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Chemistry/Preparation
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A toxoid refers to an inactivated form of a bacterial toxin. An antitoxin is an antibody or antibody fragment capable of neutralizing a toxin. Multiple injections over months of formalin-inactivated toxoid are required to effectively immunize horses against botulinum toxin and to produce equine-derived antitoxins.31 The resultant antibotulinum immunoglobulin requires several purification and preparation steps.25 H-BAT is produced by pooling plasma from horses immunized with specific botulinum toxoid subtypes (A–G), followed by pepsin digestion and blending of the seven serotype antitoxins into a heptavalent product.12 H-BAT contains anti-type A, 4500 international units (IU); anti-type B, 3300 IU; anti-type C, 3000 IU; anti-type D, 600 IU; anti-type E, 5100 IU; anti-type F, 3000 IU; and anti-type G, 600 IU.29 As the equine HBAT is “despeciated” by pepsin enzymatic cleavage and removal of the Fc fragment portion, the result is a product composed of ≥90% Fab and F(ab′)2 immunoglobulin fragments and less than 2% intact immunoglobulin G (IgG).2 This decreases the risk of immediate hypersensitivity reactions and serum sickness.
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Human whole IgG BIG-IV is derived from cold ethanol precipitation of pooled adult plasma collected from human donors immunized multiple times with PBT (A–E).37,52 The reconstituted product (50 ± 10 mg immunoglobulin/mL) contains greater than or equal to 15 IU/mL anti-type A toxin activity and greater than or equal to 4 IU/mL anti-type B toxin activity; antibody titers against botulinum neurotoxins C, D, and E remain undetermined.22,29
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Current antitoxins (whether equine or human derived) bind to and neutralize free botulinum toxin.13 Thus, antitoxins are ineffective against toxin bound to presynaptic acetylcholine release sites, toxin endocytosed by peripheral neuronal cells, and intracellular botulinum toxin light chain endopeptidase activity.10 Affected presynaptic end plates must regenerate in order to regain function.
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Pharmacokinetics and Pharmacodynamics
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Antidotal antibody fragments (eg, F{ab′}2, F{ab′}, and single-chain variable fragments {scFv}) demonstrate shorter half-lives compared to whole immunoglobulins.32 Improved renal clearance and uptake by vascular endothelium and surrounding tissues may contribute to this effect.42 While linking fragments to polyethylene glycol can extend their half-life,16 this methodology remains investigational. Compared to the half-lives for antitoxin types A, B, and E of 6.5, 7.6, and 5.3 days measured in a patient provided whole immunoglobulin trivalent antitoxin, clinical trials following single vial administration determined shorter H-BAT antitoxin half-lives of anti-A (8.64 hours), anti-B (34.20 hours), anti-C (29.60 hours), anti-D (7.51 hours), anti-E hours (7.75 hours), anti-F (14.10 hours), and anti-G (11.70 hours).11 These half-lives increased to 10.20 hours, 57.10 hours, 45.60 hours, 7.77 hours, 7.32 hours, 18.20 hours, and 14.70 hours, respectively, when two vials were administered.11,20,24 The volume of distribution ranges from 1465 mL (anti-D) to 14,172 mL (anti-E), depending on the number of vials.11 The more rapid clearance of Fab and F(ab′)2 fragments compared to whole IgG may necessitate repeat H-BAT dosing in patients with wound or intestinal colonization or other cases in which in situ botulinum toxin production continues after antitoxin clearance.2 Indeed, recurrence of paralysis was reported following H-BAT therapy in a patient with persistent type F intestinal colonization.20
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By convention, one IU of botulinum antitoxin neutralizes 10,000 mouse intraperitoneal median lethal doses (MIPLD50) of toxin types A, B, C, D, and F, or 1000 MIPLD50 of toxin type E (the IU for type G remains undefined).14 Thus, for example, in the doses found in H-BAT, the 4500 IU of anti-A, 3300 IU of anti-B, 3000 IU of anti-C, 600 IU of anti-D, 5100 IUof anti-E, and 3300 IU of anti-F would offset a total of 4.5 × 107 MIPLD50 of botulinum A toxin, 3.3 × 107 MIPLD50 of botulinum B toxin, 3.0 × 107 MIPLD50 of botulinum C toxin, 6.0 × 106 MIPLD50 of botulinum D toxin, 5.1 × 106 MIPLD50 of botulinum E toxin, and 3.0 × 107 MIPLD50 of botulinum F toxin, respectively.46 These values would be anticipated to provide significant neutralization; patient serum botulinum toxin concentrations in food-borne botulism are usually less than 10 MIPLD50/mL and rarely exceed 32 MIPLD50/mL, using a plasma volume of 3000 mL reported previously.5,24,45 In isolated outbreaks, botulinum toxin concentrations in adult serum samples collected less than 18 hours after exposure are reported to be as high as 160 MIPLD50/mL.7 Other recent cases have also yielded human serum botulinum toxin concentrations of type A of 1800 MIPLD50/mL.46 Following single vial administration, the reported H-BAT maximum concentration values12 of 2.69 IU/mL for anti-A, 1.90 IU/mL for anti-B, 2.26 IU/mL for anti-C, 0.81 IU/mL for anti-D, 0.94 IU/mL for anti-E, and 2.37 IU/mL for anti-F would be anticipated to neutralize 2.69 × 104 MIPLD50/mL of botulinum A toxin, 1.90 × 104 MIPLD50/mL of botulinum B toxin, 2.26 × 104 MIPLD50/mL of botulinum C toxin, 8.1 × 103 MIPLD50/mL of botulinum D toxin, 9.4 × 102 MIPLD50/mL of botulinum E toxin, and 2.37 × 104 MIPLD50/mL of botulinum F toxin. One study that measured four patients’ serum antitoxin concentrations following trivalent antitoxin (ABE) administration determined that those patients’ measured antitoxin titers would retain the capability to neutralize 1500, 1000, and 12 times the anticipated toxin concentrations of types A, B, and E, respectively.24 Complexity in anticipating or interpreting absolute neutralization efficacy is due to the fact that animal studies have revealed that the relationship between circulating neutralizing antibody concentrations and the amount of botulinum toxin neutralized is nonlinear, leading to a more efficacious, disproportional increase in botulinum toxin neutralization as antibody concentration is increased.45
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The half-life of BIG-IV is approximately 28 days.29 BIG-IV anti-A titers were 0.5371 ± 0.2134 IU/mL on day one. Since one IU neutralizes 10,000 mouse intraperitoneal median lethal doses (MIPLD50) of botulinum toxin A, this yields approximately titers of 5370 MIPLD50/mL.14,29 Thus, a single infusion is anticipated to neutralize all botulinum toxin that might be absorbed from an infant’s colon for several months.6
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In order to properly interpret earlier botulism studies, it is important to recall that the previously available, equine BAT-AB antitoxin contained 7500 IU of anti-A antitoxin and 5500 IU of anti-B antitoxin.41 BAT-E contained 5000 IU of antitoxin. Trivalent antitoxin (ABE) contained 7500 IU of anti-A, 5500 IU of anti-B, and 8500 IU of anti-E antitoxins.27 Investigational, whole, and fragment-derived monoclonal antibodies that have been raised in murine and equine species against types A and B are also being explored.31,32
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Investigational PBT vaccine, combining individual monovalent toxoids, was initially manufactured by Parke Davis more than 50 years ago and was subsequently produced by the Michigan Department of Public Health under contract from the US Army.21,47 A replacement vaccine developed by US Army Medical Research Institute for Infectious Diseases (USAMRIID)—recombinant botulinum vaccine (rBV A/B)—is designed to protect adults 18 to 55 years of age against botulism type A (subtype A1) and type B (subtype B1).23 It has been studied in animals and, completed human clinical trials await publication, although approval may be pursued under the FDA Animal Rule.17,23,44 Other recombinant subunit vaccines against C1, D, E, and F have demonstrated efficacy in animals.47 Humanized monoclonal antibodies, small peptides and peptide mimetics, receptor mimics, and small molecules targeting the endopeptidase activity are other avenues being explored for botulism treatment.10