- Email: [email protected]
Commercial preparations and handling of botulinum toxin type A and type B
Commercial Preparations and Handling of Botulinum Toxin Type A and Type B JENNIFER L. PARISH, MD Abstract. The recognition of botulinum toxin as a therapeutic tool has revolutionized the approach to facial esthetics. In the 1970s, Dr. Alan B. Scott pioneered the use of botulinum toxin type A (BTX-A) for strabismus.1 This discovery led to further studies examining other clinical applications of BTX-A and, more recently, botulinum toxin type B (BTX-B). Despite its widespread use and the more than two decades of experience with BTX-A, controversy remains over the preparation and handling of the toxin.
B
otulinum toxin is a potent but fragile neurotoxin produced by the bacterium, Clostridium botulinum. Although there are eight strains, only BTX-A and BTX-B are commercially available. They have similar structures and basic mechanism of action; however, each serotype is antigenically distinct, and each strain binds to a different acceptor site on the plasma membranes of a cholinergic neuronal endplate.2 In addition, the production of BTX-A involves the process of either lyophilization or vacuum-drying, which may reduce the stability and activity of the toxin. This does not occur in the preparation of BTX-B.3 These differences may render direct comparison of the potency of neurotoxins unfeasible.2,4 The choice of diluent, dilution, and storage may also affect the efficacy of the toxin. There are conflicting results from several clinical trials regarding the handling of the neurotoxin. The debate partially arises from the concern that the toxin, particularly BTX-A, is susceptible to surface denaturation and thus, loss of potency.4-10 Studies and anecdotal reports indicate that the toxin may not be as vulnerable as presumed. In addition, although the preparation of BTX-B may render it more stable than BTX-A, the paucity of information makes it difficult to determine the influence of different diluents, dilutions, and storage on its efficacy.11-13 Further clinical trials, therefore, are warranted to determine which factors influence toxin activity.
Preparations There are currently three preparations of botulinum toxin, and there are two sources of BTX-A, Botox® From the Department of Dermatology and Cutaneous Biology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania. Address correspondence to: Jennifer L. Parish, MD, 1760 Market Street, Ste. 310, Philadelphia, PA 19103. E-mail address: [email protected] © 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010
(Allergan, Inc., Irvine, California) and Dysport™ (Ipsen Ltd., Berkshire, UK). Botox® gained U.S. Food and Drug Administration approval for treatment of glabellar lines in 2002. Recently, BTX-B became available in the United States. There is only one form of BTX-B. In the United States, the trade name of BTX-B is Myobloc™ (Elan Pharmaceuticals, South San Francisco, California), and in Europe, it is Neurobloc™. BTX-B received U.S. Food and Drug Administration approval for use in cervical dystonia in 2001. Owing to the success of BTX-A, there are studies investigating the use of BTX-B in facial esthetics.11,13 This article will focus primarily on Botox® and Myobloc™, because both preparations are available in the United States. Botox® and Dysport™ do not have the same formulations or potency. Botox® is supplied in vials containing 100 U of BTX-A, 0.5 mg of human albumin, and 0.9 mg of sodium chloride in a sterile, vacuum-dried crystalline form without a preservative. The production of Botox® involves the fermentation of the Hall strain of C. botulinum serotype A, which is developed on a medium containing yeast extract and N-Z amine.9,14 After purification of the culture solution by acid precipitation and dialysis, a crystalline complex develops. The complex is dissolved in a solution containing sterile saline and albumin and sterile-filtered before vacuum-drying. Before adding the diluent, the manufacturer recommends storing the vial in a freezer at ⫺5°C or below.9 The preparation of Dysport™ is different from that of Botox®. Each vial contains 500 U of BTX-A, 125 g of human albumin, and 2.5 mg of lactose.15 The production of Dysport™ involves column-based purification instead of the precipitation method utilized for Botox®.14 The preparation of Dysport™ involves lyophilization. Before reconstituting Dysport™, the manufacturer recommends storage in a refrigerator at 2-8°C.15 The measurements of potency used for Botox® and Dysport™ are not equivalent. One unit of Botox® cor0738-081X/03/$–see front matter doi:10.1016/j.clindermatol.2003.11.009
482 PARISH
responds to the LD50, which is the lethal dose required to cause death in 50% of female Swiss-Webster mice that weigh 18-22 g each. Although the same definition of units applies to Dysport™, 1 U of Botox® is not equal in potency to 1 U of Dysport™. The method of testing the LD50 is specific to each preparation of BTX-A.9 Clinical studies have indicated that 1 U of Botox® is equivalent to 3-6 U of Dysport™16-18; nevertheless, because of the differences in testing and formulation, comparison of dosing or potency is an approximation. BTX-B differs from BTX-A not only by serotype but also by method of production and toxin activity. Unlike BTX-A, Myobloc™ is a solution available in three vial presentations: 2500 U/0.5 mL, 5000 U/1.0 mL, and 10,000 U/2.0 mL. Each vial contains 5000 U/mL of BTX-B, 0.05% human albumin, 0.1 mol/L sodium chloride, and 0.01 mol/L sodium succinate. The production of Myobloc™ involves the fermentation of the Bean strain of C. botulinum type B. After anaerobic fermentation, the neurotoxin complex, which consists of BTX-B and associated proteins, undergoes purification by means of precipitation and chromatography.3,10 The manufacturer recommends storage of Myobloc™ in a refrigerator at 2-8°C.10 The processing of BTX-B does not involve lyophilization. Lyophilization may result in a reduction of toxin activity, and therefore, BTX-B may be more stable than preparations of BTX-A.3 Myobloc™ also does not require dilution before injection, as do both commercial preparations of BTX-A. Therefore, there may be a reduced hypothetical risk of affecting the potency of the neurotoxin. There is not a single dose ratio to define the potency relationship between Botox® and Myobloc™. BTX-B and BTX-A use the same measurement for biologic activity; however, 1 U of Botox® is not equivalent to 1 U of Myobloc™.2 Because of longer clinical experience, much of the current information regarding botulinum toxin activity is based on Botox®. As a result, investigators have attempted to compare the potency of Myobloc™ to Botox®. Animal and human studies indicate that a dose ratio does not exist for Botox® and Myobloc™.2,19 Further clinical trials, therefore, are necessary to determine appropriate dosing for Myobloc™.
Diluent Proper handling of botulinum toxin is a crucial factor in maintaining toxin activity. The manufacturers of Botox® and Myobloc™ make specific recommendations regarding diluent, dilution, and storage; however, clinical studies and anecdotal reports challenge these labeling instructions. The package insert for Botox® suggests reconstitution with 0.9% sterile saline without preservative and use within 4 hours of adding diluent9 A double-blind, randomized, controlled trial compared
Clinics in Dermatology
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the use of unpreserved saline to preserved saline. Reconstitution with preserved saline reduced the pain associated with Botox® injection. The decreased discomfort may result from the anesthetic properties of the benzyl alcohol in the preserved saline. Furthermore, the investigators noted equal efficacy.7 The author of this article has also had similar experience with preserved saline. A double-blind, randomized, controlled study indicated that reconstitution with lidocaine with epinephrine does not jeopardize toxin potency. The initial onset of action of Botox® occurs 1-2 days after injection. This time course decreases the predictability of the effect of Botox®. As a result, the addition of lidocaine with epinephrine may provide the clinician with immediate feedback. The investigators of the trial demonstrated that the immediate paralysis caused by the anesthetic diluent improved the reliability of the results. In addition, the diluent did not appear to decrease toxin efficacy.8 In the literature, there is some agreement with regard to the appropriate technique for reconstituting Botox®. The toxin is fragile. Agitation of the diluted neurotoxin allegedly can cause surface denaturation, which results in deactivation of the neurotoxin. Therefore, according to the package insert, the reconstituted Botox® should not be shaken. The diluent should be added into the vial by vacuum and not by actively squirting it into the vial.5,9,14,20 There are those who remove the rubber stopper. If this is done without caution, there may be a loss in toxin activity. In addition, alcohol can inactivate the neurotoxin protein. Alcohol used to wipe the cap or the skin should be allowed to evaporate before filling of the syringe or injecting into the skin.5 Therefore, despite the choice of diluent, the potency of toxin can decrease with improper methods of reconstitution. Unlike BTX-A, Myobloc™ does not require the addition of a diluent before injection. The package insert indicates that the solution can be diluted with unpreserved saline; however, the manufacturer recommends use within 4 hours to maintain toxin activity.10,21 It is unclear whether preserved saline would affect the potency of Myobloc™. The presence of benzyl alcohol in preserved saline may decrease the pain of injection. A recent study demonstrated the efficacy of Myobloc™ in the treatment of glabellar wrinkles. The investigators used preserved saline as a diluent without apparent loss of toxin activity.13 Additional studies are necessary to assess the influence of different diluents on the potency of Myobloc™. There is little discussion in the literature regarding the process of diluting Myobloc™. The ability to inject Myobloc™ without adding a diluent may account for the paucity of information. Although BTX-B may be a more stable preparation than BTX-A, BTX-B is still a fragile neurotoxin.3 The technique for diluting BTX-B
Clinics in Dermatology
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should most likely be similar to that for BTX-A. Further studies are warranted to determine the appropriate method for dilution.
Dilution The dilution of Botox® varies. The manufacturer suggests using 2.5 mL of saline to dilute the vial.9 Most clinicians reconstitute Botox® with 1-3 mL of diluent.5,22 In the literature, there are reports of large-volume dilution up to 20 mL.23 Although a 1-mL dilution provides for easy calculation (1 U ⫽ 0.01 mL), it is difficult to deliver the accurate dose due to the small volume. There is a loss of ⬃0.07 mL toxin in the dead space of the traditional needle hub and syringe. The use of a specialized syringe, such as the Ultra-Fine II short needle 0.3-cc insulin syringe, can reduce the potential waste of toxin.24 The optimal dilution partly depends on the experience of the clinician. A correlation is apparent between the diluent volume and the intended dose response. Injection of a high concentration in small volumes results in a more localized effect.18 There is usually a dispersion area of at least 1.0 cm.14 In contrast, injection of a low concentration in large volumes results in greater diffusion of the toxin.5 The large volumes may cause paralysis of unintended muscles. For example, treatment of the glabellar region with high volumes may allow diffusion of the toxin into the levator muscle, causing blepharoptosis. In addition, some authors report that large dilutions may have a shorter duration of effect.5 Therefore, the choice of dilution should be based on the desired outcome. There is less clinical experience with the dilution of Myobloc™. The initial trials focused on the use of Myobloc™ for cervical dystonia. In those studies, the doses of BTX-B did not require dilution.25 The appropriate doses for management of facial lines are still uncertain. It appears that dilution may be necessary to achieve the desired effect. In several trials examining the cosmetic application of Myobloc™, the investigators diluted the solution to obtain a concentration of 200-300 U/0.1 mL.11,13,26 Further studies are warranted to determine optimal dilution and dosing for the use of Myobloc™ for facial rejuvenation.
Storage Some controversy exists about the storage of reconstituted Botox®. The manufacturer recommends storing the diluted vial in the refrigerator at a temperature of 2-8°C.9 The debate arises over the duration of potency of the reconstituted toxin. According to the package insert, injection of the toxin should occur within 4 hours of dilution.9 There is concern that prolonged storage will compromise toxin activity. A study by Gartlan and Hoffman27 utilized the LD50 mouse assay to determine the potency of the diluted toxin. After 12 hours, there
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was a significant loss in the activity of the reconstituted toxin. In contrast, Sloop et al6 used a human model to demonstrate that diluted Botox® stored for 2 weeks maintained its potency. Another trial indicated that diluted toxin stored for 30 days was as efficacious as freshly mixed Botox®.28 Klein5 reports that many clinicians store Botox® reconstituted with unpreserved saline for a period of 7-30 days. There is a potential risk of contamination with prolonged storage of Botox® diluted with unpreserved saline.5,18 The use of preserved saline as the diluent could eliminate the risk. In addition, there are reports that Botox® reconstituted with preserved saline is stable after 5 weeks of refrigeration.7 The ability to store Botox® for an extended period not only conserves material but also enables more convenient patient scheduling. The toxin activity of Myobloc™ is viable for ⬃21 months.10 The manufacturer suggests storing the toxin at 2-8°C; however, studies have shown that the toxin is stable at room temperature (25°C) for 9 months.21 The prolonged shelf life of Myobloc™ makes it advantageous to the clinician. There is a paucity of information regarding the storage of diluted Myobloc™. According to the package insert, Myobloc™ should be injected within 4 hours of dilution with unpreserved saline.10 The limited experience with Myobloc™ makes it unclear whether prolonged storage of the toxin after dilution jeopardizes its activity.
Conclusions The preparation and handling of Botox® and Myobloc™ influence clinical efficacy. The neurotoxin is fragile. As a result, studies have attempted to delineate which factors affect toxin activity. Although both Botox® and Myobloc™ have a similar mechanism of action, the formulations are different. In contrast to Myobloc™, the preparation of Botox® involves vacuum-drying, which may render it more vulnerable to surface denaturation. In addition, Myobloc™ does not require reconstitution, which may help to maintain the potency of the toxin. Further clinical experience is necessary to determine whether Myobloc™ can be as effective as Botox® in facial esthetics.
References 1. Scott AB. Botulinum toxin injection into extraocular muscles as an alternative to strabismus surgery. Ophthalmology 1980;87:1044-9. 2. Aoki RK. Botulinum neurotoxin serotypes A and B preparations have different safety margins in preclinical models of muscle weakening efficacy and systemic safety. Toxicon 2002;40:923-8. 3. Callaway JE, Arezzo JC, Grethlein AJ. Botulinum toxin type B: an overview of its biochemistry and preclinical pharmacology. Semin Cutan Med Surg 2001;20:127-36.
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4. Matarasso SL. Comparison of botulinum toxin types A and B: a bilateral and double-blind randomized evaluation in the treatment of canthal rhytides. Dermatol Surg 2003;29:7-13. 5. Klein AW. Dilution and storage of botulinum toxin. Dermatol Surg 1998;24:1179-80. 6. Sloop RR, Cole BA, Escutin RO. Reconstituted botulinum toxin type A does not lose potency in humans if it is refrozen or refrigerated for 2 weeks before use. Neurology 1997;48:249-53. 7. Alam M, Dover JS, Arndt KA. Pain associated with injection of botulinum A exotoxin reconstituted using isotonic sodium chloride with and without preservative: a doubleblind, randomized controlled trial. Arch Dermatol 2002; 138:510-4. 8. Gassner HG, Sherris DA. Addition of an anesthetic agent to enhance the predictability of the effects of botulinum toxin type A injections: a randomized controlled study. Mayo Clin Proc 2000;75:701-4. 9. Package insert on Botox® Cosmetic (botulinum toxin type A) purified neurotoxin complex. Irvine, CA: Allergan Inc.; 2002. 10. Package insert on Myobloc. San Diego, CA: Elan Pharmaceuticals; 2000. 11. Ramirez AL, Reeck J, Maas CS. Botulinum toxin type B (MyoBloc) in the management of hyperkinetic facial lines. Otolaryngol Head Neck Surg 2002;126:459-67. 12. Cather JC, Menter A. Update on botulinum toxin for facial aesthetics. Dermatol Clin 2002;20:749-61. 13. Sadick NS. Botulinum toxin type B for glabellar wrinkles: a prospective open-label response study. Dermatol Surg 2002;28:817-21. 14. Huang W, Foster JA, Rogachefsky AS. Pharmacology of botulinum toxin. J Am Acad Dermatol 2000;43:249-59. 15. Package insert on Dysport. Berkshire, U.K.: Ipsen Ltd.; 2001. 16. Odergren T, Hjaltason H, Kaakkola S, et al. A double blind, randomised, parallel group study to investigate the
17.
18.
19.
20. 21. 22. 23. 24.
25. 26.
27.
28.
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dose equivalence of Dysport and Botox in the treatment of cervical dystonia. J Neurol Neurosurg Psychiatry 1998;64: 6-12. Nussgens Z, Roggenkamper P. Comparison of two botulinum-toxin preparations in the treatment of essential blepharospasm. Graefes Arch Clin Exp Ophthalmol 1997; 235:197-9. Carruthers A, Carruthers J. Botulinum toxin type A: history and current cosmetic use in the upper face. Semin Cutan Med Surg 2001;20:71-84. Sloop RR, Cole BA, Escutin RO. Human response to botulinum toxin injection: type B compared with type A. Neurology 1997;49:189-94. Matarasso A, Deva AK. Botulinum toxin. Plast Reconstr Surg 2002;109:1191-7. Setler P. The biochemistry of botulinum toxin type B. Neurology 2000;55:S22-8. Benedetto AV. The cosmetic uses of botulinum toxin type A. Int J Dermatol 1999;38:641-55. Fulton JE. Botulinum toxin: the Newport Beach experience. Dermatol Surg 1998;24:1219-24. Flynn TC, Carruthers A, Carruthers J. Surgical pearl: the use of the Ultra-Fine II short needle 0.3-cc insulin syringe for botulinum toxin injections. J Am Acad Dermatol 2002; 46:931-3. Schwetz BA. From the Food and Drug Administration. JAMA 2001;285:724. Ramirez AL, Reeck J, Maas CS. Preliminary experience with botulinum toxin type B in hyperkinetic facial lines. Plast Reconstr Surg 2002;109:2154-5. Gartlan MG, Hoffman HT. Crystalline preparation of botulinum toxin type A (Botox): degradation in potency with storage. Otolaryngol Head Neck Surg 1993;108: 135-40. Garcia A, Fulton JE Jr. Cosmetic denervation of the muscles of facial expression with botulinum toxin: a doseresponse study. Dermatol Surg 1996;22:39-43.
B
otulinum toxin is a potent but fragile neurotoxin produced by the bacterium, Clostridium botulinum. Although there are eight strains, only BTX-A and BTX-B are commercially available. They have similar structures and basic mechanism of action; however, each serotype is antigenically distinct, and each strain binds to a different acceptor site on the plasma membranes of a cholinergic neuronal endplate.2 In addition, the production of BTX-A involves the process of either lyophilization or vacuum-drying, which may reduce the stability and activity of the toxin. This does not occur in the preparation of BTX-B.3 These differences may render direct comparison of the potency of neurotoxins unfeasible.2,4 The choice of diluent, dilution, and storage may also affect the efficacy of the toxin. There are conflicting results from several clinical trials regarding the handling of the neurotoxin. The debate partially arises from the concern that the toxin, particularly BTX-A, is susceptible to surface denaturation and thus, loss of potency.4-10 Studies and anecdotal reports indicate that the toxin may not be as vulnerable as presumed. In addition, although the preparation of BTX-B may render it more stable than BTX-A, the paucity of information makes it difficult to determine the influence of different diluents, dilutions, and storage on its efficacy.11-13 Further clinical trials, therefore, are warranted to determine which factors influence toxin activity.
Preparations There are currently three preparations of botulinum toxin, and there are two sources of BTX-A, Botox® From the Department of Dermatology and Cutaneous Biology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania. Address correspondence to: Jennifer L. Parish, MD, 1760 Market Street, Ste. 310, Philadelphia, PA 19103. E-mail address: [email protected] © 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010
(Allergan, Inc., Irvine, California) and Dysport™ (Ipsen Ltd., Berkshire, UK). Botox® gained U.S. Food and Drug Administration approval for treatment of glabellar lines in 2002. Recently, BTX-B became available in the United States. There is only one form of BTX-B. In the United States, the trade name of BTX-B is Myobloc™ (Elan Pharmaceuticals, South San Francisco, California), and in Europe, it is Neurobloc™. BTX-B received U.S. Food and Drug Administration approval for use in cervical dystonia in 2001. Owing to the success of BTX-A, there are studies investigating the use of BTX-B in facial esthetics.11,13 This article will focus primarily on Botox® and Myobloc™, because both preparations are available in the United States. Botox® and Dysport™ do not have the same formulations or potency. Botox® is supplied in vials containing 100 U of BTX-A, 0.5 mg of human albumin, and 0.9 mg of sodium chloride in a sterile, vacuum-dried crystalline form without a preservative. The production of Botox® involves the fermentation of the Hall strain of C. botulinum serotype A, which is developed on a medium containing yeast extract and N-Z amine.9,14 After purification of the culture solution by acid precipitation and dialysis, a crystalline complex develops. The complex is dissolved in a solution containing sterile saline and albumin and sterile-filtered before vacuum-drying. Before adding the diluent, the manufacturer recommends storing the vial in a freezer at ⫺5°C or below.9 The preparation of Dysport™ is different from that of Botox®. Each vial contains 500 U of BTX-A, 125 g of human albumin, and 2.5 mg of lactose.15 The production of Dysport™ involves column-based purification instead of the precipitation method utilized for Botox®.14 The preparation of Dysport™ involves lyophilization. Before reconstituting Dysport™, the manufacturer recommends storage in a refrigerator at 2-8°C.15 The measurements of potency used for Botox® and Dysport™ are not equivalent. One unit of Botox® cor0738-081X/03/$–see front matter doi:10.1016/j.clindermatol.2003.11.009
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responds to the LD50, which is the lethal dose required to cause death in 50% of female Swiss-Webster mice that weigh 18-22 g each. Although the same definition of units applies to Dysport™, 1 U of Botox® is not equal in potency to 1 U of Dysport™. The method of testing the LD50 is specific to each preparation of BTX-A.9 Clinical studies have indicated that 1 U of Botox® is equivalent to 3-6 U of Dysport™16-18; nevertheless, because of the differences in testing and formulation, comparison of dosing or potency is an approximation. BTX-B differs from BTX-A not only by serotype but also by method of production and toxin activity. Unlike BTX-A, Myobloc™ is a solution available in three vial presentations: 2500 U/0.5 mL, 5000 U/1.0 mL, and 10,000 U/2.0 mL. Each vial contains 5000 U/mL of BTX-B, 0.05% human albumin, 0.1 mol/L sodium chloride, and 0.01 mol/L sodium succinate. The production of Myobloc™ involves the fermentation of the Bean strain of C. botulinum type B. After anaerobic fermentation, the neurotoxin complex, which consists of BTX-B and associated proteins, undergoes purification by means of precipitation and chromatography.3,10 The manufacturer recommends storage of Myobloc™ in a refrigerator at 2-8°C.10 The processing of BTX-B does not involve lyophilization. Lyophilization may result in a reduction of toxin activity, and therefore, BTX-B may be more stable than preparations of BTX-A.3 Myobloc™ also does not require dilution before injection, as do both commercial preparations of BTX-A. Therefore, there may be a reduced hypothetical risk of affecting the potency of the neurotoxin. There is not a single dose ratio to define the potency relationship between Botox® and Myobloc™. BTX-B and BTX-A use the same measurement for biologic activity; however, 1 U of Botox® is not equivalent to 1 U of Myobloc™.2 Because of longer clinical experience, much of the current information regarding botulinum toxin activity is based on Botox®. As a result, investigators have attempted to compare the potency of Myobloc™ to Botox®. Animal and human studies indicate that a dose ratio does not exist for Botox® and Myobloc™.2,19 Further clinical trials, therefore, are necessary to determine appropriate dosing for Myobloc™.
Diluent Proper handling of botulinum toxin is a crucial factor in maintaining toxin activity. The manufacturers of Botox® and Myobloc™ make specific recommendations regarding diluent, dilution, and storage; however, clinical studies and anecdotal reports challenge these labeling instructions. The package insert for Botox® suggests reconstitution with 0.9% sterile saline without preservative and use within 4 hours of adding diluent9 A double-blind, randomized, controlled trial compared
Clinics in Dermatology
Y
2003;21:481⫺484
the use of unpreserved saline to preserved saline. Reconstitution with preserved saline reduced the pain associated with Botox® injection. The decreased discomfort may result from the anesthetic properties of the benzyl alcohol in the preserved saline. Furthermore, the investigators noted equal efficacy.7 The author of this article has also had similar experience with preserved saline. A double-blind, randomized, controlled study indicated that reconstitution with lidocaine with epinephrine does not jeopardize toxin potency. The initial onset of action of Botox® occurs 1-2 days after injection. This time course decreases the predictability of the effect of Botox®. As a result, the addition of lidocaine with epinephrine may provide the clinician with immediate feedback. The investigators of the trial demonstrated that the immediate paralysis caused by the anesthetic diluent improved the reliability of the results. In addition, the diluent did not appear to decrease toxin efficacy.8 In the literature, there is some agreement with regard to the appropriate technique for reconstituting Botox®. The toxin is fragile. Agitation of the diluted neurotoxin allegedly can cause surface denaturation, which results in deactivation of the neurotoxin. Therefore, according to the package insert, the reconstituted Botox® should not be shaken. The diluent should be added into the vial by vacuum and not by actively squirting it into the vial.5,9,14,20 There are those who remove the rubber stopper. If this is done without caution, there may be a loss in toxin activity. In addition, alcohol can inactivate the neurotoxin protein. Alcohol used to wipe the cap or the skin should be allowed to evaporate before filling of the syringe or injecting into the skin.5 Therefore, despite the choice of diluent, the potency of toxin can decrease with improper methods of reconstitution. Unlike BTX-A, Myobloc™ does not require the addition of a diluent before injection. The package insert indicates that the solution can be diluted with unpreserved saline; however, the manufacturer recommends use within 4 hours to maintain toxin activity.10,21 It is unclear whether preserved saline would affect the potency of Myobloc™. The presence of benzyl alcohol in preserved saline may decrease the pain of injection. A recent study demonstrated the efficacy of Myobloc™ in the treatment of glabellar wrinkles. The investigators used preserved saline as a diluent without apparent loss of toxin activity.13 Additional studies are necessary to assess the influence of different diluents on the potency of Myobloc™. There is little discussion in the literature regarding the process of diluting Myobloc™. The ability to inject Myobloc™ without adding a diluent may account for the paucity of information. Although BTX-B may be a more stable preparation than BTX-A, BTX-B is still a fragile neurotoxin.3 The technique for diluting BTX-B
Clinics in Dermatology
Y
2003;21:481⫺484
should most likely be similar to that for BTX-A. Further studies are warranted to determine the appropriate method for dilution.
Dilution The dilution of Botox® varies. The manufacturer suggests using 2.5 mL of saline to dilute the vial.9 Most clinicians reconstitute Botox® with 1-3 mL of diluent.5,22 In the literature, there are reports of large-volume dilution up to 20 mL.23 Although a 1-mL dilution provides for easy calculation (1 U ⫽ 0.01 mL), it is difficult to deliver the accurate dose due to the small volume. There is a loss of ⬃0.07 mL toxin in the dead space of the traditional needle hub and syringe. The use of a specialized syringe, such as the Ultra-Fine II short needle 0.3-cc insulin syringe, can reduce the potential waste of toxin.24 The optimal dilution partly depends on the experience of the clinician. A correlation is apparent between the diluent volume and the intended dose response. Injection of a high concentration in small volumes results in a more localized effect.18 There is usually a dispersion area of at least 1.0 cm.14 In contrast, injection of a low concentration in large volumes results in greater diffusion of the toxin.5 The large volumes may cause paralysis of unintended muscles. For example, treatment of the glabellar region with high volumes may allow diffusion of the toxin into the levator muscle, causing blepharoptosis. In addition, some authors report that large dilutions may have a shorter duration of effect.5 Therefore, the choice of dilution should be based on the desired outcome. There is less clinical experience with the dilution of Myobloc™. The initial trials focused on the use of Myobloc™ for cervical dystonia. In those studies, the doses of BTX-B did not require dilution.25 The appropriate doses for management of facial lines are still uncertain. It appears that dilution may be necessary to achieve the desired effect. In several trials examining the cosmetic application of Myobloc™, the investigators diluted the solution to obtain a concentration of 200-300 U/0.1 mL.11,13,26 Further studies are warranted to determine optimal dilution and dosing for the use of Myobloc™ for facial rejuvenation.
Storage Some controversy exists about the storage of reconstituted Botox®. The manufacturer recommends storing the diluted vial in the refrigerator at a temperature of 2-8°C.9 The debate arises over the duration of potency of the reconstituted toxin. According to the package insert, injection of the toxin should occur within 4 hours of dilution.9 There is concern that prolonged storage will compromise toxin activity. A study by Gartlan and Hoffman27 utilized the LD50 mouse assay to determine the potency of the diluted toxin. After 12 hours, there
COMMERCIAL PREPARATIONS AND HANDLING OF BTX-A/-B
483
was a significant loss in the activity of the reconstituted toxin. In contrast, Sloop et al6 used a human model to demonstrate that diluted Botox® stored for 2 weeks maintained its potency. Another trial indicated that diluted toxin stored for 30 days was as efficacious as freshly mixed Botox®.28 Klein5 reports that many clinicians store Botox® reconstituted with unpreserved saline for a period of 7-30 days. There is a potential risk of contamination with prolonged storage of Botox® diluted with unpreserved saline.5,18 The use of preserved saline as the diluent could eliminate the risk. In addition, there are reports that Botox® reconstituted with preserved saline is stable after 5 weeks of refrigeration.7 The ability to store Botox® for an extended period not only conserves material but also enables more convenient patient scheduling. The toxin activity of Myobloc™ is viable for ⬃21 months.10 The manufacturer suggests storing the toxin at 2-8°C; however, studies have shown that the toxin is stable at room temperature (25°C) for 9 months.21 The prolonged shelf life of Myobloc™ makes it advantageous to the clinician. There is a paucity of information regarding the storage of diluted Myobloc™. According to the package insert, Myobloc™ should be injected within 4 hours of dilution with unpreserved saline.10 The limited experience with Myobloc™ makes it unclear whether prolonged storage of the toxin after dilution jeopardizes its activity.
Conclusions The preparation and handling of Botox® and Myobloc™ influence clinical efficacy. The neurotoxin is fragile. As a result, studies have attempted to delineate which factors affect toxin activity. Although both Botox® and Myobloc™ have a similar mechanism of action, the formulations are different. In contrast to Myobloc™, the preparation of Botox® involves vacuum-drying, which may render it more vulnerable to surface denaturation. In addition, Myobloc™ does not require reconstitution, which may help to maintain the potency of the toxin. Further clinical experience is necessary to determine whether Myobloc™ can be as effective as Botox® in facial esthetics.
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