologies and limited evolutionary structural precursors (Lewis et al 2003). Venoms are here defined as biologically produced concoctions typically made up of multiple biotoxic proteinaceous peptides. The peptide nature of these toxic compounds, contained within an overall venom solution, allows them to be easily produced using biotechnological methods like cloning and cell culturing and thus studied for biochemical functioning in a laboratory (Bereki et al. 2010).
A particular genus of venom-producing predatory gastropod known as the Conus snails, (cone snails) over the last 16 years, have been a major focus for pharmacological research into novel analgesics due to their highly unique, lightning-fast mode of toxicity. Like all venoms, Cone snail venom is made up of smaller based constituents termed conotoxins. These large array of conotoxins themselves are proteonaceous peptides who are known to act upon specific binding sites, namely neuronal and muscular voltage-gated calcium channels (Lewis et al. 2003). As often found in animals who exhibit venomacous abilities, these cone snails have highly specialized radular mouth parts which have evolved into a few venom- injecting teeth known as the toxoglossate radulae (Haddad et al 2006). These harpoon-like teeth can be shot out from a proboscis into the soft tissues of its prey, allowing it to pump its conotoxins into the prey’s body subcutaneously, intramuscularly, or intravenously (Lewis et al 2003). The end result of envenomation is rapid immobilization occurring with in minutes, and often is referred to as the lightning strike event. Once the cone snail has captured its now immobilized prey, consumption begins, typically while prey remain alive (Haddad et al 2006).
The majority of cone snails feed upon other mollusks and polychaete worms, however there are a subgroup of Conus snails that are piscivorous (fish consuming). These piscivorous cone snails (examples include Conus magnus, Conus geographus, Conus textile etc.) venoms differ from the other non- piscivorous groupings in the methods of action with regards to prey envenomation especially with respect to the toxins given binding sites. These differences present themselves as a highly-toxic and highly- specialized venom which is deadly to higher-order vertebrate prey and thus present the most danger to human beings. (Haddad et al 2006). In human victims, cone snail envenomation presents as an intense debilitating burning sensation at the site of injection followed by a progressive paralysis of the body systems approximately one hour after attack leading to eventual asphyxiation and death (Haddad et al 2006). The characteristic lightning fast physiological effects of piscivorous cone snail conotoxins/conopeptides coupled with their neuronal and muscular voltage- gated calcium channel binding sites in vertebrate subjects is what has prompted them to be of such high interest to pharmacologists for potential models of analgesic drugs for the relief of chronic neurological pain sensation/perception.
In general, the neurological precession and perception of pain is highly dependent on and modulated by any given number of ion channels and receptors channels typically involved in nociceptive neurological signaling (Zamponi et al. 2009). Typically the series of events that results in the perception of pain proceeds as follows: a noxious stimulus that acts on a nociceptor and thus presents itself to the peripheral nervous system. Upon stimulation of the nociceptors in the area of stimulus, the neurons associated with those receptors are stimulated, causing a string of action potentials to travel along the axons of the primary afferent nerve fibers towards the synaptic nerve terminals in the lamina of the dorsal horn of the spinal cord. These presynaptic neurons then release pro-nociceptive neurotransmitters such as Substance P, glutamate and others which then bind post synaptic ligand gated receptor sites, induce stimulation of the post- synaptic neurons located in the spinothamalic tracts of the body as well as stimulate immune system responses. Once these signals have reached the thalamus, the cortex can begin interpreting and conveying the perception of pain. Descending pathways from the cortex through the thalamus and downward help to modulate and regulate the bodies’ responses to these pain stimuli. (Hama et al 2009).
Voltage gated calcium channels through their presynaptic involvement,with the release of neurotransmitters into the synaptic cleft,contribute to the induction of action potentials. As a result of these neurotransmitters binding to corresponding receptor sites on the post synaptic terminals, post synaptic membrane permeability to given ions can be modulated and thus inhibitory or excitatory responses can be induced which present as the transmission or cessation of such pain stimuli. T- type (Cav3.2 subtype of the α1 subunit of the channel protein) voltage gated calcium channels are often situated in the cell bodies and presynaptic nerve endings of afferent peripheral nerves, specifically found in the dorsal root ganglia. (Zamponi et al. 2009).These channels help to contribute to the procession of action potential waves by lowering action potential thresholds, increasing the bursting activity of a particular neuron and increasing synaptic excitation (Zamponi et al. 2009). This threshold lowering occurs as a result of the influx of the positive current of Ca2+ changing the cells reversal potential specifically by increasing the membrane potential closer to its threshold potential. Unlike T type calcium channel receptors, N type calcium channels tend to be located at the presynaptic junction of the nerve synaptic head. Here these channels respond to action potentials and thus mediate the amount of calcium which is released into the synaptic terminal. As a result, synaptic vesicle release is triggered which in turn results in the activation of spinothalamic neurons (Zamponi et al. 2009). Studies, in which the gene responsible for production of Cav2.3 channels has been removed using genetic clones, seem to suggest that N type calcium channels play a critical role in the transmission of neuropathic pain. Because of this, most of the treatments for neuropathic pain are designed around blocking or modifying the modes of action of the N type channels (Cav2.2 α1 subunit specifically of the Cav2 family) (Yang et al 2009).
Prialt ® is a novel drug designed from a particular conotoxin constituent (ω-MVIII) that was recently approved for use by the FDA for the treatment of neuropathic pain in cancer patients (Hama et al. 2009). Peptides of the ω- conotoxin family often show N type binding specificity and thus are common models for pharmacological studies (Yang et al 2009). Bereki et al. (2009) researched the activity of two newly-identified ω- conotoxins named CVIE and CVIF that were found in a cDNA (complimentary DNA) library of Conus catus. Their studies focused on characterizing the modes of action of these two ω-conotoxin types and attempted to look at particular subunits of the channel proteins known as the β subunit, to gain a better understanding of this subunits role in recovery from conotoxin binding (Bereki et al. 2010). The concept of compound efficiency with relation to time, namely how fast acting and permanent the compounds effects are on a given target is a key component to a pharmacological study. If a particular drug model candidate shows no reversibility after dosing, the safety of the drug must be called into question due to the potential for toxic overdose or even potential death. Candidate molecules which show too rapid reversibility or little binding action, call into question the effectiveness of the compound. Bereki et al. found that like the other members of the ω-conotoxin family, CVIE and CVIF showed high affinity for the N type channel proteins. They showed almost no activity when presented to T type channel proteins. Their experimental setup focused on measuring the amplitude of calcium current over time after dosing with CVIE and CVIF as seen in figure 6. For the studies on the β subunit variant’s roles in the reversibility of the binding of CVIF and CVIE, the amplitude of current was measured relative to time in instances of absence, dosed and swashed-out situations when the toxins were dosed upon a recombinant type Cav2.2 subtype channel. The data they found highly support the conclusion that these new ω-conotoxins are reversible and highly-specific and might prove useful for novel drug candidates pending on clinical trials. The high specificity of action was determined by measuring which voltage resulted in 50% reduction of current relative to which subunit type the toxins were dosed on. Their results also suggest that the specific subtype of the β-subunit might play a role in the reversibility of ω-conotoxins, and could prove a secondary target for drug action (Bereki et al. 2010). Research by Yan et al. 2010 like the research by Bereki et al (2010)looked into the methods of action of a new ω-conotoxin termed S0-3found from the cone snail Conus striatus. Results showed that SO-3’s method of action is identical to that of ω-MVIII due to it being almost structurally homologous. They also found that SO-3 selectively bindsCav2.2 subunits and shows extremely high potency (effectiveness) per dose and reversibility. They found that SO3 showed potent activity regardless of the carrier solution in which it was placed for the rat formalin flinch test, the acetic acid writhing test, and locomotive force test, but it also actually exhibited a supportive effect when dosed with morphine in that morphine’s analgesic effects became more potent and longer lasting. These data were obtained by inducing pain in mice via the different tests and measuring their responses to that pain when injected with various combinations of saline, SO3 and morphine in differing carrier compounds. The data highly support SO-3’s mode of action as a Cav2.2 subunit antagonist and high N size (sample size) and large spectrum of pain tests using this compound have made this an ideal study of the methods of action of this SO-3 ω-conotoxin. These results are of huge importance due to the high similarity of SO-3 with MVIII indicating it as a potential replacement for the current active ingredient in Prialt (Yan et al 2010).
When Prialt (ω-MVIII) first had clinical trials, its key characteristic was that it exhibited such high specificity to binding with Cav2.2 subunits and thus high efficacy in the reduction of neuropathic pain symptoms. Prialt has, however, shown unwanted side effects such as memory loss, unruly behavior and hypotension (Zamponi et al. 2009). Because of these unwanted side effects, research into other Cav2 family subunits roles in neuropathic pain transmission has gained popularity as potential sites of action for other drug therapies. Recent research done by Yang et al. specifically attempted to look at the viability of using the R-type Cav2.3 subunit as a target site for modulating neuropathic pain symptoms rather than the normal N type Cav2.2 subunit (Yang et al 2009). They hoped that by investigating the action of this subunit compared to the other major calcium subunits, they could shed more light onto how R type Cav2.3 is acting specifically and what its functions are in terms of chronic neuropathic pain. What they found rather was that this channel type seemed to show little effect during periods of neuropathic pain. Their results strengthened the already known data that N type Cav2.2 plays a major role in neuropathic pain modulation. This could be seen in their results from the patch clamp electrophysiology experiments that they performed. They looked at the amount of measured calcium current in combination with various calcium channel specific neurotoxins, in particular ω conotoxin, in order to study the effects of these subunit types to the overall calcium current in situations of neuropathic pain. (Yang et al 2009).
In general, the amount of sensitivity to the Cav2.2 specific neurotoxin actually increases during periods of neuropathic pain, which seemed to suggest an increase in the production of Cav2.2 subtypes by the cell relative to the normal non neuropathic situation as seen in figure 5 part C. Studies using the Cav2.3 lacking (-/-) homozygous mice, however resulted in some questionable results, particularly in the fact that knockout gene mice suffering from neuropathic pain inexplicably showed little sensitivity to toxins dosed on the Cav2.2 channel. This is unusual since it contradicts most published findings regarding Cav2.2’s role in the transmission of neuropathic pain. (e.g. Yan et al. 2010 and Klimis et al. 2011). Despite this unexplainable situation, the data presented in this paper imply that neuronal plasticity in regards to pain can be adjusted via the Cav2 family subunits during neuropathic pain states. Namely this plasticity is due to the reduction in Cav2.3 sensitivity and an increase in Cav2.2 sensitivity in wild type mice. This is a decent finding which provides future studies with a possibility of regulating the production of the Cav2.2 subunit relative to the Cav2.3 subunit as a potential action site for the fine tuning/control of pain symptoms (Yang et al 2009).
Like all pharmacological studies, the investigation into ω-conotoxin as a potential treatment for neuropathic pain has also proceeded in a stepwise fashion with particular emphasis on the affinity and effects of various ω-conotoxin family peptides to the N type Cav2.2 subunit voltage gated calcium channels (Baron et al 2010). There exists however, alternative research that delve more into other groups of conotoxins such as α-conotoxin as in the case of Klimis et al. 2010 or even other ion- blocking peptide constituents of cone snail venom such as conotokin-G when combined with ω-contoxins as in the case of Hama et al. (year).Research by Klimis et al. focus on the different analgesic properties of 3 types of α-contoxins termed: Vc1.1, AuIB, and MII. They found that Vc1.1 and Au1B provided the most potent and longest lasting relief of allodenic type pain in rats suffering from partial sciatic nerve ligation. MII only seemed to provide very ineffective relief of allodenic pain (Klimis et al 2010).
Tests were examined for the efficacy of these toxins previously established target sites, in particular the action of all three toxins on α3 nicotinic acetylcholinesterase receptor proteins (nAChRs) and Vc1.1’s effect on α9α10 type nAchRs as well as their effects on GABA receptors responsible for the normal stimulation of the voltage-gated calcium channels. Data shown in figure 4 shows that Vc1.1 has the highest paw withdrawal threshold relative to dose concentration when compared to Au1B and MII. This means that when rats were dosed with Vc1.1 conotoxin, more mechanical stimulation was required to cause the mice to withdrawal indicating the greatest relief in pain stimuli. The effectiveness of the action of Vc1.1 was primarily due to the toxins high antagonistic affinity with the GABA receptor sites responsible for Cav2.2 type voltage gated calcium channel action, and not in fact various nicotinic acytelcholinesterase receptors. These results were able to demonstrate that by antagonistically blocking the GABA receptors associated with the action of N type Calcium channels, neuropathic pain modulation can occur. This makes α-contoxins another potentially useful candidate in analgesic drug models (Klimis et al. 2010).
Like the research done by Klimis et al., work done by Hama et al. looked at the effects of other types of Conotoxin constituents on neuropathic pain modulation. Unlike the research done by Klimis et al however, Hama et al looked at the effects of neuropathic pain modulation when two different families of conotoxins were used in conjunction with one another, namely conotokin-G an antagonist of the NMDA receptor and the ω- conotoxin constituent MVIIA, in particular their effects in combination on neuropathic pain modulation from spinal cord injuries. Their experimental data indicated that when dosed concurrently, MVIIA and conotokin-G showed the greatest additive efficacy towards the relief of neuropathic pain associated with spinal cord injuries in rat models, relative to the results that were generated when each toxin was dosed independent of the other. The results were generated using multiple trials of various types of spinal cord injury coupled with the dosing of the particular toxins. The measured withdrawal threshold, defined as the amount of stimulus needed to elicit a withdrawal response of the hind leg, was taken for rats under these conditions receiving either a single toxin type or a combination type. Then these results were used to calculate the dose at which half of the withdrawal response was measured and thus neuropathic pain was reduced. From here graphical representations of the data, in this case isobolograms, were used to depict whether or not an effect was considered merely additive indicicating a weak or normal type response versus super additive indicating a desirable effect (Hama et al 2009). Data in this particular paper are lacking due to a small sample size per test (less than 10 mice) and a lack of definition of what constituted a behavior indicative of pain. As such the resulting isobolograms are thus a weak indication / poorly accurate representation of experimental data due to the data itself being general and unquantifiable. However, despite lacking in the quality and accuracy of the graphical representation of their data, the research performed by Hama et al. still has important implications. This study helps to show how toxins with differing modes of action can have an additive effect in general and perhaps lead to a lower dosed, higher efficacy analgesic for the treatment of neuropathic pain. Neuropathic pain present patients with debilitating chronic pain that often for many is worse than death. Advances in medical and pharmacological research which utilize compounds which block the N type calcium channels found in the peripheral neurons associated with neuropathic pain, have allowed for generally acceptable treatments.
In particular Prialt, a drug derived from the conotoxin ω-MVIII is highly specific and long lasting, seeming to provide the best relief to patients suffering from neuropathic pain. However the side effects that occur in patients taking Prialt as a treatment course are still considered by many to be unacceptable. Recent research into other types of conotoxins has shown that either through the modification of the Voltage gated calcium subunits themselves or through the discovery of novel conopeptides used in conjunction with already known existing peptides, potentially new treatments of neuropathic pain could be further developed. Future research will mainly focus on these specific subunits and through various gene therapies perhaps modulate the actual resting periods of these receptor types themselves thus merely lowering action rather than acting as a completely antagonistic blocker of these channels themselves.
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