By DR. BRIAN FUSELIER, DR. MELVIN FIELD, AND DR. BARRY LOUGHNER
Abstract
Following FDA approval for Botox on October 15, 2010 to treat headaches in adult patients with chronic migraine, the authors of this paper have administered Botox to over 600 co-morbid patients using off-labeled prescriptions in the trigeminal system. Taken together, the co-morbidities of these patients consisted of variances of TMJ dysfunction, facial migraine, trigeminal neuralgia type 1 and type 2, post-herpetic neuralgia, multiple sclerosis, and post-traumatic neuropathic pain. The authors have found the clinical use of these off-labeled prescriptions to be helpful as an adjunctive therapy in the setting whereby all conventional standards of medical care have failed. In some cases, the relief of pain has continued in the trigeminal system beyond the usual 3-month period. A legion of research publications and reviews have explained the motor neuron mechanism of action of Botox on the alpha motor end plate, and explained the anti-nociceptive effect on small-diameter, peripheral somatosensory neurons. Based on our review of the literature our conclusion uniquely summarizes that failed exocytosis of neurotransmitters produced by the mechanism of action of Botox is equivalent in both sensory and motor neurons in the trigeminal system. In our clinical experience, the identic action of Botox results in an advantage which creates more effective and longer lasting clinical relief in a population of co-morbid patients with different etiologies associated with pain.
Structure and Function of Botox
Neurotoxin onabotulinumtoxin A (BoNT/A) is one of seven serotypes of the core neurotoxin proteins derived from bacterium Clostridium botulinum. BoNT/A is considered to be the most potent of these neurotoxins with the most prolonged action. The therapeutic success of Botox results from its potent inhibition of neurotransmitter release in both motor neurons and sensory neurons with a duration measured in months.
Botox is a 150-kDa dichain protein composed of a 100-kDa heavy chain and a 50-kDa zinc-dependent light chain. These two chains are linked by a disulfide bond. Endocytosis of Botox into motor or sensory neurons is performed by the heavy chain. The heavy chain acts as the receptor- binding and endocytotic vehicle. The light chain is unable to be internalized into neurons without being initially bound to the heavy chain. The pharmacological action of Botox is due to the light chain which causes protease activity within the neuron which leads to inhibition of neurotransmitter exocytosis.
Molecular Mechanisms of Botox
Both, the heavy chain and the light chain consist of two distinct domains: a carboxyl terminal and an amino terminal, respectively (Figure 1). Successful endocytosis of Botox involves the carboxyl terminal of the heavy chain which binds with high affinity to glycoprotein receptors on the synaptic vesicle proteins type 2 (SV2). These specialized synaptic vesicles are expressed on the plasmalemma in areas where synaptic vesicles fuse with the neuronal membrane followed by endocytosis. SV2 is expressed abundantly throughout the nervous system. The ubiquitous nature of the SV2 receptors suggests that they perform a function common to all synaptic vesicles. The tertiary structure of the amino terminal of the heavy chain forms an iontotropic channel that allows Botox access into the SV2 synaptic vesicle. Once the Botox/SV2 complex is internalized into the neuron, it forms an endosome. The low pH conditions prevailing in the endosome cleaves the disulphide bond and allows the light chain to exit osmotically into the cytosol via the amino terminal of the heavy chain.
Botox is a 150-kDa dichain protein composed of a 100-kDa heavy chain and a 50-kDa zinc-dependent light chain which are linked by a disulfide bond. Both, the heavy chain and the light chain consists of two distinct domains: a carboxyl terminal (COOH) and an amino terminal (NH2)
The second mechanism of action of Botox is failure of neurotransmitter exocytosis. The light chain which is a highly specific endopepdidase (protease) cleaves a 25KDA synaptosome-associated protein (SNAP-25). Snap-25 is responsible for the formation of SNARE protein complexes (i.e. soluble n-ethylmaleimide-sensitive factor attachment protein receptors) in the cytosol (Figure 2).
SNARE exocytotic protein complexes in the cytosol are a universal mechanism providing a substratum network necessary for neurotransmitter release in eukaryotic cells which includes all motor and all sensory neurons. SNAP-25 proteins are essential for SNARE complexes to cause the transport of synaptic vesicles containing neurotransmitters to reach the cell surface, fuse with the neuronal membrane, and allow normal neurotransmitter release.
SNARE proteins represent a universal mechanism providing a substratum network necessary for neurotransmitter release in eukaryotic cells which includes all human motor and sensory neurons. SNAP-25 proteins are essential for SNARE complexes to cause the transport of endosomes containing neurotransmitters to reach the cell surface, then subsequently fuse with the neuronal membrane and result in normal neurotransmitter release.
The intracellular proteolytic action of the light chain of Botox cleaves SNAP-25, thus causing inactivation of the entire SNARE transport system. The result is failure of exocytotic release of neurotransmitters into the extracellular milieu. Operant neurotransmitters in the trigeminal nociceptive system include a neuropeptide (e.g. Substance P) and an excitatory amino acid (e. g. glutamate). The release of calcitonin gene-related peptide (CGRP) from cutaneous tissue innervated by the trigeminal nerve is also blocked by Botox On the other hand, Botox is unable to block proton-mediated CGRP.
Taken together, the successful endocytosis of Botox, and the failure of exocytosis of neurotransmitters incorporates the therapeutic action of Botox. The extensive distribution of SV2 proteins and SNAP-25 proteins indicate that both are expressed on motor and sensory neurons. In alpha motor neurons, Botox binds to SV2 receptor sites on motor nerve terminals, enters the nerve terminals, cleaves SNAP-25, and inhibits the release of acetylcholine (Ach). What follows is inactivity of the motor end plate and paralysis of localized muscle fibers. In somatosensory nociceptors, Botox binds to SV2 protein receptor sites on free ending terminals, enters the somatosensory terminal, cleaves SNAP-25, and prevents the release of substance P, glutamate, and CGRP which are key mediators of neurogenic inflammation (Figure 3).
Functional endocytosis and dysfunctional exocytosis of neurotransmitters incorporates the therapeutic action of Botox. In alpha motor neurons and nociceptors, Botox binds to SV2 receptor sites on nerve terminals and enters the nerve terminal (left side). The light chain exits the synaptic vesicle into the cytosol, cleaves SNAP-25, thus inhibiting the release of neurotransmitters (right side).
Skeletal muscle contains both peripheral terminals of alpha motor neurons and free ending terminals of group III and group IV nociceptors. Injection of Botox at therapeutic doses into skeletal muscle causes inhibition of Ach release from alpha motor neurons leading to partial chemical denervation of the muscle and localized reduction in muscle activity. In addition, Botox blocks the release of pro-inflammatory agents from free ending terminals of group III and IV nociceptors. Pain relief within 24 hours following treatment suggests that the anti-nociceptive effect of Botox on the sensory neuron may be independent of its motor end plate activity since Botox-induced paralysis takes at least 5 days to become clinically evident. This theory that the dual actions of Botox are independent is further supported by clinical evidence which is demonstrated by the effect of Botox on pain that occurs in areas devoid of muscle. Matthew and his colleagues (2008) published a case study of 4 patients with primary headache characterized by chronic moderate pain localized in the parietal area of the scalp that is absent of underlying muscle. Botox was focally administered in these painful parietal areas. All patients experienced a reduction in pain lasting on average approximately 14 weeks. Repeated injection gave the same degree of improvement.
Botox Effect on Central Sensitization
Not only has the analgesic effect of Botox been considered independent of its motor end plate activity, but also Botox has been demonstrated to have a more complex mechanism of action on the pain system compared to the motor system. In addition to the documented molecular mechanism of action on the peripheral nociceptive terminals, Botox has been demonstrated to have a central effect located at the first proximal neuronal interface.
Botox inhibits peripheral sensitization by inhibiting neurogenic inflammation. This mechanism of action prevents the release of pain-related neurotransmitters and neuropeptides such as substance P and glutamate from the peripheral terminals of primary trigeminal and cervical afferents, thereby, indirectly reducing central sensitization since central sensitization commences as a result of tonic nociceptive input. Therefore, the consequence of inhibiting these peripheral signals inhibits central sensitization. Moreover, Botox appears to have a direct inhibition of central sensitization. When peripherally injected into muscle, Botox is conveyed by retrograde transport via microtubule-dependent transit along sensory axons of peripheral nerves resulting in inhibitory effects at the level of the medullary dorsal horn. Retrograde transport of Botox is further supported by a formalin-induced facial pain rat model demonstrating that injection of Botox into the rat whisker pad or sensory trigeminal ganglion reduced nocifensive responses (Figure 4). When colchicines, which block microtubule-dependent transport, was administered, the antinociceptive effect of Botox was eliminated. Interestingly, three days following the subcutaneous injection of Botox into the rat whisker pad, Botox-cleaved SNAP-25 was observed in the trigeminal nucleus caudalis of the medullary dorsal horn.
One mechanism of action of Botox is failed exocytosis. Failed exocytosis inhibits peripheral sensitization at the free ending terminal by inhibiting neurogenic inflammation, thus preventing the release of pain-related neurotransmitters and neuropeptides such as substance P, glutamate and CGRP. This neuronal peripheral inhibition indirectly reduces central sensitization (left side). Botox also appears to have a direct inhibition of central sensitization. When injected into muscle, Botox is conveyed by retrograde transport via microtubule-dependent transit along axons of peripheral nerves resulting in failed exocytosis of pro-inflammatory neurotransmitters (right side) resulting in inhibition of central sensitization at the level of the medullary dorsal horn.
Botox Therapy in the Trigeminal Pain System
Botox has been used in patients suffering from a variety of pain problems originating from the trigeminal system. Three of the most common problems are temporomandibular dysfunction (TMD), migraine, and neuropathic pain.
Numerous studies have been published demonstrating the safe and successful, off-labeled use of Botox for the treatment of TMD - a group of pathological conditions affecting the function of the temporomandibular joint (TMJ) and/or muscles of mastication. The hyperactivity of one or more of the four elevator muscles of the mandible is a common clinical presentation. In an off-labeled study of 46 patients with chronic TMD for an average of 96 months were treated with 150 units of Botox. This study showed that extra-oral injection of the masseter and temporalis muscles resulted in significant improvement based on a pain visual analog scale, functional index, tenderness to palpation and interincisal oral opening. Whereas, the motor-end plate effect of Botox is transient, this study revealed that the muscular healing effect is significantly more longstanding than the average 3- month duration of Botox.
Another TMD condition occasionally observed clinically is subluxation of the mandibular condyle involving excessive anterior dislocation of the condyle beyond the crest of the articular eminence which results in a wide open locked position of the mouth. In a case study of 5 elderly patients, with persistent episodes of subluxation caused by complications of a neurological or severe systemic disease, were treated with two extraoral injections of 25-50 units of Botox into the lateral pterygoid muscle immediately following reduction of the dislocation by manual repositioning of the condyle. One injection site was 1cm below the central zygomatic arch, and the second injection site was in the area of the insertion of the lower belly of the lateral pterygoid muscle in the fovea of the condylar neck of the mandible. The depth of the Botox injection was in the range 3-4 cm as measured with CT imaging. Mandibular fixation with elastic bands was required for 4-5 days. All treatment was successful with no recurrence for 3 months to 2 years.
The FDA has approved the use of Botox for chronic migraine in patients who have failed numerous preventative protocols. Pooled data from trial 1 and 2 of the Phase III Research Evaluating Migraine Prophylaxis Therapy (PREEMPT) studies (n=1384) demonstrated a mean decrease in frequency of headache days per month compared to placebo. In addition, significant group differences favoring Botox were observed that included less hours of headache during headache days. Critical analysis of chronic migraine based on these randomized, double-blind, placebo-controlled trials showed that the group differences were modest (8.4 headache-free days/month for Botox vs. 6.6 days/month for placebo). An earlier study conducted by the Seymour Diamond group in Chicago demonstrated similar modest gains. Forty-one patients were randomized to 100 units of Botox vs. placebo using a fixed site and fixed dose paradigm. Severe headache frequency was reduced from 13.1 to 10.1 episodes/month compared with placebo-treated episodes that actually increased from 14.6 to 15.4 episodes/month. Taken together, these modest studies along with the high cost, places Botox as a second-line therapy for prophylaxis of chronic migraine.
Neuropathic pain has been defined by the International Association for the Study of Pain as "pain initiated or caused by a primary lesion or dysfunction in the nervous system." The probable effectiveness of Botox has been demonstrated in a select group of neuropathic conditions including traumatic nerve injury, post herpetic neuralgia, peripheral neuropathies, and neurovascular compression-evoked neuralgia - all of which have been observed in the trigeminal system. The postulated mechanisms of action of Botox in the treatment of neuropathic pain include blockade of substance P, glutamate, and CGRP from the peripheral primary afferent free-ending terminal. One other off-labeled trigeminal pain therapy proposed by these authors is the introduction of 25 total units of Botox into the masseter muscle, temporalis muscle insertion, and the insertion of the inferior belly of the lateral pterygoid muscle utilizing EMG guidance.
Safety and Toxicity
Botox is a relatively non-systemic protein. The pharmokinetics of Botox, using currently available analytical technology, reveals negligible titers of Botox in the peripheral blood following intramuscular injection at recommended doses. In addition, Botox is considered a low risk biologic since neutralizing antibodies that may develop are not likely to cross react with endogenous proteins, and exhibit low clinically detectable levels of antibodies when compared with other approved biologic products. On the other hand, if present in high concentrations, antibodies can inhibit the biological activity of Botox, possibly by its interaction with its neuronal receptors.
Direct effects of Botox on the CNS have not been reported, since its large size of 150-kDa cannot penetrate the blood-brain barrier. Alternatively, Botox could reach the CNS by retrograde axonal transport, since Botox has been detected at the first central synapse with radioactively labeled botulinum neurotoxin. Previously, transsynaptic transport had not been observed. Recently, however, Marino and colleagues (2014) showed that the performance of Botox on spinal events related to nociceptive processing had a direct transsynaptic effects. At present, transsynaptic effects have not been completely elucidated. Further research of the putative transsynaptic effect of Botox may be helpful in enlightening this mechanism of action, and thus maximizing its therapeutic employment and minimizing safety concerns,
Conclusion
The study of the molecular mechanisms of short-lived plasticity of synaptic modulation is important to understand pain processing in the peripheral and central nervous system. Botox modulates neuronal plasticity by impairing, on a time contingent basis, all types of neurons that express SV2 proteins on its surface and require SNAP-25 proteins for synaptic vesicle fusion. Under these conditions, Botox blocks Ach release in muscle fibers resulting in localized decreased muscle contraction. Moreover, in addition to its function as a spasmolytic, Botox also exerts its effect on peripheral sensitization by blocking the release of pro-inflammatory neurotransmitters such as substance P, glutamate, and CGRP. Botox can also be conveyed to the CNS via retrograde transport, thus mitigating the processes associated with central sensitization. The successful use of Botox as a novel analgesic is providing renewed hope in patients with refractory chronic pain involving the trigeminal system.
Brian D. Fuselier, DDS and Barry A. Loughner, DDS, MS, PhD, are members of the American Dental Association. Melvin Field, MD, FAANS is a member of the Facial Pain Association, American Association of Neurological Surgeons, North American Skull Base Society, and the World Federation of Neurologic Societies. Dr. Fuselier and Dr. Loughner are in private practice at Central Florida Oral and Maxillofacial Surgery in Orlando. Dr. Field is a partner at Orlando Neurosurgery in Winter Park, and operates at Florida Hospital Orlando and Orlando Regional Medical Center. For contact information visit their websites: Dr. Fuselier and Dr. Loughner are at www.cforalsurgery.com. Visit Dr. Field at www.orlandoneurosurgery.com
