Introduction
Botulinum toxin type A (BTA) has fundamentally changed the treatment of various neurological diseases and made a real revolution in aesthetic medicine. The list of conditions with useful and effective BTA therapy is constantly expanding. The drugs themselves are being improved, and new products with new properties appear. At the same time, our knowledge about the pharmacodynamics and especially the pharmacokinetics of botulinum toxins remain incomplete and fragmentary. This is due to significant complexity of research in this area. Indeed, both therapeutic and even toxic doses of BTA are extremely small and standard analytical methods accepted in pharmacology cannot be applied in most cases.
Diffusion, diffusion, acceptance and migration
BTA injection is followed by complex processes of its spatial distribution in tissues, acceptance by receptors and subsequent conformational and metabolic transformations. These processes are determined by 3 components: spread of solution, BTA diffusion from the injected solution and migration to distant tissues (Fig. 1).
Fig. 1. Main stages of BTA pharmacokinetics.
Spread
Solution spreading is understood as rapid physical movement of the toxin from original injection site. Dimensions of zone and spread rate depend on certain variables related to technical characteristics of injection, volume of injected solution and its correspondence to target tissue capacity [1–3], as well as anatomical and physiological characteristics of the injected zone [4]. In particular, BTA distribution is influenced by the architectonics of the muscle fiber and its spatial and functional connections with surrounding structures [4, 5], as well as the presence of intermuscular fascia and connective tissue septa [1].
Many researchers emphasize that spread of solution significantly depends on such technical parameters of injection as needle thickness, rate, direction and depth of injection, as well as distribution of BTA total dose injected into one muscle per the number of injection points [3, 6]. Finally, some additional factors also influence BTA solution spread: target tissue trauma with a thick needle, hematoma within the injection site, pressure, massage, thermal effects, etc. [1–3, 7].
Diffusion
Diffusion of BTA molecules is a relatively slow dispersion outside the solution distribution zone within several tens of minutes [8]. Toxin molecules passively move from the region of higher concentration to the region of lower concentration throughout this phase to achieve chemical balance. It should be noted that the concepts of toxin diffusion and solution spread are often confused in some reports that results terminological confusion.
Undoubtedly, concentration and dose of the drug (i.e. the number of molecules injected in a certain volume) are the most significant factors affecting BTA diffusion area. G. Borodic et al. (1994) [5] experimentally evaluated the diameter of muscle fibers and staining for acetylcholinesterase as indicators of denervation in rabbits. They found that injection of small doses of BTA (1 IU) was followed by diffusion gradient collapse within 15–30 mm. However, higher doses (5-10 IU) resulted BTA diffusion throughout the entire muscle without a visible endpoint. BTA molecules are able to penetrate through the muscle fascia into adjacent muscles and result their paralysis [9, 10]. Higher risk of side effects after injection of high doses of BTA is associated with this factor [9, 11]. Quantitative studies of BTA spread through the muscle fascia have shown that the risk of complications can be prevented by distribution of the total dose into small portions over the target muscle [1, 5, 12].
Unlike concentration and dosage, solvent volume has a greater influence on the drug spread rather its diffusion. T. Hsu et al. (2004) [13] found approximately 50% increase of the frontal muscle relaxation zone following 5-fold increase of solvent volume (with the same BTA dose (5 IU)). Enlargement of muscle relaxation zone occurred due to higher spread of solution, while decrease of toxin diffusion was not essential [13].
Do the various BTA drugs differ from each other regarding their diffusion and spread? A. De Almeida et al. (2007) [14] compared the diffusion properties of OnaBoNT-A and AboBoNT-A after intradermal frontal injections of equal volumes of toxins at a ratio of 1:2.5; 1:3 and 1:4, respectively. AboBoNT-A resulted larger zones of anhidrosis and muscle relaxation at any ratio of BT doses. The authors concluded higher diffusion properties of this drug and, consequently, higher risk of adverse events. However, A. Pickett, et al. (2008) [15] emphasized that enlargement of anhidrosis area does not indicate differences in diffusion properties. In their opinion, this is the result of injection of different BT doses.
On the other hand, equivalence coefficient can also vary significantly for different muscles and indications. Comparative analysis of national and international guidelines revealed significant deviations from the generally accepted equivalence coefficients (OnaBoNT-A: IncoBoNT-A 1: 1 and OnaBoNT-A: AboBoNT-A 1:2.5) in the recommendations for correction of various facial muscles [16].
When discussing the risks of adverse events associated with the higher diffusion capacity of a drug, it is necessary to note the recent cross-sectional analysis of the US Food and Drug Administration (FDA) on adverse events following BTA injection for aesthetic indications since January 2014 to September 2019 [17]. Eyebrow/eyelid ptosis occurred in 1,783 (6.1%) out of 29,471 adverse events associated with BTA injection for aesthetic indications. At the same time, this complication was more common after OnaBoNT-A administration (6.4%) compared to AboBoNT-A (4.2%) or IncoBoNT-A (5.7%) [17]. Thus, the incidence of this side effect is primarily associated with violations of injection technique rather the higher risk of lower molecular weight drugs per se.
The debate about certain differences in diffusion properties of BTA drugs was finally put to the end as soon as almost instantaneous dissociation of neurotoxin complex into active neurotoxin and complexing proteins during BTA drug recovery was revealed [6, 15]. No difference was found between the drugs with different neurotoxin complex dimension (OnaBoNT-A and AboBoNT-A), nor in comparison with the drug comprising only purified neurotoxin (IncoBoNT-A). Any differences between the products and their diffusion profiles can be easily eliminated by adjusting the BTA dose [18].
Diffusion assessment is another important factor influencing the results of comparative studies. The most common clinical method for analysis of diffusion is associated with dimensions of anhidrosis zone around the BTA injection point. This method is safe, simple, objective and reproducible. However, one can obtain different results depending on toxin injection depth, while these data ensure only indirect analysis of BTA spread in the muscle due to structural differences of dermis and muscle. Electromyography (EMG) is a more expensive and complex method. However, this approach ensures quantitative and accurate assessment of muscle activity [19–21]. Analysis of expression of a specific neuronal adhesion molecule (N-CAM) is one of the most sensitive and accurate methods. N-CAM is a membrane glycoprotein that is accumulated in myofibrils after denervation and absent in intact fibers. In an experimental study, this method confirmed no differences in diffusion between OnaBoNT-A, AboBoNT-A and IncoBoNT-A [22].
In addition to concentration gradient and BTA dose, accurate injection to the target muscles near target receptors is another factor influencing diffusion field [2, 3, 19]. Indeed, BTA binding with receptors limits spread area. F. Molloy et al. [23] analyzed an accuracy of BTA injection in patients with focal arm dystonia. They found that the needle reached the target muscle fibers only in 37% of cases without EMG. In case of BTA injection into the facial muscles close to the skin surface for aesthetic reasons, EMG is usually not needed.
Migration
In case of a single-stage injection of a very high dose of BTA, the third mechanism for toxin spread is possible (migration to distant parts of the body). According to many authors, possible movement of BTA light chain (LC) along the nerve processes (retrograde axonal transport) is also referred to migration [8, 10, 12, 24]. Toxin migration explains systemic and distant effects after local injection of BTA [12, 24]. On the other hand, clinical identifying the differences between local and systemic spread of BTA is not always possible. For example, headache and dysphagia can be signs of both systemic botulism and local spread of toxin after injections in the neck [17].
Hematogenous pathway followed by distant effects is possible if a large dose of toxin enters systemic circulation. This is possible only in case of injection of a very large dose of BTA or accidental intravascular injection [12]. Experimental study of pharmacokinetics and spread of BTA with a radioactive label (125I) after a single injection showed subtotal accumulation of toxin within the injection site [25]. These data confirmed its local effect on cholinergic nerve endings [26], while most cells and organs in the body do not accumulate toxin [27].
In aesthetic medicine, toxin migration has no serious practical interest [18], since these doses rarely result systemic symptoms. Few reports about systemic effects after BTA injection for cosmetic indications were the result of administration of unregistered drugs with unknown composition and activity [28]. A systematic review comprised 11 randomized controlled trials on BTA use in facial aesthetics for the period from 1977 to 2009 [29]. The authors found that the incidence of side effects associated with BTA was similar to placebo (with the exception of eyelid ptosis) and there were no systemic effects.
Back in the 70s of the XX century, several experimental studies were carried out [30, 31]. These trials confirmed retrograde transport of BTA similar to tetanus toxin [32]. However, this effect was experimentally observed only after injection of high lethal doses of the drug [31, 33]. In addition to central effects [34], retrograde axonal transport can determine the effect of BTA on antagonistic [35] or contralateral muscles [31], that is confirmed by electrophysiological studies [36, 37]. Clinical discussion of systemic effects of BTA is related to its use for neurological indications and hyperhidrosis management. These approaches imply injection of high and ultrahigh doses of BTA [10, 37–39]. Injection of BTA for aesthetic indications in appropriate doses ensures only local effect [18].
Factors influencing onset and duration of BTA effect
Whatever toxin administration route was used (oral, inhalation or injection), the BT molecule must reach the perineuronal intercellular space to trigger a sequence of events and finally block nerve impulse transmission. To date, there is no evidence that BT has any ability for selective accumulation in extracellular fluid surrounding nerve endings. However, target nerve cells have a high affinity to BT and can efficiently extract these molecules from these spaces, while other cells do not have this ability [26]. That is why BT has almost exclusively local effects after injection.
Distribution of 125I-labeled BTA in muscles was analyzed by using of magnetic resonance imaging. The authors found BTA distribution in extracellular fluid near the nerve endings along the axis of muscle fibers and subsequent fact decrease of its volume (within 12 hours) [25, 26, 40]. According to experimental studies, binding of the toxin to the nerve endings requires about 20 min even after systemic intravenous administration. In case of local intramuscular injection, this period is even shorter [41, 42]. Pharmacokinetic experiments on isolated neuromuscular specimens revealed fast elimination of toxin from perineuronal microcompartment (binding T½ about 12 min; internalization T½ about 5 min) [43]. Thus, BTA almost completely binds to neuronal receptors within the injection site. Different drugs do not differ from each other regarding distribution in the same areas if the same volumes and equivalent doses of BTA are injected [22, 25].
Onset and duration of effect after BTA administration
Onset and duration of effect are the key factors determining patient satisfaction with procedures [44–47]. A pharmacological feature of BTA is a relatively long latency period until the first signs of muscle relaxation (from several hours to several days). In addition, gradual development of paralysis complicates accurate fixation of effect onset. Typically, the researchers consider the time until initial weakening of the target muscle contraction or the time until the maximum therapeutic effect assessed clinically or by EMG [42, 48].
Anatomy and functional state of the target muscle [42, 44, 49, 50], accuracy of BTA injection near the motor end plates [7], clinical status of patients, dosage and characteristics of the drug [42, 44, 46, 50] influence development and duration of effect.
Numerous clinical studies revealed different rates of relaxation of different facial muscles in the same patient [44]. This may be due to heterogeneous physiological state of various muscles. It was found that intensely contracting [7, 49] or hypertonic muscles [42, 51] will bind BTA much more actively. Therefore, accelerated muscle relaxation and prolonged effect are expected [7, 49]. Faster effect of BTA may be explained by enhanced fusion of synaptic vesicles with presynaptic membrane that increases the number of sites of toxin binding with receptors [52]. Thus, active muscle contraction within a few minutes after injection is justified [49, 53].
According to the theory of structural face aging (“Face Recurve”) proposed by C. Le Louarn et al. (2007) [54], aging is followed by appearance of age marker fascicles in some mimic muscles. These fascicles are in a state of constant tonic tension. Logically, tense muscles will more actively capture BTA that means their faster relaxation. Moreover, facial muscles differ from each other regarding the ratio of slow (type I) and fast (types IIA and IIB) fibers [55, 56]. This can affect onset of effect and reinnervation rate after BTA injection [57, 58].
General clinical status of a patient can also influence onset and duration of effect. For example, thyroid hormones significantly affect the muscles and neuromuscular transmission [59, 60]. There is evidence that menopause and reduced level of estrogens prolong muscle relaxation after BTA injection and slow down reinnervation [58].
Literature data are very contradictory regarding the differences in onset and duration of effect after injection of different BTA drugs. According to some data, AboBoNT-A results faster and long-standing relaxation [44, 47], according to other information — after IncoBoNT-A injection [46]. However, most authors believe that there is no significant difference in onset and duration of effect between various drugs. All preparations have a well-established duration of effect from several weeks to several months. Moreover, some therapeutic effects can last up to one year [42]. Regardless the type of commercial drug, development of neuromuscular blockade and its duration are influenced by BTA dose [42]. Pharmacokinetic studies have shown the same constant of toxin delivery rate to the nerve endings. Thus, the absolute amount of toxin penetrating into the nerve endings will be increased with drug dose augmentation within any time interval. However, this will not speed up the process for each molecule [27]. Thus, higher BT dose results shorter time interval for penetration of enough amount of molecules to induce muscle relaxation.
The predicted duration of BTA effect will be determined by 4 main indicators: BT clearance from systemic circulation, BT clearance from nerve endings, intact substrate resynthesis time (SNAP-25 for BTA or synaptobrevin-2 for BT type B) and sprouting (Fig. 2).
Fig. 2. Predictors of BTA effect duration.
Intramuscular injection of the recommended doses of the drug is followed by local binding of toxin and the last one is almost absent in systemic circulation [18]. Nevertheless, one should consider the possible variants of its systemic metabolism to understand the BTA pharmacokinetics. According to modern data, active form of toxin persisted in circulatory system until it is delivered to target cells or excreted [26, 41, 61]. There is no evidence of absorption and accumulation of toxin by blood cells or its proteolytic cleavage, i.e. toxin remains structurally and functionally intact in blood [26, 41, 62, 63].
Urinary excretion is another possible way for toxin clearance. However, molecular weight limit for renal filtration is about 50–70 kDa, while molecular weight of the whole neurotoxin molecule is 150 kDa. Therefore, toxin excretion by intact kidneys is impossible.
Finally, biotransformation in liver is the third and most likely mechanism of metabolism and elimination. Experimental data do not confirm significant participation of liver and spleen in BTA biotransformation [41, 61]. Experimental intravenous administration of 125I-labeled BTA in mice revealed toxin accumulation in liver and spleen only about 7% and 1% of the total dose of the drug, respectively [61]. Toxin elimination half-life was 10 hours. Preliminary immunization of animals significantly increased excretion of complexes of neutralizing antibodies with BTA. Accumulation of complexes by the liver was up to 30–40%, by the spleen — up to 5–7% of the total dose of injected toxin [61].
Thus, the whole BTA molecule is almost not systemically metabolized and quickly eliminated from circulation with binding to receptors within neuromuscular junctions. This process is even faster and more complete after intramuscular injection. Thus, further pathways of BTA molecules after binding to receptors is of much greater interest.
Intracellular metabolism of light and heavy BT chains
BT is dissociated into two chains in axon (heavy chain (HC) and light chain (LC)). HC is a conductor for LC while the last one functions as a Zn-dependent peptidase damaging one of the proteins of SNARE complex. SNAP-25 protein is a target for BTA [26, 42, 64]. According to some studies, BTA effect duration is determined by persistence of active LC protease in nerve endings [65].
In cytosol, BTA LC is bound with the inner surface of membrane [66] and fixed near its substrate. On the one hand, this mechanism ensures more effective cleavage of SNAP-25 protein and, on the other hand, reduces availability of LC for cytosol proteolytic systems. As a result, LC remains active as enzyme for several months. Different BT serotypes have different duration of effect (from several days to several months) [67–69]. Today, we can confidently say that duration of effect of a particular serotype is largely determined by the features of proteolytic degradation of the LC [67, 70]. Maximum duration of proteolytic activity of BTA is determined by its ability to avoid ubiquitin-proteasome degradation for a long time [70-73].
Duration of effect of various BT serotypes is also influenced by their proteolytic effect. It was found that BTA LC cleaves only 9 amino acids from SNAP-25. This is fundamentally important, since insignificant damage to the SNAP-25 molecule does not disrupt its binding to other proteins of the SNARE complex. However, this process blocks neuroexocytosis [74] and complicates metabolism of the damaged protein by intracellular proteases [75, 76].
Another process affecting duration of BT effect may be metabolic pathways of HC. This protein is responsible for BT molecule binding to the nerve cell receptors and LC translocation in cytoplasm [26, 67, 77]. Metabolic pathways of HC after LC cleavage and translocation are unclear [26]. Experimental trials revealed that BT effect duration is determined only by the peculiarities of intracellular metabolism of LC, while both LC and HC are responsible for effectiveness and onset of paralytic effect [78]. According to some data, BTA HC is localized in endosomes and does not participate in cytosolic mechanism of BTA toxicity after LC internalization into neurons [79].
Innervation recovery
Sprouting (formation of new terminal processes) is traditionally considered the main mechanism for restoring neuromuscular conduction [78, 80–84]. Sprouting induction is not specific for BTA, since this process can be induced by other factors blocking nerve conduction [85, 86].
Some researchers doubt the leading role of sprouting in functional recovery of neurons [42, 83, 86]. Indeed, recovery of neurotransmitter secretion within the new processes and original terminals occurs at approximately the same time. Moreover, over 80% of acetylcholine is released from original terminals [86].
Quality of muscle innervation recovery after repeated injections of BTA is important. A. Rogozhin et al. (2008) [87] found significant functional recovery of paralyzed muscles after 3 injections of BTA with intervals of 3-4 months. However, recovery was significantly slower compared to a single injection while distribution and structure of motor end plates remained abnormal. Nevertheless, changes in neuromuscular junction plasticity under aging are still unclear [58].
Are BTs neurotoxic? Some researchers found toxicity of BT serotypes C [88, 89] and E [90] in experiments in vitro. However, these authors used BT concentrations much higher than the concentrations causing botulism. BTA cytotoxicity was not observed under experimental conditions in cell cultures [88] and during electrophysiological survey of healthy volunteers [91]. Moreover, an extensive therapeutic experience of administration of BT serotypes A and B for various indications emphasizes no signs of neuronal damage even after long-term usage [6, 73, 92–95].
Apparently, recovery of neuromuscular transmission can occur repeatedly without loss or impairment of the function of neuromuscular junction. This is rationale for safe use of repeated BTA injections in medical practice.
Conclusion
Pharmacokinetic features of BTA drugs have a great clinical importance, since these characteristics determine safe and effective BT therapy. Rate and specificity of BTA binding to the receptors, as well as reversibility of paralytic effect determine high safety of these drugs in cosmetology if BTA is used in the recommended dosages and according to indications. On the other hand, a wide therapeutic range of safety and knowledge of the features of muscle electrophysiology and anatomy make it possible to individualize the injection scheme and BT dose for each patient. Finally, analysis of subtle mechanisms underlying BTA metabolism and recovery of neuromuscular transmission is valuable for searching the methods to control these processes for prolongation and fast elimination of botulinum therapy effect. Obviously, clinical use of BT drugs is very perspective. This potential will be realized in various medical fields as new information regarding its mechanisms of action and pharmacological characteristics is obtained.
The author declares no conflicts of interest.