Complexing Protein-Free Botulinum Neurotoxin A Formulations: Implications of Excipients for Immunogenicity

Abstract

The formation of neutralizing antibodies is a growing concern in the use of botulinum neurotoxin A (BoNT/A) as it may result in secondary treatment failure. Differences in the immunogenicity of BoNT/A formulations have been attributed to the presence of pharmacologically unnecessary bacterial components. Reportedly, the rate of antibody-mediated secondary non-response is lowest in complexing protein-free (CF) IncobotulinumtoxinA (INCO). Here, the published data and literature on the composition and properties of the three commercially available CF-BoNT/A formulations, namely, INCO, Coretox^® (CORE), and DaxibotulinumtoxinA (DAXI), are reviewed to elucidate the implications for their potential immunogenicity. While all three BoNT/A formulations are free of complexing proteins and contain the core BoNT/A molecule as the active pharmaceutical ingredient, they differ in their production protocols and excipients, which may affect their immunogenicity. INCO contains only two immunologically inconspicuous excipients, namely, human serum albumin and sucrose, and has demonstrated low immunogenicity in daily practice and clinical studies for more than ten years. DAXI contains four excipients, namely, L-histidine, trehalosedihydrate, polysorbate 20, and the highly charged RTP004 peptide, of which the latter two may increase the immunogenicity of BoNT/A by introducing neo-epitopes. In early clinical studies with DAXI, antibodies against BoNT/A and RTP004 were found at low frequencies; however, the follow-up period was critically short, with a maximum of three injections. CORE contains four excipients: L-methionine, sucrose, NaCl, and polysorbate 20. Presently, no data are available on the immunogenicity of CORE in human beings. It remains to be seen whether all three CF BoNT/A formulations demonstrate the same low immunogenicity in patients over a long period of time.

Keywords: Coretox®; botulinum neurotoxin A; daxibotulinumtoxinA; excipients; immunogenicity; immunoresistance; incobotulinumtoxinA; secondary treatment failure.

Figure 1

Schematical depiction of botulinum neurotoxin A. (a) The 150 kDa neuromodulator BoNT/A consists of one heavy chain of 100 kDa (red circle) and one light chain of 50 kDa (red square) covalently linked by a disulfide bridge (yellow line). (b) The progenitor complex of 900 kDa is composed of the 150 kDa neuromodulator BoNT/A (red), a non-toxic non-hemagglutinin (NTNHA) of 150 kDA (green), and a 12-subunit complex consisting of 3 different hemagglutinins (HAs): 3 times HA70, 3 times HA 17, and 6 times HA33. The schemes were modified from the structures in [3].

Figure 2

Schematic depiction of principal differences in purity of selected BoNT/A preparations. (a) BoNT/A formulations without bacterial complexing proteins, containing only the 150 kDa neuromodulator, namely IncobotulinumtoxinA [8,9], Coretox^® [10] and DaxibotulinumtoxinA [11,12]. (b) BoNT/A formulations containing clostridial complexing proteins, such as AbobotulinumtoxinA [13], OnabotulinumtoxinA [14], PrabotulinumtoxinA [15] and LetibotulinumtoxinA [16]. Some of these also contain bacterial components such as flagellin, bacterial DNA and inactive toxin molecules [17,18].

Figure 3

Simplified model of the activation of the immune system. Two decisions have to be made by the immune system in a strictly hierarchical order before antibodies can be produced. Firstly, sentinel dendritic cells need to be fully activated by “danger signals” to become professional antigen-presenting cells. They present peptides of the antigen (here, BoNT/A) to an antigen-specific T helper lymphocyte, which can recognize the presented peptide as “foreign”. Secondly, if the peptide is foreign, such as in clostridial BoNT/A, this T helper lymphocyte becomes activated and subsequently supports antigen-specific B lymphocytes. These become activated and finally produce and release antigen-specific antibodies to BoNT/A, possibly including neutralizing antibodies.