Since its use was first described in 1977 in children with strabismus, BT has been increasingly used in various fields and diseases, from the treatment of focal dystonias, spasticity and urinary incontinence to becoming the most widely used injection in cosmetic procedures worldwide[11,12].

The use of BT for the treatment of achalasia was first described by Pasricha et al[13] in 1994 in a pilot study, which was followed by a double blinded trial[14] in which patients with achalasia were randomized to treatment either with BT injection or placebo (saline) injection into the LES. At one week, 90% of the BT injection group showed significant symptom reduction and a significant decrease in mean LES pressure. At 6 mo approximately two thirds of the patients were still in remission. Since the publication of this seminal study many studies have investigated the role of BT in the management of achalasia[15].

Every BT serotype is initially synthesized as a 150 kDa neurotoxin polypeptide chain with low intrinsic activity along with a set of neuro-toxin associated proteins (NAP), which are believed to protect the neurotoxin from proteases in the gastrointestinal tract[16]. The BT precursor is cleaved in vivo into a 100 kDa heavy chain (HC) and 50 kDa light chain (LC) linked by a disulfide bridge as well as a poorly structured protein segment called the belt. The HC can functionally be split into the heavy chain carboxy terminus (H-C) and heavy chain amino terminus (H-N) (Figure 1). The H-C, which can further be split into two subdomains, is responsible for neuronal receptor recognition and binding whilst the H-N is responsible for facilitating translocation of the LC into the cytosol[17]. The HC binds to transiently expressed specific cell receptors as well as to a polysialoganglioside, the disulfide bond is reduced and the light chain is internalized by exploiting synaptic vesicle recycling and diffusing into the cytosol. BT has a high affinity and specificity for target cells and requires two different co-receptors found on the neuronal surface, although different serotypes have different receptors[12].

Open in New Tab   Full Size Figure   Download Figure

Figure 1 Structure of botulinum toxin A. The Cα backbone is represented as ribbons with the LC in cyan, the HN in dark blue and the HC in a green to yellow gradient highlighting the HCN and HCC subdomains. The HN belt is in red. H_N + H_C: 100 kDa heavy chain; H_N: 50 kDa amino terminus; H_C: 50 kDa carboxy terminal of the heavy chain; H_CC: β-beta tree foil fold heavy chain subdomain; H_CN: β-sheet jelly roll fold heavy chain subdomain; LC: Light chain. Adapted with permission from Montal[17].

Once inside the cell, the light chain proceeds to cleave one or more of the soluble NSF-attachment protein receptor (SNARE) complex proteins, which are required for synaptic vesicle fusion with the active zone of the neuronal synapse (Figure 2). Cleavage by BT causes impairment of vesicle fusion and inhibition of synaptic activity[16].

Open in New Tab   Full Size Figure   Download Figure

Figure 2 Mechanism of action of botulinum neurotoxin. A: Release of acetylcholine at the neuromuscular junction is mediated by the assembly of a synaptic fusion complex that allows the membrane of the synaptic vesicle containing acetylcholine to fuse with the neuronal cell membrane. The synaptic fusion complex is a set of SNARE proteins, which include synaptobrevin, SNAP-25, and syntaxin. After membrane fusion, acetylcholine is released into the synaptic cleft and then bound by receptors on the muscle cell; B: Botulinum toxin binds to the neuronal cell membrane at the nerve terminus and enters the neuron by endocytosis. The light chain of botulinum toxin cleaves specific sites on the SNARE proteins, preventing complete assembly of the synaptic fusion complex and thereby blocking acetylcholine release. Botulinum toxins types B, D, F, and G cleave synaptobrevin; types A, C, and E cleave SNAP-25; and type C cleaves syntaxin. Without acetylcholine release, the muscle is unable to contract. SNARE: Soluble NSF-attachment protein receptor; NSF: N-ethylmaleimide-sensitive fusion protein; SNAP-25: Synaptosomal-associated protein of 25 kD. Reproduced with permission from Arnon et al[91].

Although the effects of BT on the nerve terminal are long lasting, they are however reversible and do not lead to neurodegeneration[18]. After inhibition of synaptic vesicle fusion by BT, neuronal sprouts begin to develop from motor nerve terminals that establish synaptic activity. Ultimately, synaptic activity at the motor neuron endplate is restored, and the neuronal sprouts retract completely[18,19].

BT is commercially produced by the anaerobic fermentation of Clostridium botulinum although in nature it is also produced by other related species such as C. barati and C. butyricum[16]. There are eight immunologically distinct serotypes of Botulinum identified, with type H being only recently discovered[20].

As of now, the Food and Drug Administration (FDA) has approved two serotypes, type A and type B for use in humans for various clinical indications. There are seven subtypes of BT (A) (A1-A7) that have been described, but all three formulations of BT (A) available for clinical use in patients with achalasia are of the A1 subtype. They include Abobotulinum (ABO; Dysport^®/Azzalure^®), Incobotulinum (INCO; Xeomin^®/Bocouture^®) and Onabotulinum (ONA; Botox^®/Vistabel^®). BT (A) is the most widely used and best studied formulations of BT[21]. Although Rimaotulinumtoxin B (Neurobloc^®/Myobloc^®) is available commercially, it has not been widely studied in patients with achalasia.

The biological activity of BT is measured in a mouse lethality assay (LD₅₀), i.e., the dose of toxin capable of killing 50% of a group of mice, and units are given in mouse units (MU)[11]. Concern for mouse welfare has prompted the investigation of more humane cell-based assays, with several being proposed such as the recently published compound muscle action potential assay[22]. The different formulations of BT (A) have varying potencies which have been compared in several studies. One MU of ONA has been shown to be equivalent to 1 MU of INCO[23-25], whilst a conversion rate of 1 MU: 2-3 MU between ONA and ABO has been proposed in various studies[26,27], as well as 1:2.5 for aesthetic indications[25].

The potency of BT (A) and BT (B) is difficulty to compare directly. BT (B) has relatively weaker motor side effects and stronger autonomic effects than BT (A), even when used at standard dose. In one study for example, patients who received BT (B) reported higher incidences of constipation and lower saliva production[28].

All of the currently available BT (A) drugs are sold in powdered form and have to be reconstituted, whereas BT (B) (Neurobloc^®/Myobloc^®) is available as a ready to use solution. In addition, only INCO can be stored at room temperatures while the other formulations need special storage temperatures[11].

In the available commercial formulations, BT is stored with excipients such as NaCl/lactose/sucrose as well as albumin to decrease the risk of inactivation during preparation and storage. Out of the three formulations of BT (A), ABO has the least amount of albumin, which may partially explain the lower amount of available toxin per injected unit, as well as the shorter shelf life and decreased duration of stability after reconstitution compared to ONA and INCO[12].

The technique used to inject BT into the LES is still largely followed as described in the pilot study by Pasricha et al[13]. The LES is visually identified during upper endoscopy and aliquots containing 20-25 U of BT (A) are injected in quadrants for a total of 80-100 U.

Several studies have used slightly different techniques in their studies, although to date there are no randomized controlled trials comparing these different methodologies. In one study, patients received two injections spaced 1 cm apart in each of four quadrants for a total of eight injections equaling 100 U of BT (A). The response rate was 89.65% at 30 d and 55.17% at one year but fell to 13.79% at 2 years[29]. In another study involving seven patients, 100 U of BT (A) was injected in eight aliquots, with four injections each at the LES and approximately 4 cm above the LES, respectively[30]. At follow up, only 28.6% of patients were in remission.

Other investigators have attempted to find the optimal dose of BT. In one study[31], 118 achalasia patients were randomized into three treatment arms to receive 50 U, 100 U or 200 U of BT (A). At 30 d, 82% of patients were considered responders, and based on symptom scores the proportion of responders was slightly higher in the higher dose group, albeit not statistically significant. Similarly, the change in LES pressure was comparable in all three groups. To determine whether the timing of administration had any effect on duration of action, responders to 100 U of BT (A) were injected with a similar dose of 100 U 30 d later. At the end of follow up, patients that received 2 doses of 100 U 30 d apart were found to be more likely in remission (19%, P < 0.04) compared to 47% and 43% in the 50 U group and single dose 200 U group respectively. This study demonstrated a statistically insignificant therapeutic effect with dose escalation of BT, but clearly showed decrease in relapse rates with repeat BT injection.

BT (A) injection into the LES is extremely well tolerated, with the most common side effect usually being transient chest pain[32]. On the other hand, BT-B, by virtue of its stronger anticholinergic effects, has been noted to cause autonomic side effects like dry mouth and jitteriness even in small doses[11].

BT injection is considered effective in the short term, but has a high rate of relapse requiring a need for reinjection. For example, one meta-analysis[33] evaluated nine studies with a total of 315 patients, and found that the rate of symptomatic improvement at one month to be 78.7%, but gradually decreased to 70% at 3 mo, 53.3% at 6 mo and 40.6% at 1 year. Furthermore, at least a second treatment was required in 46.6% of patients. Generally speaking, there is almost universal symptom relapse by two years[34], although some studies have shown continued efficacy in up to 34% of patients at two years[35]. The efficacy of BT with repeat injections decreases and is thought to be secondary to antibody formation.

Overall, pneumatic dilation and myotomy have superior long term efficacy than BT injection in treating patients with achalasia[10].

In a Cochrane review comparing BT with pneumatic dilation, there was no significant difference in rates of remission at 4 wk after intervention. However, BT was significantly less effective in maintaining symptom remission at six months and one year[33].

Similarly, one meta-analysis analyzed studies comparing pneumatic dilation with BT injection and found a remission rate of 65.8% at one year for pneumatic dilation compared to 36% for BT injection (RR = 2.20, 95%CI: 1.51-3.20, P < 0.0001)[36].

In a prospective randomized study evaluating BT injection with Heller myotomy, the authors found that results at 6 mo were comparable; however, the efficacy of BT injection decreased thereafter and the probability of being symptom free at 2 years was 87.5% after Heller myotomy and only 34% after BT injection (P < 0.05)[35].

POEM is a novel endoscopic treatment for achalasia which is a minimally invasive alternative to conventional Heller myotomy. Although to our knowledge no prospective trials have compared BT injection to POEM, various studies have compared Heller myotomy with POEM and found favorable results. One meta-analysis evaluating a total of four studies found that POEM had comparable complications [odds ratio (OR) = 1.17, 95%CI: 0.53-2.56, P = 0.70] and symptom recurrence (OR = 0.24, 95%CI: 0.04-1.55, P = 0.13) as Heller Myotomy on short term follow up[37].

The response to BT seems to be unaffected by prior therapy such as prior pneumatic dilation or myotomy, which highlights an important role for BT injection in patients who have failed prior surgical or endoscopic therapy. In one study[38], the response to BT injection in achalasia was compared in patients without prior therapy, with prior dilation and with prior myotomy. Neither LES pressures nor symptom scores differed between groups. At 6 mo the remission rate was 71.4% in those who received prior dilation, 71.4% in prior myotomy and 73.9% in prior BT injection.

Pneumatic dilation comes with recognized risks, including esophageal perforation (approximately 1.6%) as well as symptomatic heartburn in up to 45% of patients[33]. Likewise, myotomy may be associated with risks, most noticeably esophageal perforation and postoperative GERD[39], and due to the nature of the procedure may not be suitable for patients with multiple comorbidities or at high risk.

For this reason, several expert guidelines have suggested that BT injection may play a role in the elderly, in patients with extensive co-morbidities or who are poor surgical candidates and as a salvage therapy in patients who have failed other therapeutic modalities[1,39-43].

As mentioned previously, the major cause of BT’s limited long term efficacy is the sprouting phenomenon, in which the presynaptic nerve terminals begin to produce sprouting nerve collaterals with a corresponding increase in acetylcholine receptors and ultimately restoration of conduction at the neuromuscular junction (Figure 3).

Open in New Tab   Full Size Figure   Download Figure

Figure 3 Neuronal sprouting and remodeling of the neuromuscular junction. Remodeling of the neuromuscular junction in extensor digitorum longus muscle at 10 (A-C) and 21 d (D) after a single injection of botulinum neurotoxin type C. Axons and nerve terminals were immunolabelled (red). To localize the junctions, nicotinic acetylcholine receptors were stained (green). Note the sprouts that emerge from the original motor endplate and project along muscle fibers (yellow arrows). Reproduced with permission from Morbiato et al[92].

Several animal model studies investigating nerve sprouting demonstrated a complex array of neural factors and neuroreceptors upregulated and involved in the process of neuronal regeneration and sprout formation, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line derived neurotrophic factor (GDNF) and insulin-like growth factor 1 (IGF-1)[44-48].

A limited number of studies have investigated the effect of neutralizing substances against these factors on the process of sprout formation, as well as the effects of co-injection of alternate substances with BT in hopes of decreasing the rate of neuronal sprouting and thereby increasing the duration of action and efficacy of BT injection. In a murine model Streppel et al[49] demonstrated that neutralizing antibodies to NGF, BDNF, and IGF1 significantly reduced nerve sprouting. In an animal model, Harrison et al[50] demonstrated that injection of anti-IGF1 or corticotropin-releasing factor after BT injection reduced axonal sprouting, prevented the up regulation of neuromuscular junctions and increased duration of BT efficacy.

Ricin-mAb35 is an immunotoxin composed of ricin, a toxin that inhibits protein synthesis, conjugated to a monoclonal antibody to the nicotinic acetylcholine receptor. Christiansen et al[51] found that injection of BT followed by Ricin-mAB35 into animal optic muscles resulted in decreased twitch and tetanic force at 6 mo compared to BT alone. Most recently, Jiang et al[52] investigated the adjunct use of acrylamide injection to botulinum toxin injection. Acrylamide is a cumulative dose related neurotoxin that causes retrograde necrotizing neuropathy. Acrylamide injection after BT injection in the gastrocnemius muscles of rats was found to inhibit nerve sprouting as measured by electromyography and pathological observation of nerve fibers per unit. In this study, after the injection of BT (A) plus acrylamide, the increase in the number of nerve fibers was significantly reduced, the peak time of sprouting was delayed to 8-10 wk, and the peak nerve fiber counts were significantly lower than that in the BT-A group (10.65 ± 0.32 × 10⁸/m²vs 14.33 ± 0.45 × 10⁸/m², P < 0.05).

No study has yet investigated use of BT injection with sprouting inhibitors in achalasia models. Hence, it remains to be seen if this can prolong the therapeutic efficacy of BT injection in patients with achalasia.

As mentioned above, as of now studies have primarily utilized BT (A) for achalasia. Of the three formulations of BT (A) that are available on the market (ABO, ONA INCO), INCO (Xeomin^®, Boucouture^®) differs from ABO and ONA in that only the 150 kDa neurotoxin itself is included and is devoid of the NAPs that are included in the ABO and ONA formulations. NAPs are naturally produced with the neurotoxin and are thought to protect the toxin from proteolytic and acidic degradation whilst in the gastrointestinal tract[12]. However, they are not known to play a role in the neurotoxin induced blockade of cholinergic transmission. Building on previous animal studies that demonstrated increased immune response and presence of neutralizing antibodies in BT (A) complex compared to pure BT (A), Wang et al[53] recently studied the inflammatory cytokine release between human neuroblastoma cells treated with pure BT (A), BT (A) + NAPs complex or NAPs alone. They found that exposure to BT (A) + NPA complex significantly increased release of IL-6, MCP-1, IL-8, TNF-α and RANTES compared to controls.

In addition, it appears that while pure BT binds solely to neuronal cells, NAPs bind to neuronal as well as to non-neuronal cells, demonstrating that they may not simply be as passive as previously thought. It is likely that the hemagglutinins in the BT (A) complex bind to dendritic cells which act as antigen presenting cells in the early stages of the immune response. This is likely to result in increased immunogenicity and the formation of an immune response against the BT agent, although the level of antibody titers necessary to effect BT’s clinical efficacy is unknown due to heterogeneity of available studies, and their presence may or may not affect patient’s responsiveness to BT[12].

Factors that decrease the efficacy of BT over time are poorly understood. Suboptimal reconstitution of BT by physicians may decrease the efficacy of the BT preparation as shown in a recent study[54]. Similarly, it has been proposed that establishing practices to reduce the risk of neutralizing antibodies such as lower dosing frequency, longer treatment intervals, and lower number of injections may decrease the likelihood of their development[12,55].

As of yet, only serotypes A and B have been FDA approved for use in human subjects[11]. However, almost all studies investigating the use of BT in achalasia patients were done using serotype A[15]. Hence, it is unknown if the other serotypes may prove more effective by decreasing the response of neutralizing antibodies or whether their use of different neuronal surface type receptors or cleavage of different SNARE proteins may prove less likely to elicit neuronal sprouting which is what primarily contributes towards the high relapse rate seen in achalasia patients treated with BT.

Part of BT’s unique attraction is its particularly long persistence in the neuron and prolonged mode of action. Interestingly, different BT serotypes have been found to have varying duration of action. For example, in one study BT (E) was found to exert its activity for only about 2-3 wk in murine models compared to around 10 mo with BT (A)[56]. However, BT (E) cleaves more C-terminal residues from synaptosomal-associated protein of 25 kD (SNAP-25) than BT (A) causing a greater disruption of exocytosis resulting in quicker and greater neurotransmission blockade[57]. Studies have shown that the differential persistence of BT (A) vs BT (E) may be secondary to different susceptibility of the LC to various ubiquitin-dependent proteolysis pathways in the neuron through its interaction with RING finger protein TRAF2[58].

A few studies have investigated the translational applications of genetically modified BT. One study genetically fused BT (E) LC to an enzymatically inactive BT (A) mutant, resulting in an end product that had BT (A)’s stability and persistence, as well as BT (E)’s potent cleavage of SNAP-25 and resultant neuromuscular paralysis[57]. Given BT ubiquitous use in many different fields, ongoing research to produce BT mutant strains that have stronger potency and persistence may prove beneficial in diseases such as achalasia.

As of yet, the endoscopic injection procedure for achalasia has largely remained unchanged since first introduced by Pasricha et al[14]. BT injection into the LES is routinely performed during endoscopy, which may mean that precise injection into the LES may not be possible due to lack of visualization. This may contribute to the lower rates of efficacy of BT injection or early relapse in some patients.

The utility of endoscopic ultra sound (EUS) in achalasia patients was first reported by Devière et al[59] in 1989 using an early 7.5 MHz ultrasound endoscope to measure the thickness of the LES in achalasia patients. A subsequent case series demonstrated technical feasibility, safety and decrease in dysphagia scores in a series of four patients. However, the study included a very small sample size and there was no comparison between standard endoscopic injection and EUS-guided BT injection[60]. Recently, a study compared EUS-guided BT injection in achalasia patients compared to conventional endoscopic BT injection[61]. Patients that received EUS-guided injections benefited from higher rate of relief and significantly lower rate of symptomatic relapse.

In summary, there is no gold standard for injection technique or dosage of BT injected, with different authors utilizing different dosages and techniques. Randomized trials are needed to determine optimum dosage, formulation and technique of injection.

Alternative agents for the treatment of achalasia have been investigated. Ethanolamine Oleate (EO) is a sclerosant which is FDA approved for the treatment of bleeding esophageal varices, and is also used for lower extremity varicosities and vascular lesions. EO is a synthetic salt comprising of ethanolamine and oleic acid. When injected, EO produces an inflammatory response resulting in necrosis and fibrosis in the epithelium and submucosal tissue. First investigated for the use in the treatment of achalasia by Moretó et al[62] over 20 years ago, a long term study has since been published evaluating the long term efficacy of EO injection[63]. The authors included 103 patients in the study, with a mean follow up of 84.5 mo for patients who received EO. The cumulative expectancy of being free from recurrence was 90% at 50 mo.

Niknam et al[64] investigated the long term efficacy of EO in patients with achalasia who were resistant or poor candidate for pneumatic dilation or surgery. Of 220 patients who were evaluated for inclusion, thirty one patients met the inclusion criteria. EO was injected into the LES three times at two week intervals, and followed for a duration of 30.16 ± 11.3 mo with primary endpoints being symptomatic improvement on the achalasia symptom scale (ASS) and results of timed barium esophagram. At 1.5 mo post injection, the mean ASS, volume of barium and LES were significantly decreased compared to pretreatment (P = 0.0001). Symptom score and volume of barium remained significantly improved at 6 mo and one year intervals. Recently, Mikaeli et al[65] prospectively compared BT to EO in a cohort of patients who were poor candidates for pneumatic dilation or surgery. Out of the 189 patients evaluated, 10 were included in EO group vs 11 in the BT group, with a mean duration of 27.38 ± 16.49 mo of follow up. No statistically significant differences were found between either treatment groups.