The Effect of Pyridostigmine as an Antidote for Botulinum Type A: An Experimental Study on Rabbits

Huda Salim Alhasan; Jawad Hasan; Sawsan Alharoon

doi:10.12688/f1000research.127767.1

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Research Article

The Effect of Pyridostigmine as an Antidote for Botulinum Type A: An Experimental Study on Rabbits

[version 1; peer review: 2 not approved]

Huda Salim Alhasan ¹, Jawad Hasan¹, Sawsan Alharoon²

PUBLISHED 21 Dec 2022

Author details Author details

¹ Pharmacology, Basrah University, Basrah, Iraq
² Pathology, Basrah University, basrah, Iraq

Huda Salim Alhasan
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Jawad Hasan
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Visualization, Writing – Original Draft Preparation

Sawsan Alharoon
Roles: Conceptualization, Methodology, Project Administration, Resources, Software, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: Botulinum toxin (BoNT) or Botox injections are popular non-surgical and non-invasive option to optimize and change an individual’s facial appearance and achieve rejuvenation. Pyridostigmine is an acetylcholinesterase inhibitor important in clinical practice. This study evaluated the toxic effect of BoNT to demonstrate the efficacy of anticholinesterase inhibitor pyridostigmine.
Methods: We conducted an experimental interventional study to evaluate the antidote effect of pyridostigmine against BoNT. Forty rabbits (skeletally mature males, body weight 1000–2000 g) were included in the study. The rabbits were injected with Clostridium botulinum type A neurotoxin complex (BoNT or Botox), which was reconstituted with 0.9% sterile sodium chloride to a concentration of 100 U/2.5 mL. Animals were divided randomly into four groups (10 in each) as follows: Group 1 (control group), group 2 (pyridostigmine-treated group), group 3 (BoNT only group), and group 4 (BoNT + pyridostigmine treated group).
Results: In the control group, there was statistical difference between the rabbits’ weight (P=0.03). The left muscle was significantly heavier than the right (P=0.025). In the pyridostigmine only group, the rabbits’ weight had a high statistical difference during four weeks of the study (P=0.002). In the BoNT injection group, weight had high statistically significant difference four weeks of the study (P=0.033), which was more in first week (1.55±0.2 kg) and then subsequently decreased in the fourth week (1.345±0.13 kg). The mean weight of the rabbits’ right quadriceps muscle was 6.573±1.3 g, which was significantly lower than the mean weight of the rabbits’ left quadriceps muscle (8.09±1.2 g, P=0.0001).
Conclusions: To the best of our knowledge, this is the first study conducted in Iraq to investigate the antidote effect of pyridostigmine against BoNT injections. Pyridostigmine and saline caused increased weight of rabbits in comparison to rabbits that received BoNT injections. Pyridostigmine can act as strong antidote against Botox toxicity.

Keywords

BOTOX injections, Pyridostigmine, neurotoxin, clostridium botulinum type-A

Corresponding author: Huda Salim Alhasan

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2022 Salim Alhasan H et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Salim Alhasan H, Hasan J and Alharoon S. The Effect of Pyridostigmine as an Antidote for Botulinum Type A: An Experimental Study on Rabbits [version 1; peer review: 2 not approved]. F1000Research 2022, 11:1547 (https://doi.org/10.12688/f1000research.127767.1) First published: 21 Dec 2022, 11:1547 (https://doi.org/10.12688/f1000research.127767.1) Latest published: 21 Dec 2022, 11:1547 (https://doi.org/10.12688/f1000research.127767.1)

Introduction

Acetylcholinesterase inhibitors, such as pyridostigmine, are important in clinical practice, i.e., they can be used to treat the symptoms associated with myasthenia gravis, Alzheimer’s disease, and multiple sclerosis.¹^–³ Acute or prolonged exposure to reversible acetylcholinesterase inhibitors in healthy individuals, however, can have positive or negative effects on neuromuscular transmission and muscle ultrastructure.⁴^,⁵ Classically, the theory of receptor control suggests that chronic agonist stimulation of any receptor can lead to receptor desensitization and even down-regulation in a concentration- and time-dependent manner.⁶^–⁹

Clostridium botulinum is a rod-shaped, gram-positive anaerobic bacterium. It is a ubiquitous soil microorganism that can remain dormant as a spore in favorable environments. Its spores can germinate and produce a neurotoxin known as botulinum toxin.¹⁰ In 1897, Van Ermengem discovered this bacterium when Belgian patients presented with botulism. After that, several strains of the bacterium that produced serologically distinct toxin types were identified and classified into seven serotypes, A, B, C1, D, E, F, and G.¹¹ In 1928, Dr. Herman Sommer isolated the most potent serotype, botulinum neurotoxin (BoNT) type A.¹² Each serotype has its own distinct pharmacological properties,¹³^,¹⁴ and only materials containing BoNT type A or type B have been approved for human use. These neurotoxins act on the peripheral nervous system, where they inhibit the release of acetylcholine (ACh) from the synaptic terminal of neuromuscular junctions.¹⁵^,¹⁶ Currently, BoNT type A injections are a popular non-surgical and non-invasive treatment to optimize and change an individual’s facial appearance and achieve rejuvenation.¹⁷

In this study, we aimed to demonstrate the efficacy of anticholinesterase inhibitor pyridostigmine in accelerating spontaneous recovery following botulinum toxin injections in a rabbit model.

Methods

Study design

An experimental interventional study was conducted on 40 rabbits to evaluate the antidote effect of pyridostigmine against BoNT. This study was one-way one-period (28 days) from 12^th May to 11^th June 2022. Rabbits were randomly divided into four groups, and each group consisted of 10 rabbits. Control group received distal water, pyridostigmine-treated group received oral pyridostigmine, BoNT only group received injections intramuscularly with a single dose of BoNT and BoNT+pyridostigmine treated group received injections with BoNT and oral pyridostigmine, as shown in the flow chart (Figure 1).

Figure 1. The flow chart of the study.

Drugs

The drugs and their suppliers used in the study are listed in Table 1.

Table 1. Drugs used in the study.

Drugs	Trade name	Dosage form	Potency	Manufacture
Clostridium Botulinum toxin type A	HUTOX	Vial	100 unit	Huons Global Co. Korea
Pyridostigmine bromide	Mestinon	Tablet	60 mg	MEDA Pharma, Switzerland
Atropine sulfate		Vial	0.6 mg/ml	OMT Pharma India

Drug preparation

The drugs were bought from private pharmacies in Basrah city, Iraq. Tablets were crushed using a mortar and pestle, and then dissolved in distilled water to obtain a solution for administration to the rabbits. The doses of each drug administered were prepared as follows.

Pyridostigmine administration

One 60 mg tablet of pyridostigmine was dissolved in 50 mL distilled water before each administration to obtain a suspension of concentration 1.2 mg/mL. The powder was completely dissolved to give a homogenous solution in a sealed glass container. A dose of 4 mL/kg from the prepared stock solution was orally administered to each rabbit according to their body weight for an accurate dose (5 mg/kg), and then the dose was increased to 7 mg/kg after three days to avoid any unwanted cholinergic effects.

Reconstitution of botulinum toxin (BoNT) type A (Botox)

Using an appropriate-sized needle and syringe, 2.5 mL of 0.9% non-preserved sterile saline was drawn up. The needle was inserted and the saline was slowly injected into the vial. A vacuum was present in the vial, which demonstrated that it was sterile. The syringe was then disconnected from the needle and the BoNT was gently mixed with the saline by rotating the vial. The date and time of reconstitution were recorded on the label. After that, a new sterile syringe was attached and the reconstituted fluid was drawn into the syringe by angling the needle into the bottom corner of the vial to enable complete extraction. Any air bubbles in the syringe were expelled. The syringe was then disconnected from the needle used for reconstitution and a 30-gauge needle was attached for the injection. The doses of the drugs used in this study were selected according to a literature review and a pilot study,¹⁸ as follows: Pyridostigmine¹⁹ and BoNT.²⁰

Sample size

The sample size calculation of this study is based on availability of rabbit, effect of group size on the findings, and significant of this number on the experimental level.

Animals

Inclusion criteria

Forty rabbits (Oryctolagus cuniculus) (skeletally mature males) were included in the study aged from 2–4 years. They were obtained from a local market in Basrah city, and their body weights were between 1000 and 2000 g. One animal died during the study period. The rabbits were locally bred and sexually mature. Animals were housed under a controlled animal house conditioned atmosphere at a constant temperature (25°C±3) and relative humidity (50±5%) and supplied with food and water ad libitum during the study period. Animals were kept under observation for one month and fasted for three hours at the time of dosing.

Exclusion criteria

Rabbits were excluded from the study when dying, and when any adverse effect seen after administration of drugs like bleeding at site of injection, vomiting and cramping.

Preparation of animals

Rabbits were acclimatised for seven days before the study. They were allowed normal cage (65 × 345 × 330 cm³) activity and fed a standard diet (refoil and lettuce). To reduce stress, rough handling and overcrowding were avoided. During the four weeks of treatment, the body weight of each animal was recorded weekly. All authors were aware of groups during allocation, at a period of conducting the experiment, at the time of the outcomes assessment and at the time of analysis of data.

Ethical approval

This study was approved by the Ethical Committee of the Council of College of Medicine at the University of Basrah (No. 4502).

Experimental design and procedures

The animals were randomly divided into four groups (10 rabbits in each group). Each group was treated for four weeks as follows (Table 2):

• Group 1 (control group)

Table 2. Summary of drugs dosing and duration of treatment.

Groups	Treatments	Duration of treatments
Group 1 (control group)	Distilled water (4 mL/kg) orally for three days then (6 mL/kg) through the period of study & distilled water (10 U/kg) i.m. into the right quadriceps muscle (single dose)	30 days
Group 2 (pyridostigmine only group)	Pyridostigmine (5 mg/kg/day) Orally for three days, then (7 mg/kg)	30 days
Group 3 (BoNT only group)	Botulinum toxin type A (10 U/kg) i.m. into right quadriceps muscle	Single dose
Group 4 (BoNT+pyridostigmine group)	Pyridostigmine (5 mg /kg/day) Orally for three days, then (7 mg/kg) and BoNT (10 U/kg) i.m. into right quadriceps (single dose)	30 days

Rabbits in this group were treated with 4 mL/kg distilled water orally for three days. Then the dose was increased to 6 mL/kg and they were injected with distilled water (10 U/kg) in the right quadriceps muscle as a single dose and euthanized on day 30 of the study.

• Group 2 (pyridostigmine-treated group)

Rabbits in this group were orally administered pyridostigmine (4 ml/kg). Then the dose was increased to 6 mL/kg for the remaining 30 days. The rabbits were euthanized at the end of the study.

• Group 3 (BoNT only group)

Rabbits in this group were intramuscularly injected with a single dose of BoNT (10 U/kg) in the right quadriceps muscle (targeting the rectus femoris, vastus lateralis, vastus intermedius, and the vastus medialis) and euthanized on day 30 of the study.

• Group 4 (BoNT+pyridostigmine treated group)

Rabbits in this group were intramuscularly injected with BoNT (10 U/kg) into the right quadriceps muscle (single dose) and orally administered pyridostigmine (4 ml/kg) for three days. The dose was then increased to 6 mL/kg for the remaining 30 days before the rabbits were euthanized at the end of the study.

Outcomes

The following items were assessed: rabbits weight (kg) at the beginning of the study and at end of each of subsequent week, and weight of rabbit quadriceps muscle (g) after the end of the study.

Statistical analysis

The statistical analysis was performed using SPSS version 20 (IBM Inc., Chicago, IL, USA), Resource Identification Portal (RRID:SCR_004098).Descriptive statistics, consisting of numbers and percentages, are provided. The mean, median, range, minimum, maximum, and standard deviation (SD) were calculated for categorical data. Associations between groups were assessed by an unpaired t-test. Associations between variables were assessed by a chi-squared test. An ANOVA was used to describe associations between groups. A two-sided P-value of less than 0.05 was considered statistically significant. Details can be viewed at https://scicrunch.org/resources/data/record/nlx_144509-1/SCR_004098/resolver?q=SPSS%20version%2020&l=SPSS%20version%2020&i=rrid:scr_004098.

Results

Control group (A)

For the control group, the rabbits’ weight during one month of the experiment is shown in Table 3 and Figure 2. There was a statistically significant difference of weights during four weeks in the control group (P=0.03), which was more being in the third week (mean±SD=1.5±0.122 kg).

Table 3. Rabbit weight (kg) during one month of the experiment: (Control group).

No.	Group A	1^st week	2^nd week	3^rd week	4^th week
1	A1	1.6	1.55	1.6	1.6
2	A2	1.6	1.6	1.6	1.6
3	A3	1.5	1.5	1.55	1.55
4	A4	1.4	1.4	1.5	1.5
5	A5	1.2	1.4	1.5	1.5
6	A6	1.25	1.3	1.3	1.3
7	A7	1.3	1.3	1.35	1.35
8	A8	1.4	1.4	1.4	1.4
9	A9	1.7	1.7	1.7	1.7
10	A10	1.2	1.2	1.5	1.25
Mean±SD		1.415±0.18	1.435±0.15	1.5±0.122	1.475±0.145
Median		1.4	1.4	1.5	1.5
Min/Max		1.2/1.7	1.2/1.7	1.3/1.7	1.25/1.7
F=13, P=0.03

Figure 2. Box plot of the rabbit weight (kg) during one month of the experiment: (Control group).

The mean weight of the rabbits on the first day was 1.415±0.18 kg, whereas the mean weight of rabbits at the end of the fourth week was 1.475±0.145 kg, after one month of 0.9% saline injection in the control group, as shown in Table 4. There was no significant difference between groups (t=2.092, 95% CI=0.125–0.005, P=0.066).

Table 4. Weight of rabbit after month of Saline 0.9% injection (Control group).

No.	Group A	Weight on the 1^st day of experiment (kg)	Weight at the end of 4^th week (kg)
1	A1	1.6	1.6
2	A2	1.6	1.6
3	A3	1.5	1.55
4	A4	1.4	1.5
5	A5	1.2	1.5
6	A6	1.25	1.3
7	A7	1.3	1.35
8	A8	1.4	1.4
9	A9	1.7	1.7
10	A10	1.2	1.3
Mean±SD		1.415±0.18	1.475±0.145
Median		1.4	1.5
Min/Max		1.2/1.7	1.25/1.7
t-test		2.092
95% CI		0.125–0.005
P value		0.066

In this study, the mean weight of the rabbits’ right quadriceps muscle was 8.28±1.06 g, while the mean weight of the rabbits’ left quadriceps muscle was 8.385±1.06 g. The left muscle was significantly heavier than the right (t=2.677, 95%CI=0.193–0.016, P=0.025), as shown in Table 5 and Figure 3.

Table 5. Weight of rabbit quadriceps muscle: (Control group).

No	Group A	Weight of right muscle (gm)	Weight of left muscle (gm)
1	A1	9	9.2
2	A2	9.25	9.3
3	A3	8.7	8.9
4	A4	8	8.3
5	A5	8.5	8.38
6	A6	7.3	7.38
7	A7	7.25	7.34
8	A8	7	7.2
9	A9	10.3	10.38
10	A10	7.5	7.47
Mean±SD		8.28±1.06	8.385±1.06
Median		8.25	8.34
Min/Max		7/10.3	7.2/10.38
t-test		2.677
95% CI		0.193–0.016
P value		0.025

Figure 3. Weight of rabbit quadriceps muscle: (Control group).

Pyridostigmine-only group (B)

For the pyridostigmine-only group, the rabbits’ weight during one month of the study is shown in Table 6 and Figure 4. There was a high statistically significant difference among rabbits weight during four weeks of the study in this group (P=0.002), which was more being in the third week (mean±SD=1.485±0.175 kg).

Table 6. Rabbit weight (kg) during one month of the experiment: (Pyridostigmine only group).

No	Group B	1^st week	2^nd week	3^rd week	4^th week
1	B1	1.6	1.6	1.6	1.6
2	B2	1.5	1.5	1.55	1.5
3	B3	1.1	1.1	1.2	1.2
4	B4	1.6	1.55	1.55	1.55
5	B5	1.5	1.5	1.5	1.5
6	B6	1.2	1.2	1.2	1.2
7	B7	1.6	1.6	1.6	1.6
8	B8	1.7	1.7	1.7	1.7
9	B9	1.1	1.2	1.35	1.3
10	B10	1.6	1.55	1.6	1.6
Mean±SD		1.45±0.227	1.45±0.205	1.485±0.175	1.475±0.178
Median		1.55	1.525	1.55	1.525
Min/Max		1.1/1.7	1.1/1.7	1.2/1.7	1.2/1.7
F=22.685, P=0.002

Figure 4. Box plot of rabbit weight (kg) during one month of the experiment: (Pyridostigmine only group).

In the pyridostigmine-only group, the mean weight of rabbits on the first day was 1.45±0.227 kg, which was lower than the mean weight of the rabbits at the end of the fourth week, which was 1.475±0.178 kg after one month of oral pyridostigmine, as shown in Table 7. There was no significant difference between groups (t=1.103, 95%CI=0.076–0.026, P=0.299).

Table 7. Weight of rabbit after month of oral pyridostigmine dose: (Pyridostigmine only group).

No	Group B	Weight on the 1^st day of experiment (kg)	Weight at the end of 4^th week (kg)
1	B1	1.6	1.6
2	B2	1.5	1.5
3	B3	1.1	1.2
4	B4	1.6	1.55
5	B5	1.5	1.5
6	B6	1.2	1.2
7	B7	1.6	1.6
8	B8	1.7	1.7
9	B9	1.1	1.3
10	B10	1.6	1.6
Mean±SD		1.45±0.227	1.475±0.178
Median		1.55	1.525
Min/Max		1.1/1.7	1.2/1.7
t-test		1.103
95% CI		0.076–0.026
P value		0.299

In the pyridostigmine-only group, the mean weight of the rabbits’ right quadriceps muscle was 8.73±1.23 g, which was higher than the mean weight of the rabbits’ left quadriceps muscle, which was 8.635±1.14 g, but the difference was not significant (t=1.57, 95% CI=-0.042– ‐0.232, P=0.151), as shown in Table 8 and Figure 5.

Table 8. Weight of rabbit quadriceps muscle: (Pyridostigmine only group).

No	Group B	Weight of right muscle (gm) injected muscle	Weight of left muscle (gm)
1	B1	9.3	9.17
2	B2	8.3	8.38
3	B3	7.3	7.28
4	B4	8.7	8.59
5	B5	8.38	8.3
6	B6	7.34	7.38
7	B7	10.7	10.15
8	B8	10.4	10.5
9	B9	7.38	7.35
10	B10	9.5	9.25
Mean±SD		8.73±1.23	8.635±1.14
Median		8.54	8.485
Min/Max		7.3/10.7	7.28/10.5
t-test		1.57
95% CI		0.042–0.232
P value		0.151

Figure 5. Box plot of weight of rabbit quadriceps muscle: (Pyridostigmine only group).

BoNT injection group (C)

For the BoNT injection group, the rabbits’ weight during one month of the study is shown in Table 9 and Figure 6. There was a high statistically significant difference among rabbits weight during four weeks of the study in this group (P=0.033), which was more being in the first week (mean±SD=1.55±0.2 kg) but then dropped subsequently in the fourth week (mean±SD=1.345±0.13 kg).

Table 9. Rabbit weight (kg) during one month of the experiment: (BoNT injection group).

No	Group C	1^st week	2^nd week	3^rd week	4^th week
1	C1	1.6	1.5	1.4	1.4
2	C2	1.6	1.55	1.5	1.4
3	C3	1.8	1.6	1.5	1.5
4	C4	1.8	1.55	1.5	1.5
5	C5	1.3	1.2	1.1	1.1
6	C6	1.4	1.3	1.3	1.3
7	C7	1.3	1.25	1.25	1.25
8	C8	1.5	1.35	1.2	1.2
9	C9	1.4	1.4	1.4	1.4
10	C10	1.8	1.6	1.4	1.4
Mean±SD		1.55±0.2	1.43±0.15	1.355±0.14	1.345±0.13
Median		1.55	1.45	1.4	1.4
Min/Max		1.3/1.8	1.2/1.6	1.1/1.5	1.1/1.5
F=6.463, P=0.033

Figure 6. Box plot of rabbit weight (kg) during one month of the experiment: (BoNT injection group).

The mean weight of the rabbits on the first day was 1.55±0.2 kg, which was higher than the mean weight of the rabbits at the end of the fourth week, which was 1.345±0.13 kg, after one month of BoNT injections, as shown in Table 10. There was a highly significant difference between groups (t=5.156, 95% CI=0.115–0.295, P=0.001).

Table 10. Weight of rabbit after month of BoNT injection: (BoNT group).

No	Group C	Weight on the 1^st day of experiment (kg)	Weight at the end of 4^th week (kg)
1	C1	1.6	1.4
2	C2	1.6	1.4
3	C3	1.8	1.5
4	C4	1.8	1.5
5	C5	1.3	1.1
6	C6	1.4	1.3
7	C7	1.3	1.25
8	C8	1.5	1.2
9	C9	1.4	1.4
10	C10	1.8	1.4
Mean±SD		1.55±0.2	1.345±0.13
Median		1.55	1.4
Min/Max		1.3/1.8	1.1/1.5
t-test		5.156
95% CI		0.115–0.295
P value		0.001

In the Botox group, the mean weight of the rabbits’ right quadriceps muscle was 6.573±1.3 g, which was lower than the mean weight of the rabbits’ left quadriceps muscle, which was 8.09±1.2 g, a highly significant difference (t=21.795, 95% CI=1.674–1.36, P=0.0001), as shown in Table 11 and Figure 7.

Table 11. Weight of rabbit quadriceps muscle: (Botox group).

No	Group C	Weight of right muscle (gm) injected muscle	Weight of left muscle (gm)
1	C1	6.4	9
2	C2	6.14	8.4
3	C3	5	7.3
4	C4	5.2	7.6
5	C5	4.6	8.1
6	C6	5.9	8.9
7	C7	5.4	8.5
8	C8	5.2	8.4
9	C9	5.4	8.7
10	C10	6.4	8.7
Mean±SD		6.573±1.3	8.09±1.2
Median		6.25	7.9
Min/Max		4.8/8.83	6.4/10
t-test		21.795
95% CI		1.674–1.36
P value		0.0001

Figure 7. Weight of rabbit quadriceps muscle: (BoNT group).

Pyridostigmine and BoNT injection group (D)

For the pyridostigmine and BoNT injection group, the rabbits’ weight during one month of the study is shown in Table 12 and Figure 8. There was a statistically significant difference among rabbits’ weight during four weeks of the study (P=0.027), which was more being in the first week (mean±SD=1.455±0.2 kg).

Table 12. Rabbit weight (kg) during one month of the experiment: (pyridostigmine and BoNT injection group).

No	Group D	1^st week	2^nd week	3^rd week	4^th week
1	D1	1.25	1.2	1.2	1.25
2	D2	1.3	1.3	1.3	1.4
3	D3	1.3	1.25	1.2	1.25
4	D4	1.6	1.55	1.55	1.6
5	D5	1.8	1.7	1.7	1.8
6	D6	1.3	1.35	1.4	1.4
7	D7	1.4	1.4	1.4	1.4
8	D8	1.7	1.6	1.6	1.6
9	D9	1.6	1.55	1.5	1.55
10	D10	1.3	1.3	1.3	1.3
Mean±SD		1.455±0.2	1.42±0.17	1.415±0.17	1.455±0.18
Median		1.35	1.375	1.4	1.4
Min/Max		1.25/1.8	1.2/1.7	1.2/1.7	1.25/1.8
F=8.998, P=0.027

Figure 8. Box plot of rabbit weight (kg) during one month of the experiment: (pyridostigmine and BoNT injection group).

Thus, the mean weight of the rabbits on the first day was 1.455±0.2 kg, which was the same as the mean weight of rabbits at the end of the fourth week, which was 1.455±0.18 kg, after one month of BoNT injection, as shown in Table 13. There were no significant differences between the groups (t=0, 95% CI=N/A, P=1).

Table 13. Weight of rabbit after one month of BoNT injection and oral pyridostigmine dose: (BoNT injection and pyridostigmine group).

No.	Group D	Weight on the 1^st day of experiment (kg)	Weight at the end of 4^th week (kg)
1	D1	1.25	1.25
2	D2	1.3	1.4
3	D3	1.3	1.25
4	D4	1.6	1.6
5	D5	1.8	1.8
6	D6	1.3	1.4
7	D7	1.4	1.4
8	D8	1.7	1.6
9	D9	1.6	1.55
10	D10	1.3	1.3
Mean±SD		1.455±0.2	1.455±0.18
Median		1.35	1.4
Min/Max		1.25/1.8	1.25/1.8
t-test		0
95% CI		N/A
P value		1

In this study, the mean weight of the rabbits’ right quadriceps muscle was 5.564±0.615 g, which was lower than the mean weight of the rabbits’ left quadriceps muscle, which was 8.36±0.55 g, a highly statistically significant difference (t=18.656, 95% CI=3.135–2.457, P=0.0001), as shown in Table 14 and Figure 9.

Table 14. Weight of rabbit quadriceps muscle: (BoNT and pyridostigmine group).

No	Group D	Weight of right muscle (gm) injected muscle	Weight of left muscle (gm)
1	D1	5	6.7
2	D2	6	7.8
3	D3	6	7.3
4	D4	7.4	8.7
5	D5	8.83	10
6	D6	5.8	7.4
7	D7	6.5	8
8	D8	7.6	9
9	D9	7.8	9.6
10	D10	4.8	6.4
Mean±SD		5.564±0.615	8.36±0.55
Median		5.4	8.45
Min/Max		4.6/6.4	7.3/9
t-test		18.656
95% CI		3.135–2.457
P value		0.0001

Figure 9. Weight of rabbits’ quadriceps muscle (BoNT and pyridostigmine group).

ANOVA analysis

Table 15 and Figure 10 show the ANOVA analysis and linear correlation among different groups of the study in relation to the control group in the first week. There was a significant relationship between group A and B in the first week (P=0.024). There were no associations between group A and the other categories.

Table 15. ANOVA analysis of rabbit weight during 1^st week.

Control group	1^st week weight (kg)
Control group	B	C	D
ANOVA (F)	15	0.574	0.88
P value	0.024	0.743	0.593

Figure 10. Drop-line correlation of rabbit weight during 1^st week.

Table 16 and Figure 11 show the ANOVA analysis and linear correlation among different groups of the study in relation to the control group in the second week. There was a significant relationship between group A and D in the second week (P=0.047).

Table 16. ANOVA analysis of rabbit weight during 2^nd week.

Control group	2^nd week weight (kg)
Control group	B	C	D
ANOVA (F)	1.369	1.097	9.41
P value	0.43	0.511	0.047

Figure 11. Drop-line correlation of rabbit weight during 2^nd week.

Table 17 and Figure 12 show the ANOVA analysis and linear correlation among different groups of the study in relation to the control group in the third week. There was a significant relationship between group A and B in the third week (P=0.015).

Table 17. ANOVA analysis of rabbit weight during 3^rd week.

Control group	3^rd week weight (kg)
Control group	B	C	D
ANOVA (F)	21.52	0.44	1.001
P value	0.015	0.82	0.545

Figure 12. Drop-line correlation of rabbit weight during 3^rd week.

Table 18 and Figure 13 show the ANOVA analysis and linear correlation among different groups of the study in relation to the control group in the fourth week. There was no significant difference between group A and the other groups.

Table 18. ANOVA analysis of rabbit weight during 4^th week.

Control group	4^th week weight (kg)
Control group	B	C	D
ANOVA (F)	12.8	0.258	2.341
P value	0.074	0.926	0.332

Figure 13. Drop-line correlation of rabbit weight during 4^th week.

Figure 14 shows the correlation among different groups of the study in relation to the rabbits’ right quadriceps muscle weight. There was a highly statistically significant difference among groups according to the ANOVA analysis (F=18.515, P<0.0001).

Figure 14. Drop-line correlation of rabbit right quadriceps muscle weight.

Figure 15 shows the correlation among different groups of the study in relation to the rabbits’ left quadriceps muscle weight. There was no statistically significant difference among groups according to the ANOVA analysis (F=0.475, P=0.7).

Figure 15. Drop-line correlation of rabbit left quadriceps muscle weight.

Discussion

Recently, a model for BoNT intoxication was developed in rabbits for the first time.²¹ This is in accordance with the animal rule of the US FDA.²² Rabbits are the third most commonly used species for experimental research in the western countries.²³ Rabbits are larger than mice, so blood sampling and intravenous administration of drugs are easy.²⁴ Phylogenetically, rabbits are closer to humans than rodents, and due to their anatomical, physiological, genetic, and biochemical similarities, they are used as animal models for human diseases in a variety of medical research fields.²⁵

In this series, among the control group, the rabbits weight increased, reaching peak weight in the third week, with a statistically significant difference (P=0.03). The mean weight of rabbits on the first day was 1.415±0.18 kg, whereas at the end of the fourth week it was 1.475±0.145 kg, without significant difference between groups (P=0.066). Furthermore, the mean weight of the rabbits’ right quadriceps muscle was lower than the mean weight of the rabbits’ left quadriceps muscle, with a highly statistically significant difference (P=0.025). This is the same as found in Morsch et al.’s,²⁶ experiment, except they used citric acid monohydrate, sodium citrate dehydrate, methyl paraben, propyl paraben, and NaCl in sterile water (pH 5.1). However, in the saline group of Richtsfeld et al.,²⁷ there were no such changes, whereas an agreement was seen in saline injected group that showed gradual increased in the body mass of animals.²⁸

In the pyridostigmine only group, the weight during the study peaked in the third week, with a highly statistically significant difference (P=0.002). The mean weight of rabbits on the first day was lower than the mean weight of rabbits at the end of the fourth week. The mean weight of the rabbits’ right quadriceps muscle was higher than the mean weight of the rabbits’ left quadriceps muscle, with no significant difference (P=0.151). This agrees with the findings of Morsch et al.²⁶ They delivered pyridostigmine to animals systemically and continuously for seven to nine days via a minipump. Haigh et al. found that the effect of different doses of pyridostigmine on whole-blood acetylcholinesterase activity was recorded seven days after ingestion.²⁹ Morsch et al.²⁶ found rabbits treated with pyridostigmine lost weight and developed severe muscle weakness within two weeks. Hence, rather than preventing muscle weakness, pyridostigmine was capable of precipitating this weakness. This is in disagreement with what was reported by Richtsfeld et al., who found that animals in the pyridostigmine group lost 10% of their initial body weight during administration of pyridostigmine.²⁷

Morsch et al. concluded that pyridostigmine remains the only approved symptomatic drug for the treatment of BoNT intoxication, and one week of ingestion was enough to exacerbate neuromuscular impairment.²⁶

In the BoNT injection group, the rabbits’ weight was more in the first week (mean±SD=1.55±0.2 kg) but then subsequently decreased by the fourth week (mean±SD=1.345±0.13 kg); this was a highly statistically significant difference (P=0.033). Although, the mean weight on the first day (1.55±0.2 kg) was higher than the mean weight at the end of the fourth week (1.345±0.13 kg) of the study; this was a high significant difference (P=0.001). The mean weight of the rabbits’ right quadriceps muscle was 6.573±1.3 g, which was lower than the mean weight of the left quadriceps muscle (8.09±1.2 g), with a highly significant difference (P=0.0001). In the pyridostigmine and BoNT injection group, the rabbits’ weight gradually declined during subsequent injections. There was a statistically significant difference between the rabbits’ weights (P=0.027). Thus, the mean weight of rabbits on the first day was 1.455±0.2 kg, which had decreased at the end of the fourth week to 1.455±0.18 kg, with no significant between groups (t=0, 95% CI=N/A, P=1). In addition, the mean weight of the rabbits’ right quadriceps muscle was lower than the left quadriceps muscle, with a highly statistically significant difference (P=0.0001). Similarly, the animals lost weight and became weak after two weeks in Morsch et al.’s study.²⁶ Also, our study’s results are consistent with previous experiments,³⁰ and an agreement has been seen in Botox mice by Warner et al.²⁸

Torgeman et al. used different symptomatic rabbits dosing group with increased in a dose-dependent manner: 50%, 75%, and 80% with toxin doses of 0.5 ng/kg, 0.65 ng/kg, and 0.75 ng/kg, respectively. They found that 100% of the rabbits became symptomatic only in the 0.85 ng/kg dose, despite its lethal exposure dose.²¹

In our ANOVA analysis of this study, we showed the following findings between the control group and the other groups, such as a significant relationship between group A and B in the first week (P=0.024). There was a significant relationship between group A and D in the second week (P=0.047). There was a significant relationship between group A and B in the third week (P=0.015). The correlation among different groups in relation to the rabbits’ right quadriceps muscle weights had a highly statistically significant difference among groups according to the ANOVA analysis (P<0.0001). However, there was no statistically significant difference in the correlation with the rabbits’ left quadriceps muscle weight (P=0.7). Similarly, there were significant differences in the weights of the tibialis muscle between groups in different study periods.²⁷

In mice series, BoNT treatment significantly diminished right hindlimb muscle mass in both the quadriceps (102.9±31.2 vs. 195.3±13.9 mg, −47.3%, P<0.001) and calf (58.4±13.3 vs. 145.0±3.9 mg, −59.7%, P<0.001) compared to saline mice. Muscle mass in the contralateral hindlimb (left) of the BoNT-treated mice was also significantly diminished compared with that of the saline-treated mice, but to a lesser extent (quadriceps: 166.0±5.9 vs. 199.9±2.7 mg, minus 17.0%; calf: 120.8±4.2 vs. 145.0±1.6 mg, minus 16.7%, both P<0.001),²⁸ which seem similar to the current study’s findings.

Increasing evidence indicates that prolonged exposure to pyridostigmine was an etiological factor for Gulf War syndrome, which includes skeletal muscle symptoms.³¹ This could be explained by the fact that certain muscles appear to be affected more than others, for reasons unknown, but possibly due to quantitative differences in etiology.³⁰^,³²^,³³

Conclusions

To the best of our knowledge, this is the first study conducted in Iraq to investigate the antidote effect of pyridostigmine against BoNT injections. Pyridostigmine and saline caused increased weight in rabbits in comparison to BoNT-injected rabbits. No significant effect was seen on muscles after pyridostigmine administration, while the inverse was observed in the BoNT group. Pyridostigmine can act as a strong antidote against BoNT intoxication.

Data availability

Alhasan Huda Salim, Jawad K. Hasan, & Sawsan S. Al-Haroon. (2022). The effect of Pyridostigmine As Antidote for Botulinum type A. https://doi.org/10.5281/zenodo.7250444.³⁴

Reporting guidelines

The ARRIVE guidelines 2.0: author https://arriveguidelines.org/sites/arrive/files/documents/Author%20Checklist%20-%20Full.pdf

Huda Salim, Jawad Hasan, & Sawsan Alharoon. (2022). ARRIVE checklist of Pyridostigmine and BoNT. Zenodo. https://doi.org/10.5281/zenodo.7362881.³⁵

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

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Version 1

VERSION 1 PUBLISHED 21 Dec 2022

Author details Author details

¹ Pharmacology, Basrah University, Basrah, Iraq
² Pathology, Basrah University, basrah, Iraq

Huda Salim Alhasan
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Jawad Hasan
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Visualization, Writing – Original Draft Preparation

Sawsan Alharoon
Roles: Conceptualization, Methodology, Project Administration, Resources, Software, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

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Published: 21 Dec 2022, 11:1547

https://doi.org/10.12688/f1000research.127767.1

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Salim Alhasan H, Hasan J and Alharoon S. The Effect of Pyridostigmine as an Antidote for Botulinum Type A: An Experimental Study on Rabbits [version 1; peer review: 2 not approved]. F1000Research 2022, 11:1547 (https://doi.org/10.12688/f1000research.127767.1)

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Reviewer Report 17 Aug 2023

Lidija Bach-Rojecky, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia

Not Approved

https://doi.org/10.5256/f1000research.140307.r190325

Abstract:

BOTOX is not the only botulinum toxin type A product, so it should be deleted from the first sentence
Methods: It should be stated how the toxin was injected (site)

Abstract:

BOTOX is not the only botulinum toxin type A product, so it should be deleted from the first sentence
Methods: It should be stated how the toxin was injected (site) and what amount (dose and volume) of the toxin was injected.
Results: The results presentation is unclear and confusing.
Conclusion: The sentence “Pyridostigmine and saline caused the increased weight of rabbits in comparison to rabbits that received BoNT“ should be changed to „Pyridostigmine and saline didn't affect weight gain of rabbits in contrast to BoNT treated group“
From these results certainly cannot be concluded that „Pyridostigmine can act as a strong antidote against Botox toxicity“.
Keywords: delete „Botox injections“ and neurotoxin; add: rabbit, antidote

Introduction:

Comment to sentence: “Acetylcholinesterase inhibitors, such as pyridostigmine, are important in clinical practice, i.e., they can be used to treat the symptoms associated with myasthenia gravis, Alzheimer’s disease, and multiple sclerosis“ - Pyridostigmine due to its structure does not cross the blood-brain barrier and is not used in Alzheimer disease or for any cognitive dysfunction.
The mechanism of BoNT action should be described
BoNT has many various indications that should be mentioned

Methods:

This section needs significant editing and improvement
Animals should have been purchased from the registered breeder-supplier
The sex of the rabbits and method of randomization should be stated; persons involved in performing experiments and data analysis were not blinded to treatments
Table 1 – atropin sulfate was not used in the experiments and is mentioned in the table
Table 1 – column related to the potency of BoNT – 100 Units is contained in one vial; It is important to know the quantity of botulinum toxin that corresponds to 1 U. Is the titer of 1 U HUTOX equal to that of 1 U BOTOX?
The method of euthanasia is not stated
How was oral administration performed?
Were the animals inspected for signs of suffering and deteriorated health status?
For the control group the authors state that distilled water (10 U/kg) was injected. 10U/kg should be deleted (the same refers to Table 2)
Outcomes: It is a pity that different parameters and signs of potential toxicity were not examined. Weight loss is the only sign of systemic toxicity that was checked (respiration symptoms, salivary secretion, etc.), while muscle weight loss is a direct consequence of peripheral BoNT action in neuromuscular junctions.

Results:

This section needs complete re-structure and rewriting.
The results should be presented to avoid repetition of the same data in tables and figures. Also, data (for rabbits’ weight and muscle weights) for all experimental groups should be presented in one figure for easier comparison of the results.
It is unusual and unnecessary to present data for every experimental group in a separate table/figure. The results are shown in 15 figures and 18 tables, which is too much and hard to follow.
Also, the textual description of the results is confusing, For example, the authors state that “There was no significant difference between groups“ when describing one concrete experimental group (i.e. control group). I suppose the authors mean that there was no difference between the data at different time points.
Results of ANOVA analysis should be described in more detail.

Discussion:

The second, third, and fourth sentences are not relevant to the present study. They could be omitted.
Results are once again repeated in the discussion section; there is no need to repeat the same sentences from the results section
The sentence "However, in the saline group of Richtsfeld et al (27) there were no such changes, whereas an agreement was seen in the saline-injected group that showed a gradual increase in the body mass of animals (28)“ is unclear and needs rewriting. The authors should be careful regarding the citation of other authors' studies and be clear about the interpretation of the results.
For example, the study of Morsc et al (26) is wrongly cited and interpreted. The authors state "Morsch et al. concluded that pyridostigmine remains the only approved symptomatic drug for the treatment of BoNT intoxication, and one week of ingestion was enough to exacerbate neuromuscular impairment (26)“. This statement cannot be found in the cited publication.
Another miscitation refers to reference 27. The authors state “Similarly, there were significant differences in the weights of the tibialis muscle between groups in different study periods“, while in the cited study (27) five times higher dose of pyridostigmine (25 mg/kg) was applied via infusion pump and did not measure tibialis muscle weight.
How does the last paragraph in the Discussion section relate to the aim and the results of the present study?

Conclusion:

The conclusion must include novelty - how the study contributes to what is known in the field, study limitations, and the potential clinical significance of the results.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Neuropharmacology, neuroscience, pain research, botulinum toxin investigation

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Reviewer Report 26 Apr 2023

Christine Rasetti-Escargueil, Institut Pasteur, Paris, Île-de-France, France

Not Approved

https://doi.org/10.5256/f1000research.140307.r169144

Abstract:
-------------

The "Botox" is not the only approved medicine containing Botulinum toxin. Please remove "Botox" or add: "Botox like" products.

Please state briefly the main uses of pyridostigmine in the clinic.
... Continue reading

Abstract:
-------------

The "Botox" is not the only approved medicine containing Botulinum toxin. Please remove "Botox" or add: "Botox like" products.

Please state briefly the main uses of pyridostigmine in the clinic.

Please modify the third sentence to: "This study evaluated the efficacy of pyridostigmine against toxic effects of BoNT".

Results: Please state if differences in rabbit weight was between controls and treated or within the control group?

Please state what difference in weight occurred in the pyridostigmine only group, also for the Botox group: please only state that there was a statistically significant decrease of weight after 4 weeks of treatment.

Introduction:
------------------

Please detail BoNT mechanism of action and how the pyridostigmine could counteract the mechanism of BoNT effects on the neuromuscular junction.

Also, please give more details on the therapeutic uses of BoNT. There are a lot of diverse therapeutic indications.

Methods:
-------------

Flow chart: the control group should receive saline solution, not distilled water. This is not recommended for animal welfare.
Did you see any detrimental effects (pain) during water injections?

Drugs:
Please explain how the Units were calculated for the Botulinum toxin A (Hutox).

Please indicate Hutox instead of Botox since Hutox was used for reconstitution (not the product Botox). Only sterile saline should be used for injection into the rabbits due to infection risks.

Sample size: please clarify the sentence: "significant of this number on the experimental level".

Inclusion criteria: the rabbits should be obtained from an approved supplier to avoid risks of disease in the rabbits that can influence the results of the study.

Exclusion criteria:
The death, vomiting or cramping should not justify exclusion from the study since they are probably due to the treatments: Hutox or pyridostigmine.

It is important to take into account all the effects to assess true efficacy.

Outcome:
Other typical symptoms of botulism should be assessed like altered breathing, weakness, paralysis or vomiting.

Results:
------------
Control group
Table 4: please clarify if the saline solution or distilled water was injected into rabbits of the control group?

Figure 3: considering the high variability of the difference in quadriceps weight, this is unlikely to be significant.

Pyridostigmine group:
Please clarify the sentence:
"In the pyridostigmine-only group, the mean weight of the rabbits’ right quadriceps muscle was higher than the mean weight of the rabbits’ left quadriceps muscle (8.73±1.23 g for the right quadriceps versus 8.635±1.14 g for the left quadriceps),"

BoNT group:
Please state: "There was a dramatic decrease in rabbit weight after BoNT injections during the four weeks of the study. The decrease was highly significant."

"Similarly, there was a significant decrease of the right quadriceps muscle that was injected with BoNT, compared to the left muscle (not injected)."

It would be good to describe symptoms following BoNT injection and compare with other treated groups and control group.

Pyridostigmine and BoNT injection group
It is unexpected to see a statistically significant difference among rabbits’ weight during four weeks of the study considering the level of variability (0.2kg) compared to the very small differences (1.455kg versus 1.42kg: 0.035kg).

Comments for the Box plots of rabbit quadriceps weight: please remove "injected" for the left muscles as only right muscles were treated.

ANOVA analysis

Please provide detailed analysis of the ANOVA analysis specifically regarding the drop-line correlation of rabbit weight for each week as it is unclear at present how to interpret the drop-line plots. Please provide a definition of x and y axis for this type of plots.

Discussion
-----------------
This is not the first paper describing a rabbit model. Please see for example:
"A Novel Rabbit Spirometry Model of Type E Botulism and Its Use for the Evaluation of Post-symptom Antitoxin Efficacy"; February 2018; Antimicrobial Agents and Chemotherapy 62(4):AAC.02379-17
DOI:10.1128/AAC.02379-17.

Please explain why the right quadriceps muscle is smaller than the left muscle in the control group.

Please correct increased with "increase" in:
"whereas an agreement was seen in saline injected group that showed gradual increased in the body mass of animals.^28"

Please explain why Morsh et al. found different effects of pyridostigmine on rabbit weight.

Please clarify the sentence for BoNT group:
"In the BoNT injection group, the rabbits’ weight significantly decreased by the fourth week (in the first week: mean±SD=1.55±0.2 kg versus mean±SD=1.345±0.13 kg on the fourth week); this was a highly statistically significant difference (P=0.033)".

This sentence is not in line with the data of BoNT group: "Thus, the mean weight of rabbits on the first day was 1.455±0.2 kg, which had decreased at the end of the fourth week to 1.455±0.18 kg, with no significant between groups (t=0, 95% CI=N/A, P=1). "

"Torgeman et al. used different symptomatic rabbits dosing group with increase in a dose-dependent manner".

The Anova analysis deserve more explanations as it is not clear at present for non statisticians. Please detail how the correlations were done and which parameters were used.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

References

1. Diamant E, Pass A, Rosen O, Ben David A, et al.: A Novel Rabbit Spirometry Model of Type E Botulism and Its Use for the Evaluation of Postsymptom Antitoxin Efficacy.Antimicrob Agents Chemother. 2018; 62 (4). PubMed Abstract | Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Pharmacology, neurotoxins, botulinum, antitoxins, ex vivo, in vivo, in vitro models, stem cells.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Version 1 21 Dec 22	read	read

Christine Rasetti-Escargueil, Institut Pasteur, Paris, France
Lidija Bach-Rojecky, University of Zagreb, Zagreb, Croatia

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Reviewer Report

11 Views

17 Aug 2023 | for Version 1

Lidija Bach-Rojecky, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia

11 Views Cite this report Responses(0)

Not Approved

Abstract:

BOTOX is not the only botulinum toxin type A product, so it should be deleted from the first sentence
Methods: It should be stated how the toxin was injected (site) and what amount (dose and volume) of the toxin was injected.
Results: The results presentation is unclear and confusing.
Conclusion: The sentence “Pyridostigmine and saline caused the increased weight of rabbits in comparison to rabbits that received BoNT“ should be changed to „Pyridostigmine and saline didn't affect weight gain of rabbits in contrast to BoNT treated group“
From these results certainly cannot be concluded that „Pyridostigmine can act as a strong antidote against Botox toxicity“.
Keywords: delete „Botox injections“ and neurotoxin; add: rabbit, antidote

Introduction:

Comment to sentence: “Acetylcholinesterase inhibitors, such as pyridostigmine, are important in clinical practice, i.e., they can be used to treat the symptoms associated with myasthenia gravis, Alzheimer’s disease, and multiple sclerosis“ - Pyridostigmine due to its structure does not cross the blood-brain barrier and is not used in Alzheimer disease or for any cognitive dysfunction.
The mechanism of BoNT action should be described
BoNT has many various indications that should be mentioned

Methods:

This section needs significant editing and improvement
Animals should have been purchased from the registered breeder-supplier
The sex of the rabbits and method of randomization should be stated; persons involved in performing experiments and data analysis were not blinded to treatments
Table 1 – atropin sulfate was not used in the experiments and is mentioned in the table
Table 1 – column related to the potency of BoNT – 100 Units is contained in one vial; It is important to know the quantity of botulinum toxin that corresponds to 1 U. Is the titer of 1 U HUTOX equal to that of 1 U BOTOX?
The method of euthanasia is not stated
How was oral administration performed?
Were the animals inspected for signs of suffering and deteriorated health status?
For the control group the authors state that distilled water (10 U/kg) was injected. 10U/kg should be deleted (the same refers to Table 2)
Outcomes: It is a pity that different parameters and signs of potential toxicity were not examined. Weight loss is the only sign of systemic toxicity that was checked (respiration symptoms, salivary secretion, etc.), while muscle weight loss is a direct consequence of peripheral BoNT action in neuromuscular junctions.

Results:

This section needs complete re-structure and rewriting.
The results should be presented to avoid repetition of the same data in tables and figures. Also, data (for rabbits’ weight and muscle weights) for all experimental groups should be presented in one figure for easier comparison of the results.
It is unusual and unnecessary to present data for every experimental group in a separate table/figure. The results are shown in 15 figures and 18 tables, which is too much and hard to follow.
Also, the textual description of the results is confusing, For example, the authors state that “There was no significant difference between groups“ when describing one concrete experimental group (i.e. control group). I suppose the authors mean that there was no difference between the data at different time points.
Results of ANOVA analysis should be described in more detail.

Discussion:

The second, third, and fourth sentences are not relevant to the present study. They could be omitted.
Results are once again repeated in the discussion section; there is no need to repeat the same sentences from the results section
The sentence "However, in the saline group of Richtsfeld et al (27) there were no such changes, whereas an agreement was seen in the saline-injected group that showed a gradual increase in the body mass of animals (28)“ is unclear and needs rewriting. The authors should be careful regarding the citation of other authors' studies and be clear about the interpretation of the results.
For example, the study of Morsc et al (26) is wrongly cited and interpreted. The authors state "Morsch et al. concluded that pyridostigmine remains the only approved symptomatic drug for the treatment of BoNT intoxication, and one week of ingestion was enough to exacerbate neuromuscular impairment (26)“. This statement cannot be found in the cited publication.
Another miscitation refers to reference 27. The authors state “Similarly, there were significant differences in the weights of the tibialis muscle between groups in different study periods“, while in the cited study (27) five times higher dose of pyridostigmine (25 mg/kg) was applied via infusion pump and did not measure tibialis muscle weight.
How does the last paragraph in the Discussion section relate to the aim and the results of the present study?

Conclusion:

The conclusion must include novelty - how the study contributes to what is known in the field, study limitations, and the potential clinical significance of the results.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Neuropharmacology, neuroscience, pain research, botulinum toxin investigation

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Reviewer Report

12 Views

26 Apr 2023 | for Version 1

Christine Rasetti-Escargueil, Institut Pasteur, Paris, Île-de-France, France

12 Views Cite this report Responses(0)

Not Approved

Abstract:
-------------

The "Botox" is not the only approved medicine containing Botulinum toxin. Please remove "Botox" or add: "Botox like" products.

Please state briefly the main uses of pyridostigmine in the clinic.

Please modify the third sentence to: "This study evaluated the efficacy of pyridostigmine against toxic effects of BoNT".

Results: Please state if differences in rabbit weight was between controls and treated or within the control group?

Please state what difference in weight occurred in the pyridostigmine only group, also for the Botox group: please only state that there was a statistically significant decrease of weight after 4 weeks of treatment.

Introduction:
------------------

Please detail BoNT mechanism of action and how the pyridostigmine could counteract the mechanism of BoNT effects on the neuromuscular junction.

Also, please give more details on the therapeutic uses of BoNT. There are a lot of diverse therapeutic indications.

Methods:
-------------

Flow chart: the control group should receive saline solution, not distilled water. This is not recommended for animal welfare.
Did you see any detrimental effects (pain) during water injections?

Drugs:
Please explain how the Units were calculated for the Botulinum toxin A (Hutox).

Please indicate Hutox instead of Botox since Hutox was used for reconstitution (not the product Botox). Only sterile saline should be used for injection into the rabbits due to infection risks.

Sample size: please clarify the sentence: "significant of this number on the experimental level".

Inclusion criteria: the rabbits should be obtained from an approved supplier to avoid risks of disease in the rabbits that can influence the results of the study.

Exclusion criteria:
The death, vomiting or cramping should not justify exclusion from the study since they are probably due to the treatments: Hutox or pyridostigmine.

It is important to take into account all the effects to assess true efficacy.

Outcome:
Other typical symptoms of botulism should be assessed like altered breathing, weakness, paralysis or vomiting.

Results:
------------
Control group
Table 4: please clarify if the saline solution or distilled water was injected into rabbits of the control group?

Figure 3: considering the high variability of the difference in quadriceps weight, this is unlikely to be significant.

Pyridostigmine group:
Please clarify the sentence:
"In the pyridostigmine-only group, the mean weight of the rabbits’ right quadriceps muscle was higher than the mean weight of the rabbits’ left quadriceps muscle (8.73±1.23 g for the right quadriceps versus 8.635±1.14 g for the left quadriceps),"

BoNT group:
Please state: "There was a dramatic decrease in rabbit weight after BoNT injections during the four weeks of the study. The decrease was highly significant."

"Similarly, there was a significant decrease of the right quadriceps muscle that was injected with BoNT, compared to the left muscle (not injected)."

It would be good to describe symptoms following BoNT injection and compare with other treated groups and control group.

Pyridostigmine and BoNT injection group
It is unexpected to see a statistically significant difference among rabbits’ weight during four weeks of the study considering the level of variability (0.2kg) compared to the very small differences (1.455kg versus 1.42kg: 0.035kg).

Comments for the Box plots of rabbit quadriceps weight: please remove "injected" for the left muscles as only right muscles were treated.

ANOVA analysis

Please provide detailed analysis of the ANOVA analysis specifically regarding the drop-line correlation of rabbit weight for each week as it is unclear at present how to interpret the drop-line plots. Please provide a definition of x and y axis for this type of plots.

Discussion
-----------------
This is not the first paper describing a rabbit model. Please see for example:
"A Novel Rabbit Spirometry Model of Type E Botulism and Its Use for the Evaluation of Post-symptom Antitoxin Efficacy"; February 2018; Antimicrobial Agents and Chemotherapy 62(4):AAC.02379-17
DOI:10.1128/AAC.02379-17.

Please explain why the right quadriceps muscle is smaller than the left muscle in the control group.

Please correct increased with "increase" in:
"whereas an agreement was seen in saline injected group that showed gradual increased in the body mass of animals.^28"

Please explain why Morsh et al. found different effects of pyridostigmine on rabbit weight.

Please clarify the sentence for BoNT group:
"In the BoNT injection group, the rabbits’ weight significantly decreased by the fourth week (in the first week: mean±SD=1.55±0.2 kg versus mean±SD=1.345±0.13 kg on the fourth week); this was a highly statistically significant difference (P=0.033)".

This sentence is not in line with the data of BoNT group: "Thus, the mean weight of rabbits on the first day was 1.455±0.2 kg, which had decreased at the end of the fourth week to 1.455±0.18 kg, with no significant between groups (t=0, 95% CI=N/A, P=1). "

"Torgeman et al. used different symptomatic rabbits dosing group with increase in a dose-dependent manner".

The Anova analysis deserve more explanations as it is not clear at present for non statisticians. Please detail how the correlations were done and which parameters were used.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

References

1. Diamant E, Pass A, Rosen O, Ben David A, et al.: A Novel Rabbit Spirometry Model of Type E Botulism and Its Use for the Evaluation of Postsymptom Antitoxin Efficacy.Antimicrob Agents Chemother. 2018; 62 (4). PubMed Abstract | Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Pharmacology, neurotoxins, botulinum, antitoxins, ex vivo, in vivo, in vitro models, stem cells.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Respond to this report

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The Effect of Pyridostigmine as an Antidote for Botulinum Type A: An Experimental Study on Rabbits

Abstract

Keywords

Introduction

Methods

Study design

Figure 1. The flow chart of the study.

Drugs

Table 1. Drugs used in the study.

Drug preparation

Pyridostigmine administration

Reconstitution of botulinum toxin (BoNT) type A (Botox)

Sample size

Animals

Preparation of animals

Ethical approval

Experimental design and procedures

Table 2. Summary of drugs dosing and duration of treatment.

Outcomes

Statistical analysis

Results

Control group (A)

Table 3. Rabbit weight (kg) during one month of the experiment: (Control group).

Figure 2. Box plot of the rabbit weight (kg) during one month of the experiment: (Control group).

Table 4. Weight of rabbit after month of Saline 0.9% injection (Control group).

Table 5. Weight of rabbit quadriceps muscle: (Control group).

Figure 3. Weight of rabbit quadriceps muscle: (Control group).

Pyridostigmine-only group (B)

Table 6. Rabbit weight (kg) during one month of the experiment: (Pyridostigmine only group).

Figure 4. Box plot of rabbit weight (kg) during one month of the experiment: (Pyridostigmine only group).

Table 7. Weight of rabbit after month of oral pyridostigmine dose: (Pyridostigmine only group).

Table 8. Weight of rabbit quadriceps muscle: (Pyridostigmine only group).

Figure 5. Box plot of weight of rabbit quadriceps muscle: (Pyridostigmine only group).

BoNT injection group (C)

Table 9. Rabbit weight (kg) during one month of the experiment: (BoNT injection group).

Figure 6. Box plot of rabbit weight (kg) during one month of the experiment: (BoNT injection group).

Table 10. Weight of rabbit after month of BoNT injection: (BoNT group).

Table 11. Weight of rabbit quadriceps muscle: (Botox group).

Figure 7. Weight of rabbit quadriceps muscle: (BoNT group).

Pyridostigmine and BoNT injection group (D)

Table 12. Rabbit weight (kg) during one month of the experiment: (pyridostigmine and BoNT injection group).

Figure 8. Box plot of rabbit weight (kg) during one month of the experiment: (pyridostigmine and BoNT injection group).

Table 13. Weight of rabbit after one month of BoNT injection and oral pyridostigmine dose: (BoNT injection and pyridostigmine group).

Table 14. Weight of rabbit quadriceps muscle: (BoNT and pyridostigmine group).

Figure 9. Weight of rabbits’ quadriceps muscle (BoNT and pyridostigmine group).

ANOVA analysis

Table 15. ANOVA analysis of rabbit weight during 1st week.

Figure 10. Drop-line correlation of rabbit weight during 1st week.

Table 16. ANOVA analysis of rabbit weight during 2nd week.

Figure 11. Drop-line correlation of rabbit weight during 2nd week.

Table 17. ANOVA analysis of rabbit weight during 3rd week.

Figure 12. Drop-line correlation of rabbit weight during 3rd week.

Table 18. ANOVA analysis of rabbit weight during 4th week.

Figure 13. Drop-line correlation of rabbit weight during 4th week.

Figure 14. Drop-line correlation of rabbit right quadriceps muscle weight.

Figure 15. Drop-line correlation of rabbit left quadriceps muscle weight.

Discussion

Conclusions

Data availability

Reporting guidelines

References

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Table 15. ANOVA analysis of rabbit weight during 1^st week.

Figure 10. Drop-line correlation of rabbit weight during 1^st week.

Table 16. ANOVA analysis of rabbit weight during 2^nd week.

Figure 11. Drop-line correlation of rabbit weight during 2^nd week.

Table 17. ANOVA analysis of rabbit weight during 3^rd week.

Figure 12. Drop-line correlation of rabbit weight during 3^rd week.

Table 18. ANOVA analysis of rabbit weight during 4^th week.

Figure 13. Drop-line correlation of rabbit weight during 4^th week.