Abstract
Most botulinum toxin (BT) drugs are stored as powders which need to be reconstituted with normal saline before clinical use. As botulinum neurotoxin (BNT), the therapeutically active ingredient, is a large double-stranded protein the process of reconstitution should be performed with special attention to mechanical stress applied. We wanted to test the mechanical stability of BNT during the reconstitution process. For this, 100 MU onabotulinumtoxinA (Botox®, Irvine, CA, USA) was reconstituted with 2.0 ml of NaCl/H2O. Gentle reconstitution (GR) was performed with a 5 ml syringe, a 0.90 × 70 mm injection needle, one cycle of injection-aspiration-injection and two gentle shakes of the vial. Aggressive reconstitution (AR) was performed with a 5 ml syringe, a 0.40 × 40 mm injection needle, ten injection-aspiration-injection cycles and 30 s of continuous shaking of the vial. AR increased the time to paralysis in the mouse hemidiaphragm assay (HDA) from 72.0 ± 4.6 to 106.0 ± 16.0 min (*p = 0.002, two-tailed t test after Kolmogorov-Smirnova test with Lilliefors correction for normal distribution). Construction of a calibration curve revealed that the increase in the time to paralysis was correlated with a loss of potency of from 100 to 58 MU (−42 %). BT users should use large diameter injection needles for reconstitution, apply two or three injection-aspiration-injection cycles and, maybe, shake the vials a few times to rinse the entire glass wall. Aggressive reconstitution with small diameter needles, prolonged injection-aspiration-injection and violent shaking should be avoided.
Similar content being viewed by others
References
Charm SE, Wong BL (1970) Shear degradation of fibrinogen in circulation. Science 170:466–468
Charm SE, Wong BL (1975) Shear inactivation of heparin. Biorheology 12:275–278
Goschel H, Wohlfarth K, Frevert J, Dengler R, Bigalke H (1997) Botulinum A toxin therapy: neutralizing and nonneutralizing antibodies -therapeutic consequences. Exp Neurol 147:96–102
Harano Y, Yoshidome T, Kinoshita M (2008) Molecular mechanism of pressure denaturation of proteins. J Chem Phys 129:145103
http://hcp.botoxforoab.com/Content/assets/pdf/Reconstitution_Brochure.pdf
Jaspe J, Hagen SJ (2006) Do protein molecules unfold in a simple shear flow? Biophys J 91:3415–3424
Ohnishi T, Asakura T (1976) Denaturation of oxyhemoglobulinS by mechanical shaking. Biochim Biophys Acta 453:93–100
van der Veen ME, van Iersel DG, van der Goot AJ, Boom RM (2004) Shear-induced inactivation of a-amylase in a plain shear field. Biotechnol Prog 20:1140–1145
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Disclosures
DD received honoraria for consultations from Allergan, Ipsen, IAB-Interdisciplinary Working Group for Movement Disorders, Merz and Syntaxin. He is a shareholder of Allergan. He holds patents in botulinum toxin research. HB is founder and co-owner of Toxogen.
Rights and permissions
About this article
Cite this article
Dressler, D., Bigalke, H. Reconstituting botulinum toxin drugs: shaking, stirring or what?. J Neural Transm 123, 523–525 (2016). https://doi.org/10.1007/s00702-016-1538-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00702-016-1538-1