Article
`AbobotulinumtoxinA (Dysport®),
`OnabotulinumtoxinA (Botox®), and
`IncobotulinumtoxinA (Xeomin®) Neurotoxin Content
`and Potential Implications for Duration of Response
`in Patients
`
`Malgorzata Field 1, Andrew Splevins 1, Philippe Picaut 2, Marcel van der Schans 3,
`Jan Langenberg 3, Daan Noort 3 and Keith Foster 1,*
`1
`Ipsen Bioinnovation, Abingdon OX14 4RY, UK; gosia.olszowka@gmail.com (M.F.);
`andrew.splevins@ipsen.com (A.S.)
`Ipsen Pharma, Cambridge, MA 02142, USA; philippe.picaut@ipsen.com
`TNO—CBRN Protection, 2288GJ Rijswijk, The Netherlands; marcel.vanderschans@tno.nl (M.v.d.S.);
`jan.langenberg@tno.nl (J.L.); daan.noort@tno.nl (D.N.)
`* Correspondence: keith.foster@ipsen.com
`
`2
`3
`
`Received: 18 October 2018; Accepted: 11 December 2018; Published: 13 December 2018
`
`Abstract: Botulinum neurotoxin type-A (BoNT-A) blocks the release of acetylcholine from peripheral
`cholinergic nerve terminals and is an important option for the treatment of disorders characterised by
`excessive cholinergic neuronal activity. Several BoNT-A products are currently marketed, each with
`unique manufacturing processes, excipients, formulation, and non-interchangeable potency units.
`Nevertheless, the effects of all the products are mediated by the 150 kDa BoNT-A neurotoxin.
`We assessed the quantity and light chain (LC) activity of BoNT-A in three commercial BoNT-A
`products (Dysport®; Botox®; Xeomin®). We quantified 150 kDa BoNT-A by sandwich ELISA and
`assessed LC activity by EndoPep assay. In both assays, we assessed the results for the commercial
`products against recombinant 150 kDa BoNT-A. The mean 150 kDa BoNT-A content per vial measured
`by ELISA was 2.69 ng/500 U vial Dysport®, 0.90 ng/100 U vial Botox®, and 0.40 ng/100 U vial
`Xeomin®. To present clinically relevant results, we calculated the 150 kDa BoNT-A/US Food and
`Drug Administration (FDA)-approved dose in adult upper limb spasticity: 5.38 ng Dysport® (1000 U;
`2 × 500 U vials), 3.60 ng Botox® (400 U; 4 × 100 U vials), and 1.61 ng Xeomin® (400 U; 4 × 100 U vials).
`EndoPep assay showed similar LC activity among BoNT-A products. Thus, greater amounts of active
`neurotoxin are injected with Dysport®, at FDA-approved doses, than with other products. This fact
`might explain the long duration of action reported across multiple indications, which benefits patients,
`caregivers, clinicians, and healthcare systems.
`
`Keywords: botulinum toxin; BoNT; spasticity; Dysport®, abobotulinumtoxinA; glabellar lines
`
`Key Contribution: The higher quantity of neurotoxin in Dysport® than in Botox® or Xeomin® at
`the total FDA-approved dose could explain the prolonged duration of action exhibited by Dysport®
`compared with other BoNT-A products.
`
`1. Introduction
`
`Botulinum neurotoxins (BoNTs) are a well-established treatment option for disorders such as
`dystonia or spasticity [1], as well as for aesthetic facial treatments [2]. BoNTs cause temporary muscle
`relaxation, which can ease symptoms and aid rehabilitation [3] when injected into specific muscles of
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`patients suffering from movement disorders. This action is a result of their potent ability to inhibit
`neurotransmitter release, which causes flaccid paralysis [4–6]. This action underpins the therapeutic
`and aesthetic use of the toxin. [5,6]. BoNT also inhibits acetylcholine release at autonomic nerve
`terminals and is used in hyperhidrotic and urological disorders [7].
`There are multiple serotypes of BoNTs. Currently, all but one of the therapeutic BoNT products
`are botulinum neurotoxin type-A (BoNT-A). At present, there are three BoNT-A products available
`worldwide: abobotulinumtoxinA (Dysport®, Ipsen, Paris, France), onabotulinumtoxinA (Botox®,
`Allergan, Irvine, CA, USA), and incobotulinumtoxinA (Xeomin®, Merz Pharmaceuticals GmbH,
`Frankfurt, Germany) [4]. There are also other BoNT products, but they are currently only available
`in limited markets or territories around the world [8]. In this study, we have only compared the
`three BoNT-A products that are available worldwide. Each of these BoNT-A products has a unique
`manufacturing process and contains different excipients [8].
`Despite differences among these three products, the therapeutic effects are mediated in all
`instances by the 150 kDa BoNT-A neurotoxin, which consists of a light chain (LC) and a heavy
`chain (HC). The HC drives neuron-specific binding (HC domain) and translocation (HN domain) of
`the toxin LC into the neuronal cytosol. The LC contains the proteolytic domain and is responsible for
`the catalytic activity and substrate specificity of the toxin within the neuron [6,9].
`Due to the differences in BoNT-A products, each has its own dosing guidelines based on
`potency units, originally defined through murine lethality tests [10]. The lethality tests underpinning
`unit definitions differ for the various products and are sensitive to various factors, including the
`mouse strain used, their age and sex, the animal housing conditions (such as light/dark cycles and
`feeding times), experimental injection time, and diluent buffer [11,12]. This variability has resulted
`in potency units that are specific to each product and are not interchangeable [11,13–15]. Due to this
`non-equivalence, the recommended number of units varies greatly between products. For example,
`in adult upper limb spasticity (AUL), the total recommended doses according to US Food and Drug
`Administration (FDA) labels are as follows: Dysport® 1000 U, Botox® 400 U, and Xeomin® 400 U;
`for the aesthetic glabellar lines (GL) indication, approved doses are Dysport® 50 U, Botox® Cosmetic
`20 U, and Xeomin® 20 U [13–15].
`The effectiveness of BoNT-A treatment in both therapeutic and aesthetic indications is the result
`of many factors, including the extent of disease severity or patient disability and treatment objectives,
`as well as injection technique (including dose and number of injected muscles), injector experience,
`and the holistic treatment approach, ensuring sufficient rehabilitation and support alongside BoNT-A
`injections [16–20]. At the molecular level, there are other factors that may affect treatment effectiveness.
`The ability of the BoNT-A to bind, internalise, and deliver the LC into the target neuron plays a
`role, as does the proteolytic activity of the LC (the rate at which it cleaves its SNARE (Soluble NSF
`Attachment Protein) REceptor) protein target and thus blocks neurotransmitter release). The quantity
`of BoNT-A available also significantly impacts therapeutic effectiveness: the greater the number
`of 150 kDa BoNT-A molecules, the greater the ability to cleave SNARE substrates [21]. The total
`Clostridial protein content of the three main commercially available BoNT-A products—Dysport®,
`Botox®, and Xeomin®—has been previously reported in the literature, giving values of 5 ng per 100 U
`vial of Botox, 4.35 ng per 500 U vial of Dysport (correcting an earlier publication stating 12.5 ng per
`500 U vial), and Xeomin containing 0.6 ng per 100 U vial [13,22,23]. As noted by Frevert [24], however,
`these values represent not only the core neurotoxin but also the complexing proteins when present.
`With regard to neurotoxin activity and, therefore, clinical activity, it is the core 150 kDa BoNT-A content
`that is important. The 150 kDa BoNT-A content in Dysport, Botox, and Xeomin was first measured and
`reported by Frevert [24] using a sensitive sandwich ELISA. Our research extends the work of Frevert
`by comparing both BoNT-A quantity and proteolytic activity among the three main commercially
`available BoNT-A products. Other aspects of neurotoxin function are not assessed in this study.
`The differences in potency units mean that the quantity of neurotoxin in each product cannot
`be directly compared, since one Dysport® unit does not equal one Botox® unit, which does not
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`equal one Xeomin® unit. Each BoNT-A product does, however, contain the 150 kDa neurotoxin,
`with different products containing different amounts. To make a direct comparison of the amount of
`neurotoxin (150 kDa BoNT-A) in each product, we assayed the quantity of 150 kDa neurotoxin protein
`(in nanograms) in each vial of Dysport®, Botox®, and Xeomin® using a sandwich enzyme-linked
`immunosorbent assay (ELISA) with BoLISA® antibodies, and compared this with the labelled
`unit strength.
`To investigate the LC activity of neurotoxin within each BoNT-A product, we used the EndoPep
`method in which a specific substrate peptide is cleaved by the toxin [25]. The concentration of cleaved
`peptide (product peptide) after a certain incubation time is measured and the concentration of product
`peptide obtained with the BoNT-A products versus different concentrations of reference BoNT-A is
`compared [26]. The quantity of reference BoNT-A giving an equivalent level of cleavage to that by a
`product BoNT-A is then taken as a measure for the activity of the LC in the product BoNT-A. In the
`original paper from Kalb et al. [26], BoNTs were isolated from food matrixes using antibody-loaded
`magnetic beads, followed by the EndoPep reaction. In our study, we decided to skip the isolation step
`for a number of reasons:
`
`1. We anticipated that the concentration of BoNT-A in the samples would be high enough that a
`sample enrichment would not be necessary.
`The matrix of the samples is relatively clean, making an extra clean-up step redundant.
`The isolation of BoNT-A with antibodies introduces another possible source for variation, as it is
`unknown whether the affinity of the antibody for BoNT-A in the different samples is affected by
`the different compositions of the product formulations, and an additional sample manipulation.
`
`2.
`3.
`
`Therefore, the solutions of the toxin products were mixed directly in the EndoPep mixture.
`We also decided to assay the cleaved peptides with capillary electrophoresis and laser-induced
`fluorescence (CE-LIF) detection, using fluorescently labelled substrate peptides, rather than by using a
`mass spectrometric read-out. We use this method routinely in our laboratory (see Van Uhm et al., [27])
`because of its robustness, ease of operation, and easily quantifiable spectrometric read-out.
`Our results revealed greater amounts of BoNT-A neurotoxin with Dysport®, compared with
`Botox® and Xeomin®, when used at the FDA-recommended doses for the treatment of AUL,
`adult lower limb spasticity (ALL), and GL. Since all three BoNT-A products are approved for use in
`these indications [13–15], we were able to do a direct comparison of approved doses. Furthermore,
`the EndoPep assay showed no significant differences in LC activity between the BoNT-A present
`in Dysport®, Botox®, and Xeomin®. Therefore, when Dysport® is used at the recommended doses,
`more active neurotoxin is administered than when using Botox® or Xeomin®. Given that the duration
`of BoNT-A muscle paralysis is known to be dependent on the quantity of active neurotoxin applied [28],
`this greater amount of active BoNT-A in Dysport® at the recommended dose potentially prolongs
`post-injection denervation and duration of action, relative to other products.
`
`2. Results
`
`2.1. Quantity of 150 kDa BoNT-A
`We analysed three batches of Dysport®, Botox®, and Xeomin® by sandwich ELISA on three
`separate occasions using a pair of antibodies specific to the 150 kDa BoNT-A protein. We measured
`the quantity of BoNT-A by extrapolation against a standard curve of recombinant BoNT-A (rBoNT-A)
`analysed in the same assay. We have previously demonstrated that the activity of this rBoNT-A is
`identical to that of purified natural BoNT-A obtained from commercial sources [29,30].
`Table 1 shows the mean quantity of 150 kDa neurotoxin per vial measured for each product and
`each batch, tested over three runs. The average amount of 150 kDa neurotoxin for each product is
`also shown. In a 500 U Dysport® vial, there was 2.69 ± 0.03 ng of BoNT-A; in addition, there were
`0.90 ± 0.03 ng of BoNT-A in a 100 U vial of Botox® and 0.40 ± 0.01 ng in a 100 U vial of Xeomin®.
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`Table 1. Quantity (ng) of 150 kDa BoNT-A in Dysport®, Botox®, and Xeomin®, analysed by ELISA
`using BoLISA® antibodies.
`
`Product
`
`Batch
`
`Expiry Date
`
`Dysport® 500 U
`
`Quantity of BoNT-A (ng/vial)
`Batch, Mean (SD)
`Product, Mean (SD)
`2.73 (0.20)
`Dec. 2018
`M00405
`2.66 (0.20)
`Dec. 2018
`L24950
`2.68 (0.23)
`Oct. 2018
`L22072
`0.89 (0.12)
`Sep. 2019
`C4321C3
`0.89 (0.10)
`Sep. 2019
`C4289C3
`0.94 (0.09)
`Aug. 2019
`C4270C3
`0.41 (0.07)
`Jul. 2019
`694458
`0.40 (0.05)
`Sep. 2019
`696232
`0.40 (0.05)
`Sep. 2019
`694788
`BoNT-A, Botulinum neurotoxin type-A; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation;
`U, units.
`
`Botox® 100 U
`
`Xeomin® 100 U
`
`2.69 (0.03)
`
`0.90 (0.03)
`
`0.40 (0.01)
`
`We used the mean ng/vial values to calculate the amount of toxin (pg) per potency unit. The
`mean pg per unit value (±SD) was 5.38 ± 0.07 for Dysport®, 9.04 ± 0.3 for Botox®, and 4.03 ± 0.06 for
`Xeomin® (Table 2).
`
`Table 2. Quantity of 150 kDa BoNT-A (pg) per manufacturer-assigned unit in Dysport®, Botox®,
`and Xeomin®.
`
`Product
`
`Dysport® 500 U
`
`Botox® 100 U
`
`Xeomin® 100 U
`
`Batch
`
`Expiry Date
`
`Quantity of BoNT-A per Product Unit (pg/unit)
`Calculated for Batch
`Product, Mean (SD)
`5.45
`Dec. 2018
`M00405
`5.32
`Dec. 2018
`L24950
`5.36
`Oct. 2018
`L22072
`8.86
`Sep. 2019
`C4321C3
`8.88
`Sep. 2019
`C4289C3
`9.38
`Aug. 2019
`C4270C3
`4.09
`Jul. 2019
`694458
`4.01
`Sep. 2019
`696232
`3.97
`Sep. 2019
`694788
`BoNT-A, botulinum neurotoxin type-A; SD, standard deviation; U, units.
`
`5.38 (0.07)
`
`9.04 (0.30)
`
`4.03 (0.06)
`
`To present results in a clinically relevant manner, we calculated the amount of BoNT-A in the
`maximum recommended dose for AUL and ALL spasticity and for GL. For AUL, this equated to 5.38 ng
`Dysport® (1000 U dose; 2 × 500 U vials), 3.62 ng Botox® (400 U dose; 4 × 100 U vials), and 1.61 ng
`Xeomin® (400 U dose: 4 × 100 U vials). In GL, this equated to 0.27 ng Dysport® (50 U dose), 0.18 ng
`Botox® (20 U dose), and 0.08 ng Xeomin® (20 U dose). For these and other calculated quantities,
`please refer to Table 3.
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`Table 3.
`Total quantity of active 150 kDa BoNT-A in maximum recommended doses of
`BoNT-A products.
`
`Indication
`
`Product
`
`A—Total Recommended
`Dosage a, Product Units
`
`B—Amount of
`Neurotoxin Per
`Product Unit, pg
`
`1000
`400
`400
`1500
`400
`
`C—Total Amount of Active
`BoNT-A (ng) Injected at the
`Recommended Dose,
`C = A × B
`5.38
`3.62
`1.61
`8.07
`3.62
`
`AUL
`
`ALL
`
`GL
`
`Dysport®
`5.38
`Botox®
`9.04
`Xeomin®
`4.03
`Dysport®
`5.38
`Botox®
`9.04
`Xeomin®
`4.03
`Dysport®
`0.27
`5.38
`50
`Botox® *
`0.18
`9.04
`20
`Xeomin®
`0.08
`4.03
`20
`* Botox Cosmetic; a according to prescribing information [13–15,31]. ALL, adult lower limb; AUL, adult upper limb;
`BoNT-A, botulinum neurotoxin type-A; GL, glabellar lines.
`
`2.2. Light Chain Activity of BoNT-A Products
`We analysed three vials from one batch each of Dysport® (batch L23919, expiry date December
`2018), Botox® (batch C4289C3, expiry date September 2019), and Xeomin® (batch 694458, expiry date
`July 2019) to assess LC activity using the EndoPep method, in which each BoNT-A product is incubated
`with a fluorescent-labelled substrate peptide. On this occasion, we chose to use Dysport® vials from a
`300 U batch so that the amount of toxin in the vial was more similar to those in the Botox and Xeomin
`vials, as established in the quantitation work. After 4 h of incubation, we measured LC activity by
`looking at the concentration of cleaved target peptide by CE-LIF detection. We quantified LC activity
`against a standard curve of a rBoNT-A protein control, allowing the derivation of the quantity of
`recombinant neurotoxin (in ng) required to achieve equivalent LC activity to one vial of each product.
`We also analysed the same batches of each BoNT-A product by sandwich ELISA using a pair
`of antibodies specific to the 150 kDa BoNT-A protein and the quantity of BoNT-A measured by
`extrapolation against a standard curve of rBoNT-A analysed in the same assay, as detailed above.
`The results are shown in Table 4.
`The resulting ratio of activity quantity (the relative quantity of rBoNT-A giving the same protease
`activity assessed by the EndoPep method) to protein quantity (ELISA) was 0.79 (±0.17) for Dysport®,
`1.08 (±0.23) for Botox®, and 0.79 (±0.05) for Xeomin® (given as the mean (±SD)). We assessed these
`ratios statistically for any differences between the products in case of differing specific LC activities.
`Table 5 summarises the relative LC activity per nanogram of neurotoxin among the BoNT-A
`products. Mean differences in activity vary only slightly, from 0 ± 0.18 to 0.29 ± 0.29, and all differences
`were non-significant. Thus, these results demonstrate that there are no significant differences in LC
`activity among the BoNT-A products, and the 150 kDa neurotoxin molecules in each product are
`equally active.
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`Table 4. Quantity (ng) of 150 kDa BoNT-A in Dysport®, Botox®, and Xeomin®, analysed by ELISA
`using BoLISA® antibodies, compared to an equivalent quantity using the EndoPep method®.
`
`Product
`
`Vial
`
`Quantity of BoNT-A (ng/Vial) ELISA
`
`Product, Mean (SD)
`
`Quantity of BoNT-A (ng/Vial)
`EndoPep
`Product, Mean (SD)
`
`Dysport® 300 U
`
`1.81 (0.12)
`
`1.42 (0.05)
`
`Botox® 100 U
`
`0.89 (0.10)
`
`0.96 (0.10)
`
`Xeomin® 100 U
`
`0.44 (0.02)
`
`0.35 (0.04)
`
`Per Vial
`Per Vial
`1.45
`1.87
`1
`1.45
`1.88
`2
`1.37
`1.67
`3
`0.85
`0.97
`1
`0.98
`0.92
`2
`1.05
`0.78
`3
`0.39
`0.46
`1
`0.33
`0.44
`2
`0.32
`0.41
`3
`BoNT-A, botulinum neurotoxin type-A; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation;
`U, units.
`
`Table 5. Relative light chain activity per ng of Dysport®, Botox®, and Xeomin® analysed using the
`EndoPep method.
`
`Product
`
`Comparator
`
`Dysport®
`Dysport®
`Xeomin®
`
`Botox®
`Xeomin®
`Botox®
`
`3. Discussion
`
`p value
`
`Z Score
`
`Difference in LC
`Activity, Mean (SE)
`0.3028
`1.0304
`0.293 (0.285)
`0.9989
`0.0014
`0.000 (0.176)
`0.2159
`1.2375
`0.293 (0.237)
`LC, light chain; NS, not significant; SE, standard error.
`
`p value
`Adjusted
`0.6611
`1.0000
`0.5179
`
`Significance
`
`NS
`NS
`NS
`
`Our analyses reveal that there are notable differences in the quantity of neurotoxin in each
`potency unit of different commercial BoNT-A products (Dysport®, Botox®, and Xeomin®), which,
`in turn, results in large differences in the amount of BoNT-A 150 kDa protein in a vial of each product.
`This work confirms the differences and non-interchangeability of BoNT-A product potency units.
`We determined the LC activity of BoNT-A products by the EndoPep method, using CE-LIF for
`analysis of the cleaved substrate peptides [27]. CE-LIF proved to be sufficiently sensitive, and we
`were able to inject the EndoPep mixture without any further sample preparation. By measuring
`the amount of the peptide cleaved by each product, we were able to compare the relative LC
`activities across products. The original paper from Kalb et al. [26] used a mass spectrometric analysis,
`namely MALDI-TOF. MALDI-TOF has some attractive aspects, such as a fast analysis and a high
`specificity. In our hands, however, MALDI-TOF was not sensitive enough for these samples using the
`substrate peptide described in Kalb et al. [26]. Recently, Wang et al. [32] published a paper with peptide
`substrate sequences that improve the sensitivity of the detection of BoNT/A. At the time that the Wang
`et al. paper appeared, however, we were already conducting studies with the CE-LIF method, and the
`substrate used provided the necessary sensitivity for this purpose. In future studies it may be worth
`assessing the further optimised substrate peptides mentioned in [32] with MALDI-TOF detection as
`well. Alternatively, the samples could be analysed with liquid chromatography–mass spectrometry.
`The large content of bovine serum albumin (BSA) in the EndoPep assay sample and longer time of
`analysis, however, made this option less suitable. To make a true comparison among the various
`BoNT-A products (which are each formulated in differing excipients), we designed the incubation and
`sampling scheme in such a way that all products were in an identical incubation mixture during the
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`EndoPep assay. In this way, the relative enhancing or inhibitory effect of a buffer constituent or other
`excipient is equally possible for all products tested.
`The FDA approves doses of BoNT-A products based on the doses that have been shown to be
`safe and effective in clinical trials. At the FDA-approved doses, the data in Table 3 show that a greater
`amount of neurotoxin is injected with Dysport® than with other BoNT-A products. For example,
`the amount of BoNT-A per FDA maximum approved dose for AUL spasticity equates to 5.38 ng
`Dysport® (in 1000 U: 2 × 500 U vials), 3.62 ng Botox® (in 400 U: 4 × 100 U vials), or 1.61 ng Xeomin®
`(in 400 U: 4 × 100 U vials). We also observed similar differences at the approved doses in the aesthetic
`indication of GL. Despite these differences in BoNT-A, when used at the FDA-recommended doses,
`all three BoNT-A products have a similar safety profile [13–15]. We were not able to compare all
`products for adult lower limb spasticity, since Xeomin® is not currently approved in the U.S. for this
`indication, following results from a recent Phase 3 study (NCT01464307) [31].
`We measured the quantity of BoNT-A in both 300 and 500 U vials of Dysport® and found that the
`values obtained were consistent: 5.38 (±0.07) pg/U for the 500 U vial and 6.02 (±0.39) pg/U for the
`300 U vial. Thus, conclusions regarding the quantity of toxin injected relative to other products based
`on values obtained for the 500 U vial are also applicable to the 300 U vial.
`Our analyses also revealed that the 150 kDa neurotoxin molecules within the different BoNT-A
`products had equivalent LC activities. Thus, one molecule of Dysport® had the same LC activity as one
`molecule of Botox® or Xeomin®. This is perhaps unsurprising given that all products were produced
`by a Hall strain of Clostridium botulinum bacteria and contain the A1 subtype of BoNT-A. Given these
`results, we can discount differences in LC activity as being responsible for observed differences
`between the BoNT-A products. Potential effects of the different formulations upon the proteolytic
`activity measured in this study, which we addressed by standardising the composition of the assay
`buffer among the BoNT-A samples measured, are unlikely to be of relevance to in vivo activity in
`the therapeutic context due to the dilution and diffusion of the formulation components likely to
`occur upon reconstitution and injection into the tissue. Although we did not measure other aspects of
`BoNT-A activity, we believe that our results are consistent with there not being significant differences
`in BoNT-A activity between the products. In this regard, our data build on those published by Frevert
`in 2010 [24]. The amount of neurotoxin per vial shown in our analysis confirms the data published
`by Frevert. Our experiments, however, extend this analysis by also investigating the LC activity of
`neurotoxin in the various products. The conclusions in the Frevert paper are not supported by the data
`obtained in this study, where no differences in specific LC activity were observed between the products.
`Frevert, in contrast, proposed that based upon the unit activity of the products, Xeomin® had a greater
`specific activity than the other products. Given the uniqueness of potency units to each BoNT-A
`product, however, specific activities cannot be calculated and compared among products based upon
`the unit activities of the different products. A standardised assay of the full toxin functionality per
`mass unit is required for this assessment. Our results indicate that the LC activity of the BoNT-A
`in each of the three main products does not significantly differ; in other words, each product has
`equivalent LC activity per quantity of BoNT-A.
`During clinical trials of Dysport® in adults with upper limb or lower limb spasticity and in
`children with lower limb spasticity, repeated injections of Dysport® resulted in improvements in active
`movement and function [33–37]. These improvements are rarely seen in the BoNT literature. The safety
`profile demonstrated in these studies was similar to those observed with other BoNT-A products
`when dosed as per their labels. It is important, therefore, that our comparisons of levels of BoNT-A
`injected in various clinical indications between the different products is performed in the context of
`the recommended dose. Similarly, the context of the recommended dose also ensures that there is no
`increased immunogenicity risk based on the quantities of BoNT-A being administered, as it has been
`shown that the incidence of neutralising antibody formation is low for all of the products when dosed
`as per their label [38].
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`The current BoNT-A prescribing information states that administration should not be performed
`at intervals of less than 12 weeks [13–15,39], but few studies have carefully assessed the time to
`retreatment following repeated injections of BoNT-A. Both of the recent trials in patients with adult
`spasticity mentioned above had flexible reinjection visits, and many patients treated with Dysport® did
`not require reinjection until much later than the standard 12 weeks (36.9% of patients in the open-label
`adult upper limb spasticity trial; 20.1% of patients in the open-label adult lower limb spasticity
`trial) [33–35], a result also observed in the recent trial of Dysport® in children with lower limb spasticity
`(74.0% of patients in the double-blind, paediatric, lower limb spasticity trial) [36]. It is well known that
`the amount of active BoNT-A injected is positively correlated to the duration of action (muscle paralysis)
`of the treatment. This dose–duration relationship has been clearly demonstrated in animal models [28],
`and a dose–efficacy relationship has been seen in patients with upper limb spasticity, though duration
`of response was not explored in this study [40]. Other clinical studies from the field of aesthetic
`medicine, however, have provided evidence for a dose–duration relationship [41–43]. Given this
`relationship, and the results shown here, we hypothesise that the greater number of 150 kDa neurotoxin
`molecules of BoNT-A in the doses of Dysport® injected during the aforementioned studies allowed a
`prolonged duration of action. In turn, this led to a requirement for less frequent injections. Importantly,
`clinical improvements and a prolonged duration of action were observed at well-tolerated doses in
`these studies.
`These findings may have important consequences for patients who are currently treated with
`BoNT-A, since a long duration of response with Dysport® has many potential benefits. Importantly,
`with a long duration of response, patients could receive a longer period of sustained symptom relief
`between injections, rather than experiencing a waning of clinical effect and reoccurrence of symptoms
`in the weeks preceding a repeat injection. In some cases, a long duration of response may enable
`the injection of additional muscles at Week 12, when the original muscles are still benefiting from
`the previous injection. Finally, patients may require less frequent and fewer injections, resulting in
`less disruption to work and social lives. The reduced cost burden on healthcare systems is also
`worth noting.
`
`4. Conclusions
`There are notably greater amounts of active neurotoxin in Dysport® when used at the total
`FDA-recommended dose for AUL, ALL, and GL than in Botox® and Xeomin®. This greater amount of
`active neurotoxin may prolong the block of neurotransmitter release at the neuromuscular junction
`following Dysport® injection and may result in a clinically longer duration of action that benefits
`patients, caregivers, and healthcare systems.
`
`5. Materials and Methods
`
`5.1. General Methods
`Capture and detection antibodies were purchased from BioSentinel, Inc. (BoLISA® #A1029)
`(Madison, WI, USA). Plates were washed three times between incubations using a buffered solution
`made from tablets comprising 0.01 M phosphate, 0.0027 M KCl, 0.137 M sodium chloride (NaCl), pH 7.4,
`and 0.05% phosphate-buffered saline with Tween®-20 (PBS-T). Recombinant BoNT-A1 was expressed
`and purified in-house. DNA encoding rBoNT-A1 (NCBI Reference Sequence accession number:
`WP_011948511.1; UniParc identifier: UPI0000001386) was synthesised and optimised for expression in
`Escherichia coli. This was then transformed into BLR (DE3) Escherichia coli cells (Novagen Cat# 69053-3,
`Lot D00088845) (Novagen, Madison, WI, USA) cultured in 1 litre batches. Purification of rBoNT-A1
`(SXN102342) was achieved by selective precipitation, followed by hydrophobic interaction and ion
`exchange column chromatography, coupled with an endoproteinase activation step. The activation
`stage cleaves the expressed single-chain, precursor protein at a specific recognition site and produces
`the fully active di-chain rBoNT-A1 product, the light and heavy chains being joined by a disulphide
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`bond. Hydrophobic interaction chromatography was used to separate any residual endoproteinase
`from the target product.
`
`5.2. BoLISA® Procedure
`The mass quantity (ng) of 150 kDa BoNT-A in commercial products (Dysport®, Botox®,
`and Xeomin®) was determined using a sensitive sandwich ELISA (BoLISA®) using a pair of mouse
`monoclonal anti-BoNT-A antibodies, one of them biotinylated.
`A microwell plate (Thermo Scientific Nunc® #439454) (Abingdon, Oxon, UK) was coated with
`BoLISA® capture antibody diluted to 2 µg/mL in phosphate-buffered saline (PBS, Gibco™ #10010023)
`(Thermo Fisher, Abingdon, Oxon, UK) and incubated for 1 h at 37 ◦C. After incubation, the plate
`was washed three times with PBS-T and incubated with blocking reagent (Blocker™ Casein in PBS,
`Thermo Scientific #37528) for 1 h at 37 ◦C. During the incubation time, samples and standards were
`prepared. Samples were prepared by reconstitution of the lyophilised products in the vial. Vials of
`Dysport® (500 U and 300 U) and Botox® (100 U) were reconstituted with 1 mL of blocking reagent
`and Xeomin® (100 U) with 0.5 mL. Xeomin was reconstituted in a smaller volume to ensure there was
`sufficient concentration of the product to be in the quantitation range for the assay. Standards were
`prepared by serial dilution of the stock rBoNT-A (2.4 mg/mL) with the blocking reagent. Intra-assay
`quality controls were prepared and run alongside the test samples to assess the accuracy of the
`assay. Test samples, standards, and quality control samples were pipetted in triplicate onto the assay
`plate and incubated for 1 h at 37 ◦C. Wells were washed a further three times with PBS-T, and the
`plate was incubated with the biotinylated detection BoLISA® antibody (BioSentinel Inc., Madison,
`WI, USA) and diluted to 2 µg/mL in blocking reagent for 1 h at 37 ◦C. Excess detection antibody
`was removed using another set of three washes, and horseradish peroxidase (HRP)-conjugated
`streptavidin (Pierce™ Streptavidin Poly-HRP, Thermo Scientific #21140) at 0.2 µg/mL, diluted in
`blocking reagent, was added onto the plate. After the final wash step, colour reactions were
`developed using 3,3(cid:48),5,5(cid:48)-tetramethylbenzidine (TMB) One Component HRP Microwell Substrate
`(BioFX Laboratories #TMBW-1000-01) (Owings Mills, MA, USA) and subsequently stopped after 2
`minutes with the addition of 450 nm Stop Reagent for TMB (BioFX Laboratories #STPR-1000-01).
`The absorbance of each well was read at 450 nm using a microwell plate reader (Synergy HT, BioTek,
`Winooski, VT, USA). The amount of the 150 kDa neurotoxin in the three commercial products was
`determined from the mean of three independent assays.
`
`5.3. Standard Curve of the Sandwich ELISA
`
`Recombinant BoNT-A1 was used to generate a standard curve from 20 to 0.05 ng/mL. Data were
`analysed using GraphPad Prism software (version 7.04, GraphPad Software, Inc, San Diego, CA,
`USA) and fitted to a Sigmoidal 4PL curve. A representative standard curve is presented in Figure 1.
`The quantities of 150 kDa BoNT-A1 in each sample were determined by interpolation of the sample
`absorbance at 450 nm against the standard curve in each assay. The means and SDs of three results
`were calculated from these determined values (Tables 1 and 2).
`
`5.4. ELISA Accuracy and Specificity
`An assay acc