We review the findings of publications relevant to the prevalence of SNR [4-8], etiology of nonresponse to BoNT-A [6,9-18], key trigger factors in SNR [2,6,11,14,15,19-26], BoNT-A formulations composition [4,8,11,23,27-52] and immunogenicity [1,3-6,10,11,15,17,18,28,31,35-38,44,48,49,53-67], insights into mechanisms of SNR [1,3,7,14,15,23,68,69], SNR management [1,3,6,7,14,23,25,26,46,66,70-81], and data gaps and limitations [3-6,10,11,14,15,26,28,40,49,63,82-84].

The prevalence of SNR in therapeutic applications of BoNT-A varies among conditions treated and is often correlated with the toxin dose used. Detection of NAbs correlated to nonresponsiveness in therapeutic applications [4], with its global prevalence estimated at 0.3%‐27.6% [5]. Limited data exist regarding its prevalence in aesthetics, which is partly due to the diverse treatment approaches used and difficulties in quantifying the cosmetic effect [6]. In a recent survey among 673 Korean aesthetic providers, 53.9% reported experiencing BoNT-A resistance. Of those, 59% providers indicated the resistance rate as <1%, and 36% providers reported as approximately 1‐25% [7]. In the same study, 23.8% of respondents continued using the same product but at a higher dose when they suspected that a patient might be experiencing BoNT-A resistance. Therefore, the prevalence of resistance is likely underreported, as many providers are unaware and may solely increase the BoNT-A dose in subsequent sessions following a partial response [5,8].

All BoNT-A formulations contain the same 150-kDa core neurotoxin derived from the Clostridium botulinum Hall A strain [11,27,28]. The 150-kDa core neurotoxin contains a 100-kDa heavy chain and 50-kDa light chain, linked by a disulfide bond. BoNT-A formulations vary in purity, specific bioactivity, complexing proteins, and excipient content (Table 1), all of which can influence their potential to elicit an immune response.

First-generation BoNT-A formulations such as onaBoNT-A and aboBoNT-A contain pharmacologically unnecessary components such as complexing accessory clostridial proteins, inactive neurotoxin, clostridial DNA, and excipients (Table 1) that may increase the risk of immune response [8,23,28]. The accessory proteins assemble into a supramolecular structure that serves two main functions: protecting the core neurotoxin from low pH conditions when ingested orally and facilitating its absorption in the gastrointestinal tract [27]. The protective function is mediated via the nontoxic nonhemagglutinin protein and the absorption function via hemagglutinin proteins [11]. Importantly, the accessory proteins rapidly dissociate from the core neurotoxin at neutral pH [27,52].

Second-generation Bo-NT-As, such as incoBoNT-A and daxiBoNT-A lack accessory proteins because of their removal during purification [11]. DaxiBoNT-A contains an HIV-derived, highly charged peptide (RTP004) which, according to the manufacturer, binds noncovalently to the negatively charged BoNT-A molecule and stabilizes it by preventing protein aggregation [28]. Additionally, the peptide may bind to negatively charged neuronal surfaces, which could enhance the internalization of the neurotoxin. However, Martin et al [28] reported that the binding of RTP004 to negatively charged neuronal surfaces should not be considered selective, as all cell types are negatively charged due to the terminal sialic acid residues on surface glycoproteins.

Case studies of BoNT-A use for aesthetic purposes demonstrated both SNR and NAb development over time with onaBoNT-A and aboBoNT-A [6,15,48,63]. In general, prevalences of NAb development and SNR are lower in aesthetic indications (overall NAb rate estimated at 0.2%‐0.4%) [5], which may reflect the lower doses employed and minimal long-term data [15,49,64]. Thirteen cases of NAb-related SNR emerging during aesthetic BoNT-A treatments [1,3,6,15,18,65,66] were identified in case reports or series. Key observations of this review are presented in Textbox 1. Complete SNR is usually preceded by partial SNR in the patient [6,17,18]. Complete SNR usually occurs after more than two injection series [6,17]. It can occur as long as after 5 years of treatment [6,17]. In a small sample study, 30% of patients who did not respond to onaBoNT-A cosmetic treatments responded when switched to incoBoNT-A therapy, which did not provoke immune responses [69].

Retrospective studies suggest an association between higher protein exposure and increased risk of Ab formation [14,70,71]. The precise mechanisms leading to resistance are still unknown, as the pure 150-kDa neurotoxin has low immunogenicity without any known associated pattern recognition receptors or toll-like receptors on dendritic cells. Park et al [23] suggested that when adjuvants in the BoNT formulation are injected alongside the 150-kDa neurotoxin, they can activate dendritic cells that may internalize the neurotoxin and present it to T-helper lymphocytes, resulting in NAb formation. Exogenous factors such as environmental allergens (eg, COVID-19 vaccine) may prime NAbs [72,73]. Specific immune system activation by a wasp sting was proposed as a contributing factor for BoNT-A Ab formation [74].

Alternate explanations for resistance to BoNT-A include muscle injection fibrosis, BoNT receptor downregulation, dynamic line depth worsening, and interactions with drugs like aminoglycosides and quinolones [3]. Intradermal injections are thought to carry a higher risk of developing resistance to BoNT-A compared to intramuscular injections, as the dermis is rich in antigen-presenting dendritic cells [5,7]. A phenomenon of decreased responsiveness after many years of BoNT-A therapy, known as tachyphylaxis, has been reported [1]. In such cases, the clinical effect is mitigating despite the absence of NAbs. Nevertheless, it is still uncertain whether this phenomenon has an immunologic basis and whether low-titer or poorly binding antibodies might play a role.

Early diagnosis is crucial, particularly as an increase in NAb formation must be addressed promptly. A patient’s aesthetic journey, especially a need for increasing BoNT-A doses and more frequent treatments, should alert the provider of possible SNR. Accurately detecting and quantifying NAbs supports the diagnosis. Structural assays such as ELISA and immunoprecipitation assays are sensitive for detecting BoNT Abs, but do not discriminate between NAbs and non-neutralizing Abs [14,15,23]. Bioassays such as the MPA or MHDA use animal models to identify NAbs. The MHDA, the only assay approved by the FDA, uses ex vivo testing for NAbs [14].

Most clinicians do not have access to the above assays and use clinical resistance tests to confirm the diagnosis of SNR [1,14]. One such test is the unilateral brow injection, which involves injecting a standard amount of BoNT-A, such as 20IU onaBoNT-A, into the right (by convention) medial eyebrow [14]. After allowing sufficient time for the toxin to take effect (typically 1‐3 weeks), the frowning facial expression is evaluated. Since nearly all individuals usually frown symmetrically, asymmetric frowning indicates responsiveness to the injected BoNT-A that has weakened the right corrugator or procerus muscles. In contrast, symmetric frowning indicates that the injected muscles were not weakened; therefore, the patient is likely resistant to that specific type of BoNT-A.

Several authors have advocated for using a highly purified toxin that demonstrates the least immunogenicity, such as incoBoNT-A [15,23]. This is especially important in large-dose injections and while treating younger patients who will accumulate higher lifetime doses [23]. Most experts recommend using the smallest BoNT-A dose that achieves the desired clinical effect, avoiding booster injections, and waiting at least 3 months between treatments [6,7,15]. Regarding maximum dose, 56.5% of aesthetic providers responded that BoNT-A dose should be limited to <100 IU per day, and 97.3% reported using <300 IU in total [7]. Such total doses are unlikely for wrinkle reduction but are possible with some off-label indications such as muscle size reduction. In body indications, higher doses of BoNT-A are injected, increasing a patient’s exposure to foreign proteins and their risk of NAB formation. Consequently, it is advisable to use a highly purified BoNT-A when treating body indications.

Increasing the efficacy and longevity of outcomes of BoNT-A treatments leads to decreased frequency of such treatments, which can help prevent resistance. Several authors recommend using toxins that offer improved longevity for cosmetic results, such as daxiBoNT-A [46]. In two of three randomized controlled trials, coadministration with oral zinc supplementation enhanced the longevity of BoNT-A outcomes [75-77]; however, the available data are limited. Hyaluronidase is a known tissue permeability modifier that increases the dispersion of drugs [78]. In a small pilot study on axillary hyperhidrosis, the coadministration of BoNT-A with hyaluronidase allowed for a reduction in the BoNT-A dose needed to achieve a similar effect compared to BoNT-A injections administered alone [79]. Notably, in one patient, the right side of the forehead–treated with both BoNT-A and hyaluronidase–exhibited a larger area of effect than the left side, which received only BoNT-A, across all postinjection evaluations. The authors suggested that the reduced dose of BoNT-A required when used alongside hyaluronidase may be attributed to the enhanced dispersion of the toxin facilitated by hyaluronidase. This approach could help avoid the use of high toxin doses that may lead to nonresponsiveness over time. However, more data are needed to confirm these findings.

Switching to a highly purified toxin such as incoBoNT-A once partial SNR is noted, has been advocated [14,23,66], especially as this was associated with a downward trend in NAb titers [66,80]. This switch was associated with clinical response in a study of patients with cervical dystonia [81,82] and another involving onaBoNT-A cosmetic treatments [69]. Nevertheless, in our review of aesthetic treatments (Textbox 1), this switch was not associated with short-term SNR resolution [3,6,15,65]. Longer follow-up is required for aesthetic applications in patients with SNR switching to incoBoNT-A. A switch to daxiBoNT-A may also be considered given its low immunogenicity in limited studies [46], but more data is required. The first author successfully used a short course of low-dose oral methotrexate immediately before BoNT-A injection to mitigate an immune response leading to further reduction of clinical efficacy in patients who experienced partial SNR. Patients with prior complete or partial SNR to onaBoNT-A may benefit from anti-calcitonin gene-related peptide monoclonal Ab therapy [83].

For complete nonresponse, many experts advise offering a 12- to 18-month “drug holiday,” and then resuming with a highly purified toxin. This suggestion is based on the medical applications of BoNT-A [67,84] and aims to normalize NAb levels before administering BoNT-A again. The duration of the “drug holiday” should be determined by measuring NAb levels. However, other experts argue against offering a “drug holiday,” noting that switching to incoBoNT-A results in most patients’ NAb titers returning to negative, similar to those who stopped receiving BoNT-A treatment altogether [80]. Moreover, switching to incoBoNT-A may be the only option for patients whose NAb titers take longer to become negative [67,80].

Switching to a different BoNT serotype, such as type B (BoNT-B), has been attempted. For cervical dystonia, switching to BoNT-B (rimabotulinumtoxinB), was beneficial [62]. In two patients reviewed here, after SNR developed, injection of BoNT-B showed a normal therapeutic effect [1,6]. However, patients who switched from BoNT-A to BoNT-B after developing NAbs and SNR may subsequently develop NAbs to BoNT-B due to the 30% structural homology in the heavy chains of BoNT-A and BoNT-B [26]. Several studies have demonstrated that patients who initially respond to BoNT-B after developing SNR to BoNT-A are likely to eventually develop SNR to BoNT-B as well [67,68]. Additionally, injecting BoNT-B, an off-label toxin in aesthetics presents challenges, including suboptimal longevity and adverse effects such as an intense stinging sensation on injection [85,86].

Aesthetic studies on NAb formation and SNR have been limited and have primarily focused on approved indications [4,6,63,87], while off-label applications involving higher BoNT-A doses have not been investigated. Additionally, the follow-up periods in these studies were relatively short (4-16 months), although NAbs usually develop over a more extended period, often spanning several years [5,40]. The frequency of NAb formation and SNR in real-world aesthetic practice may be higher than published estimates [5], likely due to extensive off-label use and the lack of a commercially available test for measuring NAb levels [11].

Detecting NAbs depends on the specific assay used, as there can be significant variability in sensitivity and specificity [10,11]. It also depends on the assay methodology, handling, and timing of collection of samples, and concurrent use of medications. Although the MHDA is the most sensitive bioassay, it is semiquantitative and not widely available. However, this assay has raised concerns about false-positive results and may detect subclinical Ab titers that do not result in treatment failure [1,14,15]. A quantitative, FDA-approved, commercially available assay to measure NAbs is needed to study the temporal variations in Ab titers [11]. This limitation prevents robust conclusions regarding the relationship of NAbs with nonresponsiveness. A lack of studies comparing BoNT-A formulations with a standardized NAb assay hinders reliable comparisons. Finally, it remains unclear to what extent the accessory proteins, inactive neurotoxin, and excipients may trigger the immune system, especially since the time frame for developing Ab-mediated SNR was short in most studies (ie, up to three injection cycles) [28]. This hampers our ability to draw firm conclusions regarding the excipients’ impact on the BoNT-A formulation’s immunogenicity.

A key uncertainty involves the relationship between NAbs and SNR [14]. Some patients with detectable NAbs retain their clinical responsiveness, while others without detectable NAbs have been nonresponsive to BoNT-As [14,49]. This indicates that there is no absolute correlation between NAb detection and nonresponse [88], and no established threshold for NAb titer reliably predicts clinical resistance to BoNT-A [3]. However, a correlation between responsiveness and NAb titers has been proposed [6,89]. Further complicating patient responses, variations in target binding site and binding affinity result in anti-BoNT-A Abs with variable neutralizing effects [10,26]. These observations highlight the complexity of BoNT-A immunogenicity and the variability in individual patient responses [14].

Nonresponse to BoNT-A is becoming increasingly important in aesthetics, particularly as many patients undergo lifelong treatments. Preventing SNR is crucial given the lack of solid data on effective treatments. When choosing a BoNT-A formulation, considering the potential for immunogenicity is essential. Aesthetic providers should perform comprehensive clinical assessments, inform patients about the associated risks, and develop strategies to minimize immunogenicity in their treatment protocols.