Core Neurotoxin and Accessory Proteins

In its natural form, BoNTA is produced by the bacterium, Clostridium botulinum . In humans, BoNTA’s light chain binds to synaptosomal-associated protein 25 on the presynaptic membranes of neurons, preventing the release of the neurotransmitter acetylcholine, resulting in muscle paralysis.

All commercially available BoNTA contain an identical core 150 kDa (kilodalton) neurotoxin isolated from C . botulinum. Differences in BoNTAs lie entirely in their patented manufacturing process [ ]. Specifications of protein purification, stabilization, and excipients added during production determine the distinctive physical properties of BoNTAs and render important differences in their clinical application ( Table 1 ). Understanding how these differences influence the clinical profile of each BoNTA formulation allows clinicians to tailor treatments to their specific patient’s needs.

In some formulations (Ona-BoNTA, Abo-BoNTA, and Pra-BoNTA), the core 150-kDa neurotoxin is electrostatically complexed with accessory proteins (APs) that are natively expressed in the bacterial cell. These APs contribute to the overall molecular weight of the active product (see Table 1 ). During reconstitution with bacteriostatic saline, pH changes result in the disruption of weak electrostatic connections between the core toxin and APs, leading to dissociation.

Immunogenicity

Although APs do not influence BoNTA durability or diffusion, they have been linked to the development of neutralizing antibodies against BoNTA [ ]. This mechanism is thought to drive the clinical phenomenon of BoNTA “nonresponders” or reduction in response or durability of BoNTA overtime [ ]. For patients experiencing a loss of response or durability to BoNTA, or for patients who plan to have serial injections for medical indications (eg, migraines), BoNTA formulations lacking associated proteins may prove beneficial. Inco-BoNTA and Daxi-BoNTA are the only 2 commercially available products that do not contain APs (see Table 1 ).

Excipients

BoNTA products also differ in their excipients, which are inactive components added to stabilize the active product. Human serum albumin (HSA) is a shared excipient of all BoNTA formulations with the important exception of Daxi-BoNTA, contributing to many of its attractive clinical attributes [ , ]. HAS prevents aggregation of BoNTA to itself and the glass vial, preserving activity. Other common excipients include sugars, surfactants, sodium chloride, and buffers (see Table 1 ).

Potency

During manufacturing, purification processes remove bacterial cell contaminants and are followed by a finishing method that produces the BoNTA into a powdered form (see Table 1 ). Each BoNTA product must be evaluated to quantify its potency, which does not necessarily correlate to quantity of toxin protein. BoNTA potency is expressed in units (U) and is defined as the biological activity of a given amount of nanogram (ng) toxin protein [ ]. BoNTA manufacturers each have unique assays to measure and quantify potency (U) and package their product (as U/vial) based on their specific assay [ ].

It is imperative to understand that no standardization exists for 1U of BoNTA, and units for each BoNTA formulation are noninterchangeable [ ]. For example, 1U of Abo-BoNTA is not the same as a 1U of Ona-BoNTA or Inco-BoNTA. To mitigate this lack of standardization, nanogram of active toxin protein per dose may be used to compare BoNTA formulations. However, this method is imprecise because quantity of protein product does not necessarily correlate to potency [ ].

RTP004

As mentioned, Daxi-BoNTA is the only available BoNTA without the stabilizing excipient, HAS [ , ]. Instead, RTP004, a novel proprietary 5-kDa synthetic peptide, acts as its stabilizing excipient. RT004 contains 35 amino acids with 15 consecutive lysine residues, which form strong electrostatic bonds with Daxi-BoNTA. Flanking these lysine residues is a 10 amino acid protein transduction domain (PTD). Engineered from the human immunodeficiency virus protein, PTD provides the excipient with cell-penetrating abilities, which have been used for intracellular molecular delivery in other pharmaceuticals [ ].

RTP004 prevents aggregation of toxin to glass vial and ensures sustained retention of toxin during manufacturing and transport [ , ]. Importantly, its synthetic derivation precludes RTP004 from exhibiting immunogenicity [ ].

Improved Efficacy and Durability

Following local injection, effective binding of BoNTA to the nerve terminal of the intended muscle is an intricate process. The small concentration of BoNTA must diffuse through the extracellular space to locate and selectively bind to the target nerve terminal. Negatively charged glycosphingolipids in the nerve terminal’s cellular membranes serve as binding sites for positively charged BoNTA residues [ ]. Binding of BoNTA to glycosphingolipids permits translocation of neurotoxin and contributes to durability and duration of action as BoNTA is held and accumulated at the nerve terminal [ ].

All BoNTAs contain positively charged amino acids that bind to glycosphingolipids. However, the RTP004 excipient of Daxi-BoNTA, with its positively charged lysine residues and flanking PTD, is postulated to facilitate and intensify its binding to glycosphingolipids [ ]. This is supported by findings from preclinical studies that BoNTA with RTP004 excipient had superior binding to nerve terminals compared with identical BoNTA without RTP004 [ ].

Increased binding of Daxi-BoNTA to the nerve terminal reduces loss of Daxi-BoNTA and potentiates the availability of BoNTA for endocytosis into the neuron. Furthermore, the accumulation of more product at the nerve terminal allows for continued endocytosis overtime, explaining its extended duration of action compared with other commercially available BoNTAs (see Table 1 ) [ ].

Superior Diffusion

Diffusion is defined as the ability of toxin to passively move along a concentration gradient within the target muscle. Diffusion is different from spread , defined as the inadvertent migration of toxin to unintended treatment areas, which may lead to adverse events such as ptosis.

BoNTA typically has the greatest clinical effect at the point of injection with tapering, subtle effects on the surrounding muscle fibers due to diffusion limitations. This may result in uneven muscle paralysis and a patchwork appearance of intervening retained rhytids within a treated area [ ]. This phenomenon is commonly experienced when treating the broad frontalis muscle, where injections are avoided in the inferior portion of the muscle to reduce risk of spread and brow ptosis [ ]. Although diffusion from superior frontalis injections is intended to treat the inferior portion of the frontalis, diffusion is occasionally suboptimal leading to compensatory recruitment and worsening of inferior frontalis lines above the brow.

Anecdotally, Daxi-BoNTA seems to exhibit superior diffusion similar to that of Abo-BoNTA. This is attributed to RTP004’s ability to improve the binding and retention of Daxi-BoNTA at nerve terminals, facilitating its passive movement along the treated muscle and wider extension of clinical effect [ ]. Optimal diffusion contributes to the more even “field effect” clinically described, likely most beneficial when treating broad muscles such as the frontalis or orbicularis oculi [ ]. For example, optimal diffusion along the entire orbicularis oculi may explain the reduction in recruitment of compensatory medial muscle movements following Daxi-BoNTA treatment of lateral canthal lines (LCL; Fig. 1 A and B ). Superior diffusion may also contribute to the reduced need for “touch-ups” observed with Daxi-BoNTA.

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