Peripheral TMD Pain Mechanisms and the Effect by Botulinum Toxin A

Study Overview
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Brief Summary
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The goal of this clinical trial is to investigate the effect of botulinum toxin on neurons' plasticity in the masseter muscle in humans with and without painful myogenous temporomandibular disorders (TMDM). The main questions it aims to answer are: does treatment with botulinum toxin alter gene expressions, epigenetic signatures, and cells plasticity in the masseter muscles of TMDM patients? do any such changes differ between patients with local and regional TMDM? does treatment with botulinum toxin influence pain characteristics (intensity, frequency, and sensibility) and other variables in patients with TMDM and are there correlations between significantly changed expression of biomarkers and other variables? Participants will be examined with a questionnaire, clinical examination, and biopsy sampling from one of the masseter and are then randomized to treatment with botulinum toxin or control (isotonic saline). Follow-ups occur after one, three, and six months with questionnaire, clinical examination, and collection of post-treatment microbiopsies to see if botulinum toxin alter peripheral molecular events and clinical variables.

Detailed Description
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Patient recruitment: The patients will be recruited among those referred to the Specialist Clinic for Orofacial Pain and Jaw Function at the University Dental Clinic, Karolinska Institutet, Huddinge, Sweden, or via advertisement. Randomization: Patients will be randomly assigned to treatment by a random generator (www.randomization.com). For each participant the treatment will be written on a note and placed in a sealed envelope by a researcher not involved in any other part of data collection. A second person not involved in patient examination will open the envelope, prepare the syringe with the randomized solution, and place it in the examination room before the examiner and patient enters it. Botulinum toxin and saline have identical appearance so the participant and investigator will be masked to treatment assignment. Procedure: The participants complete an extended and slightly modified version of the Swedish Axis II questionnaire included in the Diagnostic Criteria for TMD (DC/TMD) before the first visit. The Axis II questionnaire contains sociodemographic question, questions regarding TMD symptoms and headache (presence, duration, and frequency) used for diagnostic purposes (Symptom Questionnaire), and validated instruments to assess physical and emotional function. A clinical examination is then performed according to the DC/TMD Axis I which also includes recordings of pressure pain threshold, temporal summation pain, and conditioned pain modulation. After having ensured that the participant fulfils the eligible criteria biopsies are obtained from a painful and a non-painful site for later analysis. At least one week hereafter treatment are given. Biopsies: Microbiopsies will be obtained from the masseter and anterior tibialis muscle (a non-treated pain-free internal control). The microbiopsies are taken through the skin overlaying the most prominent part of the muscle under skin surface anesthesia. A disposable biopsy instrument with a penetration depth of 11 mm and a diameter of 18 gauche (G) (masseter) or 16G (tibialis) will be used. The biopsy instrument will be guided with a coaxial needle, that will be inserted to a depth of 10 mm. Three to four microbiopsies will be taken from each muscle to ensure that sufficient muscle tissue is obtained. The coaxial needle will be inserted along the long axis of the muscles until the fascia is penetrated and the biopsy instrument is then inserted through the coaxial needle and a piece of the muscle with a size of approximately 0.12 cm * 1.1 cm is collected. The muscle section will be removed from the biopsy instrument using a sterile probe and immediately put in a cryotube which is snap frozen in liquid nitrogen. After removal of the muscle section the biopsy instrument will be rinsed with isotonic saline. This procedure will be repeated two to three times. Each time the biopsy instrument will be rotated 45° so that the microbiopsy would be taken from a new portion of the muscle. The biopsies will be stored (-80º) in Karolinska Institutet's dental biobank until analysis. Analyses of biopsies: The biopsies will be analyzed with bulk ribonucleic acid sequencing (RNA-seq), Multiome (combined single-cell Assay for Transposase-Accessible Chromatin (ATAC) and RNA-seq), immunohistochemistry (IHC), light sheet microscopy, and flow cytometry (FC) at University of Texas Health Science Center in San Antonio (UTHSCSA) and at the Live Cell Imaging Facility at Karolinska Institutet, Huddinge, Sweden. Data of the non-painful site (anterior tibialis will be subtracted from data of the painful site (masseter) for every patient. Then, these data can be clustered. For bulk RNA seq, differentially expressed genes (DEGs) will be selected and then paired analysis will be run within empirical analysis of DEGs in R (edgeR) software to select significantly different (p<0.05) DEGs. This is an established and well standardized approach. Selection criteria for further analysis are fold charge (FC)>1.5 and Reads Per Kilobase of transcript per Million mapped reads (RPKM)>10. Finally, biological processes will be defined by www.pantherdb.org on base of DEGs. Substation of internal controls from experimental data will also be done, followed by clustering of patients using DEGs. Both approaches produce similar results. Power analysis based on preliminary results from skin biopsies showed that 19 paired samples will obtain 83.6% power with a standard deviation (SD) of 0.8. However, RNA from muscle tissue is usually of higher quality than skin samples. Hence, the investigators anticipate that bulk RNA-seq will have lesser SD and require 14 paired samples per group; this includes samples after treatments. Combined gene and epigenetic signature plasticity on cellular levels will be evaluated by Multiome using Genomic x10 platform. Multiome analysis is standardized by Genomic x10. For visual presentation Seurat will be used. Power analysis for Multiome showed that 3-4 paired samples per group is needed. Currently accepted analysis is that paired samples belonging to the same participant group combined and multiple t-test-like analysis for each cell cluster run against another group. The IHC analysis will focus on sensory neurite plasticity using markers for subsets of sensory neurons and use Light Sheet microscopy to measure neurite length and branching for whole biopsies. The investigators also plan to use the Glyclick method which generates stable and homogenous antibody conjugates for immunoglobin G from several species and subclasses. If possible, also proteomics will be done. FC will be used to profile immune and vasculature cells. Power analysis showed that 8 samples per group will be needed for IHC and FC. Analyses of clinical data: P<0.05 will be used as significance level. The Shapiro-Wilk's test will test for normality of data. Mean (SD) or median (interquartile range, IQR), depending on normality, will be used to describe the data. For normally distributed and continuous data repeated measures Analysis of Variance (ANOVA) will be used to analyze differences in treatment effect with Group and Time as factors. If significant differences are found these will be further analyzed with Dunnet's posthoc test. Data that are skewed or ordinal will be analyzed with Friedman ANOVA for each group separately with Dunn's test as posthoc test for time differences. The Mann-Whitney U-test will be used to analyze differences between groups at different time points, using Bonferroni correction for multiple testing. Multivariate statistics will be used to identify correlations between significantly changed biomarkers and other variables. Principal component analysis (PCA) will be used to identify moderate or strong outliers. Orthogonal partial least squares discriminant analysis (OPLS-DA) will then be used to regress group membership using the biomarkers as regressors. For correlations between biomarkers and clinical variables OPLS modelling will be performed. The Variable Influence on Projection (VIP) value indicates the relevance of each X-variable pooled over all dimensions and the Y-variables indicate the group of variables that best explain Y. VIP > 1.0 is considered significant. R2 describes the goodness of fit, i.e., the fraction of sum of squares of all the variables explained by a principal component whereas Q2 describes the goodness of prediction, i.e., the fraction of the total variation of the variables that can be predicted by a principal component using cross validation methods. R2 should not be considerably higher than Q2, if substantially higher (>0.3) the robustness of the model is poor. To validate the model, an obtained cross-validated analysis of variance (CV-ANOVA) p-value will be used. The OPLS-DA model is considered of significant importance if the CV-ANOVA has a p-value < 0.05.

Official Title
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Peripheral TMD Pain Mechanisms and the Effect by Botulinum Toxin A. A Randomized, Controlled, Double-blind Study

Conditions
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Temporomandibular Disorders

Intervention / Treatment
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* Drug: Botulinum toxin type A
* Drug: Isotonic saline 0,9%


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: a diagnosis of TMDM myalgia or myofascial pain according to the DC/TMD criteria females with adequate contraceptives and a negative pregnancy test pain upon digital palpation of at least one of the masseter muscle a characteristic pain intensity of > 40/100. Exclusion Criteria: difficulties understanding the Swedish language systemic inflammatory connective tissue diseases widespread pain neuromuscular disorders diagnosed or severe psychiatric disease neuropathic pain pain of dental origin history of trauma to the face, head or neck pregnancy or nursing known allergy to botulinum toxin or antibiotics use of muscle relaxants, antidepressant, neuropsychiatric, anticoagulant drugs, or aminoglycoside antibiotics previous treatment with botulinum toxin during the last 12 months use of analgesic or anti-inflammatory medication during the 48 hours preceding biopsy skin infection over injection/biopsy site

Ages Eligible for Study
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Minimum Age: 20 Years
Maximum Age: 45 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Diagnostic
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: Botulinum toxin<br>Single bilateral injection into three standardized points of the masseter muscles and two of the anterior temporalis muscle. The total dose administered is 100 U (10 U/point) which is dissolved in 1 mL of isotonic saline. Thus, 0.1 ml of botulinum toxin solution is injected in each point. | Drug: Botulinum toxin type A<br>* Randomized double-blind administration<br>* Other names: Botox®️ (100 U, AbbVie AB, Solna, Sweden);|
| Placebo Comparator: Isotonic saline<br>Single injection of 1 mL isotonic saline (0.9 mg/mL) into the same five points per side in a similar manner as for botulinum toxin. | Drug: Isotonic saline 0,9%<br>* Randomized double-blind administration<br>* Other names: Natriumklorid Braun (0,9 mg/mL, B. Braun Medical AB, Danderyd, Sweden);|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change of gene expression measured with bulk RNA-seq | Gene expression in the anterior tibialis muscle (internal control) will be subtracted from the masseter and the patients will then be clustered according to DEGs and compared to baseline. | 1-6 months | 
| Change of epigenetic signature measured with ATAC | The epigenetic signature in the anterior tibialis muscle (internal control) will be subtracted from the masseter and the patients will then be clustered according to DEGs and compared to baseline. | 1-6 months | 
| Change of expression of sensory neuron markers measured with IHC | The expression will be measured as the change of frequency (%) of selected sensory neuron markers. | 1-6 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change of pain intensity compared to baseline using 0-10 numeric rating scales (NRS) | Assessed with the Brief Pain Inventory and includes assessment of the current and the worst, average, and least pain intensity during the last week, each scored on 0-10 numeric rating scale. The worst, average, and current pain can be used to calculate the Characteristic Pain Intensity (0-100) which is the mean score of the three assessments multiplied with ten. | 1-3 months | 
| Global improvement using Patient Global Impression of Change Scale (PGIC) | Assessed with the 7-point PGIC with the alternatives: 0 = eliminated, 1 = much improved, 2 = improved, 3 = unchanged, 4 = impaired, 5 =much impaired and 6 = very much impaired. | 1-3 months | 
| Change of pain quality compared to baseline using the McGill Pain questionnaire (MPQ) | Assessed with the MPQ Short-Form which includes 15 adjectives that can be used to describe pain, each scored according to severity (0-3). The total score is calculated. | 1-3 months | 
| Number of participants with adverse events assessed with questionnaire | The participant lists any adverse event occurring during the week after injection and rate them as mild, moderate or severe. | 1 week | 
| Number of participants with adverse events assessed with questionnaire | The participant lists any adverse event occurring since the last visit and rate them as mild, moderate or severe. | 1-3 months | 
| Change of physical function assessed with questionnaire | Assessed with the Axis II questionnaire included in the DC/TMD. Includes the The Brief Pain Inventory (7 items, score ranges 0-100 for each), the Jaw Function Limitation Scale (20 items, score ranges 0-10), and the Oral Behavior Checklist (21 items, score ranges 0-84). A lower score indicates a better outcome for all instruments. | 1-3 months | 
| Change of emotional function assessed with questionnaire | Assessed with the Axis II questionnaire included in the DC/TMD. Includes the Beck's Depression inventory (21 items, score ranges 0-63), the Generalized Anxiety Disorder (7 items, score ranges 0-21), the Patient Health Questionnaire (15 items, score ranges 0-30), the Pain Catastrophizing Scale (13 items, score ranges 0-52), the Perceived Stress Scale (1o items, score ranges 0-40), and the Insomnia Severity Index (7 items, score ranges 0-28). A lower score indicates a better outcome for all instruments. | 1-3 months | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Temporomandibular Disorders, Botulinum Toxin A, Myalgia, Randomized, Clinical Trial, Pain, Gene, Neural Plasticity