Reconstitution / Dilution

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11 Reconstitution / Dilution

Ada R. Trindade de Almeida, Letícia Cardoso Secco

Summary and Key Features

• Prescribing information on all available products recommends reconstitution in unpreserved saline

• Studies in the literature suggest that other solutions can be used such as preserved saline, anesthetics (lidocaine or bupivacaine), with or without epinephrine and albumin

• In vivo studies demonstrate that onabotulinumtoxinA is not as fragile as originally thought and that foam during reconstitution does not inactivate the toxin

• Although manufacturers recommend storage at 2–8°C in the refrigerator, toxin administration within 4–24 hours, and avoiding freezing after reconstitution, it appears that some products can be stored for longer periods of time after reconstitution, in the refrigerator or the freezer, without loss of efficacy or contamination

• In regard to the best dilution to be used, when treating limb muscles, perhaps as a result of greater dispersion of the toxin, higher dilutions might be beneficial

• For facial muscles, more concentrated volumes are preferred, since they produce less discomfort for patients

• The literature suggests that toxins may be more sturdy and resistant to degradation than previously understood

Several situations have been implicated in affecting the potency of BoNT. For this reason, different recommendations have been made aiming at not interfering with its efficacy or duration. In this chapter, published information on various methods of reconstitution, including mixtures of toxins with several substances, agitation or foam formation, will be reviewed as well as the impact of volume on botulinum toxin performance.

Reported substances used in the reconstitution process

OnabotulinumtoxinA is provided as a vacuum-dried powder, whereas incobotulinumtoxinA, abobotulinumtoxinA, and BoNT-A from Lanzhou (China) are presented as lyophilized powders (Fig. 11.1). RimabotulinumtoxinB is provided as a ready-to-use sterile liquid and no reconstitution is required.

image

Figure 11.1 Appearance of various vacuum-dried and lyophilized BoNT-A powder products, before reconstitution.

Unpreserved saline

Most manufacturers and also an international consensus on the use of abobotulinumtoxinA recommend reconstitution of botulinum toxin A using unpreserved saline.

Preserved saline

Preserved saline contains benzyl alcohol. Three trials including 213 patients (for cosmetic, blepharospasm, and axillary hyperhidrosis indications) demonstrated that it does not affect the potency of onabotulinumtoxinA and it made injections less painful.

It was chosen by an onabotulinumtoxinA consensus panel in 2004 as the preferred diluent for reconstitution, and another abobotulinumtoxinA expert panel recognized that benzyl alcohol may act as a local anesthetic, but this effect is considered negligible at low volumes.

Hyaluronidase

Goodman tested the viability of onabotulinumtoxinA reconstituted with saline plus hyaluronidase. Results after 2 weeks showed maintenance of efficacy and a greater diffusion of the product. No long-term results were evaluated.

Sterile water

BoNT-A still works if dissolved in sterile water, but intense short-lived pain at injection site will occur.

Epinephrine and / or lidocaine

Hantash et al tested the effect of epinephrine (EPI) 1 : 100 000 on onabotulinumtoxinA efficacy in periorbital rhytides in 14 patients, evaluated for up to 6 months. They found that EPI promoted a faster onset of action and enhanced short-term efficacy of onabotulinumtoxinA. Gassner & Sherris, in a double-blind, randomized, controlled study with 10 volunteers for cosmetic indication, showed that when onabotulinumtoxinA was reconstituted with 1% lidocaine with 1 : 100 000 epinephrine, all components retained their function. Similar results were found by Haubner in two cases, using 2% lidocaine with 1 : 200 000 epinephrine.

A cocktail composed of abobotulinumtoxinA, 2% lidocaine with 1 : 100 000 epinephrine and hyaluronic acid (Perlane®, Medicis, Scottsdale, AZ) was recently described. The combined product was injected in five patients for periocular and / or glabellar areas and the author reported no compromise in efficacy or safety, but only periocular post-treatment photographs were showed.

A study conducted in 29 patients, compared side by side, onabotulinumtoxinA reconstituted in 2% lidocaine or normal saline for axillary hyperhidrosis and found similar effectiveness in both the short and long term. As the lidocaine-admixed solution was associated with significant pain reduction, it might be preferable for treating axillary hyperhidrosis.

A fatal case of anaphylaxis after the injection of onabotulinumtoxinA and lidocaine mixture in a woman for chronic neck and back pain was also reported. Since it was not possible to determine which drug was responsible for the reaction, it is advisable to consider that lidocaine may increase the possibility of an anaphylactic reaction.

Pearl 1

The use of local anesthetics for reconstitution does not affect botulinum toxin potency, but may increase the possibility of an anaphylactic reaction.

Bupivacaine

Yen et al, in a randomized, double-blind, split-face study with 16 patients with glabellar wrinkles, compared onabotulinumtoxinA reconstituted with 0.75% bupivacaine or with unpreserved saline on either side of their face. The bupivacaine-reconstituted toxin treated sides made the procedure less painful and showed greater muscle weakness at 1 week, but there were no differences at 1 and 3 months. The explanation for the faster onset of the paresis might be a synergistic effect of bupivacaine inducing myotoxicity.

Albumin

Bigalke et al demonstrated that human albumin supplementation of abobotulinumtoxinA could allow dose reduction without decrease of therapeutic efficacy or safety. Their findings were confirmed by another study with 106 patients treated with abobotulinumtoxinA, for cervical dystonia, blepharospasm, and hemifacial spasm, over 5–10 years.

Pearl 2

Albumin supplementation of abobotulinumtoxin could allow dose reduction without decrease of therapeutic efficacy or safety.

Box 11.1 summarizes substances reported not to inactivate BoNT-A.

Box 11.1

Substances reported not to inactivate BoNT-A

image Unpreserved saline

image Preserved saline

image Hyaluronidase

image Distilled water

image Epinephrine

image Lidocaine

image Bupivacaine

image Albumin

image Hyaluronic acid

Variations in the reconstitution process

Foam during reconstitution

Toth et al evaluated in vitro the stability of fragments of BoNT (light chain, LcA) and the binding domain of the heavy chain (Hc) to mild agitation, and found that LcA fragments rapidly lost their secondary structures and Hc domains were denatured by mild stirring. They speculated that BoNT domains underwent surface denaturation owing to rapid exposure of hydrophobic residues by mechanical agitation.

A recent experimental study evaluated the effect of onabotulinumtoxinA after continuous inversion and straightening of the vial, 30 times a minute, for up to 6 weeks. Eight mice were each injected intraperitoneally with 1 U of the agitated onabotulinumtoxinA on days 1, 3, 5, 7, 14, 21, 28, and 42. The main outcome measure was death of the mice, demonstrating toxin efficacy. At the end of the study, half of each group of mice (4 of 8 mice) died within 48 hours of the injection (range 16–48 hours), demonstrating maintenance of the onabobotulinumtoxinA effect.

These results confirmed previous studies in clinical settings: a split-face study comparing muscle paralysis in periocular and glabellar areas, after injection of onabotulinumtoxinA gently reconstituted on one side and onabotulinumtoxinA manually shaken on the other side, showed no differences in short or long term effects. Kazim et al reached similar conclusions after 6 months evaluating frontalis muscle injections of onabotulinumtoxinA, reconstituted gently on one side of the face and vigorously (placed on a vortex Touch mixer at maximum speed for 30 seconds) on the other.

Differences between in vitro and in vivo studies may be explained by the presence of associated proteins inside the complex of onabotulinumtoxinA, which in addition to stabilizing the neurotoxin molecule may further protect it from degradation.

Storage

RimabotulinumtoxinB must be stored at 2–8°C. At this temperature, no significant loss of activity has been found after 30 months, but at room temperature (25°C) it dropped to 9 months.

Regarding reconstituted BoNT-A vials, manufacturers recommend administration within 4–24 hours, storage at 2–8°C in the refrigerator, and avoidance of freezing after reconstitution, but several publications suggest that these recommendations may in fact be excessively strict.

Shelf life after reconstitution

A multicenter, double-blind study in 88 patients demonstrated that reconstitution with non-preserved saline up to 6 weeks before use did not reduce efficacy in treating glabellar frown lines for up to 4 months, findings that were confirmed by a recent experimental trial in mice. In the 2004 consensus panel, 73% of panelists stored onabotulinumtoxinA for more than 4 weeks.

For crow’s feet lines, one study used 1-week-reconstituted onabotulinumtoxinA in 30 patients, and another used onabotulinumtoxinA after 2 weeks of refrigeration. Both found similar clinical results to fresh product.

Another trial using 2-week-reconstituted abobotulinumtoxinA found no difference in safety or efficacy, while a consensus panel in 2010 considered that it can be used within 2–3 weeks without adverse effects.

Fresh or frozen

Parsa et al compared reconstituted onabotulinumtoxinA frozen for up to 6 months with fresh onabotulinumtoxinA used within 4 hours after reconstitution, in 118 sites of 80 patients; they concluded that it may be frozen, thawed, and injected without losing its potency for up to 6 months. Regarding abobotulinumtoxinA, a prospective, double-blinded, randomized controlled trial by Yang et al, with 40 subjects treated for horizontal forehead rhytides, found no difference in efficacy after 2 weeks of refrigeration or freezing compared with fresh product.

Pearl 3

Fresh versus frozen / thawed reconstituted abobotulinumtoxinA and onabotulinumtoxinA showed no difference in efficacy.

Sterility

Since storing and reusing reconstituted toxin for some days or weeks has become a frequent practice, sterility assessment is an important issue addressed by some authors. Alam et al evaluated 127 vials of onabotulinumtoxinA that were reconstituted with preservative-containing saline, stored and reused, simulating routine clinical practice. Each vial underwent an average of 4.5 access procedures during a period of up to 7 weeks, and there was no evidence found of contamination in sterility analysis. Menon analyzed 11 consecutive bottles of abobotulinum toxinA left for 4 hours at room temperature and found no microbial growth; there were similar findings in 8 vials of this product kept refrigerated for 15 days after reconstitution.

Pearl 4

There was no evidence of contamination in vials stored and reused for up to 2 weeks.

Dilution issues

There is considerable discussion about the effect of dilution of BoNTs on clinical effect, duration and diffusion. Table 11.1 shows the BoNT-A manufacturers’ recommended volumes for reconstitution.

Table 11.1 BoNT-A manufacturers’ dilution recommendations

Toxin Dilution range Concentration
OnabotulinumtoxinA 1–8 mL of saline 12.5–100 U/mL
AbobotulinumtoxinA 300 U in 0.6–2.5 mL 120–500 U/mL
IncobotulinumtoxinA 1–8 mL 12.5–100 U/mL

Rimabotulinumtoxin B can be diluted up to sixfold using normal saline, but a more concentrated solution cannot be generated.

Some researchers believe that higher dilutions could be associated with a higher risk of diffusion to unwanted sites, unsatisfactory results or shorter duration, but this is not unanimous in the literature. To facilitate the understanding of the dilution impact on diffusion, adverse effects and success, the available studies were reviewed by the authors in this regard.

Facial muscles

Cosmetic indications

Expert consensus panels for cosmetic indications recommend dilutions between 1 and 3 mL of saline for onabotulinumtoxinA and between 1.5 and 2.5 mL for abobotulinumtoxinA.

In the past, a group reported the use of large volumes of low-dose BoNT-A (up to 20 mL/vial) and described efficacy, but of short duration. The need for frequent treatments to maintain results led to a great number of individuals resistant to toxin. Therefore, higher dilutions are no longer recommended.

Hankins et al evaluated 46 patients with glabellar wrinkles injected with different concentrations of onabotulinumtoxinA (10 or 100 U/mL) for up to 21 weeks and found no differences in efficacy or duration, but larger volumes were associated with more discomfort.

In a study with 10 volunteers, Hsu et al injected the forehead with 5 U of onabotulinumtoxinA diluted to 20 (5 mL) or 100 U/mL (1 mL). They found that higher-volume injections resulted in greater diffusion and a larger affected area. Carruthers et al studied the effect of different dilutions of onabotulinumtoxinA in the treatment of glabellar rhytides. Eighty patients were treated with dilutions of 10, 20, 33.3, or 100 U/mL (20/group) and followed for 48 weeks. No statistical differences in treatment success or adverse effect were reported, although the six cases of eyebrow ptosis were all in the higher-dilution group.

Another trial treated 20 patients for lateral orbital rhytides, observed over 90 days. Dilutions of onabotulinumtoxinA were 20 or 100 U/mL. No statistically significant differences in response or adverse effects were detected, even though the lower-dilution group showed a slightly better response.

For incobotulinumtoxinA, two different dilutions (25 and 40 U/mL) were evaluated in a split-face study in 40 patients with glabellar lines; it found no significant differences between sides.

Pearl 5

For cosmetic indications (i.e. facial muscles), concentrated solutions seem better than diluted ones because they produce less discomfort for patients.

Blepharospasm

Sixteen benign essential blepharospasm patients were treated with concentrations of 10 or 100 U/mL, and followed for 8 months. No significant difference between dilutions was reported with respect to either efficacy or complications.

Non-facial muscles

Experimental studies

Shaari et al treated the tibialis anterior muscle in rats and found that a constant dose of 0.2 U of botulinum toxin resulted in greater paralysis when injected in larger volumes. Kim et al injected 16 rabbits with 10 U of onabotulinumtoxinA at concentrations of 20 or 100 U/mL and drew the same conclusions.

Limb muscle dystonias / spasticity

A group from Korea injected a fixed dose of 2.5 U of onabotulinumtoxinA to human extensor digitorum brevis muscle in two dilutions (5 and 25 U/mL) and concluded that a fivefold increase in dilution volume did not enhance the paralyzing effect of onabotulinumtoxinA. Francisco et al treated 13 patients with 60 U diluted to 50 or 100 U/mL for wrist and finger flexor spasticity and also found no significant differences.

In contrast, Gracies et al used 160 U of onabotulinumtoxinA at two different dilutions (20 and 100 U/mL), in a double-blind randomized controlled trial with 21 individuals with spastic elbow flexors. They found that the higher-volume group (20 U/mL) achieved greater neuromuscular blockade and spasticity reduction, compared with the concentrated solution.

Two studies were conducted in children with cerebral palsy and limb spasticity. One used onabotulinumtoxinA in 38 children at concentrations of 12.5 (n = 19) and 50 U/mL (n = 19) respectively in the gastrocnemius muscles for reducing ankle plantar flexor spasticity. Although no significant differences were found in physical evaluation or gait analysis, the larger-volume group had more frequent side effects, making the more concentrated solution a better option. In another randomized controlled trial, 22 children with cerebral palsy received a fixed dose of abobotulinumtoxinA. Dilutions of 500 U/5 mL and 500 U/1 mL respectively were injected in each gastrocnemius muscle. The authors considered the high-volume preparation more effective.

Pearl 6

When treating limb muscles, higher dilutions might be beneficial, perhaps due to a greater dispersion of the toxin.

Hyperhidrosis

There is no standardized dilution for botulinum toxin treatment of focal hyperhidrosis. Dilutions reported for hyperhidrosis are detailed in Table 11.2.

Table 11.2 Dilutions reported for hyperhidrosis

Toxin Dilution range Most used dilution
OnabotulinumtoxinA 1–10 mL of saline 2–5 mL
AbobotulinumtoxinA 1.25–10 mL 2.5–5 mL
IncobotulinumtoxinA 1–10 mL of saline 10 mL (one paper)

Open comparative trials in hyperhidrotic areas assessed by iodine starch tests were not conclusive: a study of 9 volunteers injected in the axillary region with 3 U of onabotuliumtoxinA diluted in 1–5 mL of saline solution reported no difference in anhydrotic halos; in addition 3 patients with compensatory hyperhidrosis received injections of 5 U of abobotulinumtoxinA in the back, in three dilutions of 0.2, 0.4, or 0.6 mL. Although larger anhidrotic halos were found with the more diluted abobotulinumtoxinA group, the authors considered the differences to be irrelevant.

We reviewed 13 comparative dilution studies: five of these reported a greater diffusion or enhanced effect with higher volumes, whereas nine found no difference in efficacy. Three trials reported more discomfort with higher dilutions, even though these were not statistically significant. Hence this topic remains controversial. For small muscles, such as those located on the face or hand, it seems that there is no difference in results with the addition of higher volumes. For large muscles of the limbs, though, this might be advantageous. More studies, with larger samples, are needed to clarify this matter.

Many of the precautions surrounding BoNT use, often recommended by the manufacturers, are described in the clinical literature as very restrictive. The current literature suggests rather that toxins may be more sturdy and resistant to degradation than was previously understood.

Further reading

Asher B, Talarico S, Casuto D, et al. International consensus recommendations on the aesthetic usage of botulinum toxin type A (Speywood Unit) – part I: upper facial wrinkles. Journal of the European Academy of Dermatology and Venereology. 2010;24(11):1285–1295.

Boyle MH, McGwin, Flanagan CE, et al. High versus low concentration botulinum toxin A for benign essential blepharospasm: does dilution make a difference? Ophthalmic Plastic and Reconstructive Surgery. 2009;25:81–84.

Carruthers J, Carruthers A. Botulinumtoxin in facial rejuvenation: an update. Dermatologic Clinics. 2009;27:417–425.

Carruthers A, Carruthers J, Cohen J. Dilution volume of botulinum toxin type a for the treatment of glabellar rhytides: does it matter? Dermatologic Surgery. 2007;33:S97–S104.

Carruthers J, Fagien S, Matarasso SL. Consensus recommendations on the use of botulinum toxin Type A in facial aesthetics. Plastic and Reconstructive Surgery. 2004;114(6 suppl):1S–22S.

Gracies JM, Lugassy M, Weisz DJ, et al. Botulinum toxin dilution and endplate targeting in spasticity: a double-blind controlled study. Archives of Physical Medicine and Rehabilitation. 2009;90:9–16.

Hexsel DM, Trindade de Almeida AR, Rutowithsch M, et al. Multicenter, double blind study of the efficacy of injections with botulinum toxin type A reconstituted up to six consecutive weeks before application. Dermatologic Surgery. 2003;29:523.

Hu GC, Chuang YC, Liu JP, et al. Botulinum toxin (Dysport) treatment of the spastic gastrocnemius muscle in children with cerebral palsy: a randomized trial comparing two injection volumes. Clinical Rehabilitation. 2009;23(1):64–71.

Kane M, Donofrio L, Ascher B, et al. Expanding the use of neurotoxins in facial aesthetics: A consensus panel’s assessment and recommendations. Journal of Drugs in Dermatology. 2010;9(1):S3–S22.

Kazim NA, Black EH. Botox: shaken, not stirred. Ophthalmic Plastic and Reconstructive Surgery. 2008;24(1):10–12.

Lee LR, Chuang YC, Yang BJ, et al. Botulinum toxin for lower limb spasticity in children with cerebral palsy: a single-blinded trial comparing dilution techniques. American Journal of Physical Medicine and Rehabilitation. 2004;83:766–773.

Mohammadi B, Kollewe K, Wegener M, et al. Experience with long-term treatment with albumin-supplemented botulinum toxin type A. Journal of Neural Transmission. 2009;116:437–441.

Parsa AA, et al. Reconstituted botulinum type A neurotoxin: clinical efficacy after long-term freezing before use. Aesthetic Plastic Surgery. 2007;31:188–191.

Shome D, Nair AG, Kapoor R, et al. Botulinum toxin A: is it really a fragile molecule? Dermatologic Surgery. 2010;36:2106–2110.

Toth SI, Smith LA, Ahmed SA. Extreme sensitivity of botulinum neurotoxin domains towards mild agitation. Journal of Pharmaceutical Sciences. 2009;98(9):3302–3311.

Trindade de Almeida AR, Kadunc BV, Di Chiacchio N, et al. Foam during reconstitution does not affect the potency of botulinum toxin A. Dermatologic Surgery. 2003;29:530.

Trindade de Almeida AR, Secco LC, Carruthers A. Handling botulinum toxins: an update literature review. Dermatologic Surgery. 2011;37(11):1553–1565.

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