Students' guide to documentation in clinical trials.

PURPOSE: In the wake of the coronavirus disease 2019 (COVID-19) pandemic, support in clinical trials by students of human medicine and related disciplines has become of even greater importance than in pre-pandemic times. Documentation in clinical trials adheres to the principles of Good Clinical Practice (GCP), and healthcare professionals involved in the conduct of clinical trials-including students-are obliged to perform documentation in accordance with GCP principles. Unprecedented challenges have arisen with regard to the appropriate training of students as training courses in presence had largely to be suspended due to social-distancing regulations during the heyday of the COVID-19 pandemic. Therefore, novel training formats and self-study training materials for students working in clinical trials are urgently warranted. METHODS: To overcome this shortcoming and to define a common quality standard, an interdisciplinary, multiprofessional (physicians, study nurses, medical students), and binational (Germany, The Netherlands) expert panel convened and devised the Students' guide to documentation in clinical trials. RESULTS: Following a brief description of the different roles in clinical trials (e.g., sponsor, (principal) investigator, monitor) and an introduction into the principles of GCP, the documentation of adverse events, concomitant medication, medical history, and quality control are comprehensively discussed. The Guide concludes with a trilingual medical dictionary (English, German, Dutch) and with recommendations of pertinent literature for further reading. CONCLUSION: Serving both as textbook for self-training and as (quick-) reference work for the daily routine, the Guide has specifically been designed to complement, but not to replace practical training courses for students. While primarily addressed at students of human medicine and related disciplines, the Guide can also be of high relevance and utility to other healthcare professionals involved in the conduct of clinical trials.

Functional and Molecular Properties of DYT-SGCE Myoclonus-Dystonia Patient-Derived Striatal Medium Spiny Neurons.

Myoclonus-dystonia (DYT-SGCE, formerly DYT11) is characterized by alcohol-sensitive, myoclonic-like appearance of fast dystonic movements. It is caused by mutations in the SGCE gene encoding ε-sarcoglycan leading to a dysfunction of this transmembrane protein, alterations in the cerebello-thalamic pathway and impaired striatal plasticity. To elucidate underlying pathogenic mechanisms, we investigated induced pluripotent stem cell (iPSC)-derived striatal medium spiny neurons (MSNs) from two myoclonus-dystonia patients carrying a heterozygous mutation in the SGCE gene (c.298T>G and c.304C>T with protein changes W100G and R102X) in comparison to two matched healthy control lines. Calcium imaging showed significantly elevated basal intracellular Ca2+ content and lower frequency of spontaneous Ca2+ signals in SGCE MSNs. Blocking of voltage-gated Ca2+ channels by verapamil was less efficient in suppressing KCl-induced Ca2+ peaks of SGCE MSNs. Ca2+ amplitudes upon glycine and acetylcholine applications were increased in SGCE MSNs, but not after GABA or glutamate applications. Expression of voltage-gated Ca2+ channels and most ionotropic receptor subunits was not altered. SGCE MSNs showed significantly reduced GABAergic synaptic density. Whole-cell patch-clamp recordings displayed elevated amplitudes of miniature postsynaptic currents and action potentials in SGCE MSNs. Our data contribute to a better understanding of the pathophysiology and the development of novel therapeutic strategies for myoclonus-dystonia.

Dysphagia in cervical dystonia patients receiving optimised botulinum toxin therapy: a single-center retrospective cohort study.

To explore the correlations of botulinum toxin (BT) therapy with dysphagia, we wanted to study a group of cervical dystonia (CD) patients with optimised BT therapy during a prolonged period of time to record their dysphagia frequency, severity and duration, to study potential risk factors and try to avoid it by BT application with ultrasound guidance. BT therapy of 75 CD patients (23 males, 52 females, age 60 ± 12 years, BT total dose 303.5 ± 101.5 uMU) was retrospectively analysed for 1 year. BT therapy was optimised prior to the observation period. Dysphagia was noticed by one fifth of the patients. In those patients, it only occurred in about one third of the injection series. It was never associated with a functional deficit and lasted several days to 2 weeks. It was not related to patient age or gender, BT total dose, BT dose in the sternocleidomastoid muscle, BT dose in the sternocleidomastoid and scalenii muscles, by BT therapy with bilateral sternocleidomastoid muscle injections or BT therapy with abobotulinumtoxinA. Ultrasound guidance was not able to prevent it. Further prospective studies will be necessary to study underlying dystonia associated swallowing abnormalities as a potentially predisposing factor.

Intramuscular tetanus neurotoxin reverses muscle atrophy: a randomized controlled trial in dogs with spinal cord injury.

BACKGROUND: Motor symptoms of spinal cord injury (SCI) considerably impair quality of life and are associated with a high risk of secondary diseases. So far, no pharmacological treatment is available for these symptoms. Therefore, we conducted a randomized, double-blinded, placebo-controlled study in dogs with spontaneous SCI due to disc herniation to test whether a reduction of spinal inhibitory activity by intramuscular injections of tetanus neurotoxin (TeNT) alleviates motor symptoms such as muscle atrophy or gait function. METHODS: To this end, 25 dogs were treated with injections of either TeNT or placebo into their paretic hindlimb muscles. Effects of TeNT on muscle thickness were assessed by ultrasound, while effects on gait function were measured using the modified functional scoring system in dogs. RESULTS: Four weeks after the TeNT injections, muscle thickness of the gluteus medius muscle (before median 1.56 cm [inter-quartile range {IQR} 1.34-1.71 cm] and after median 1.56 cm [IQR 1.37-1.85 cm], P-value 0.0133) as well as of the rectus femoris muscle (before median 0.76 cm [IQR 0.60-0.98 cm] and after median 0.93 cm [IQR 0.65-1.05 cm], P-value 0.0033) significantly increased in the TeNT group. However, there was no difference in gait function between the TeNT and placebo groups. The treatment was well tolerated by all dogs without any signs of generalized tetanus symptoms or any spreading of effects beyond the lumbar level of the injected hindlimbs. CONCLUSIONS: With regard to the beneficial effects on muscle thickness, intramuscular injections of TeNT represent the first pharmacological approach that focally reverses muscle atrophy in SCI. Moreover, the study data support the safety of this treatment when TeNT is used at low dose.

Botulinum toxin-induced focal paresis in mice is unaffected by muscle activity.

INTRODUCTION: To test the hypothesis that the efficacy of botulinum toxin depends on the activity of the neuromuscular junction, we developed an in vivo paradigm to determine the degree and duration of low-dose botulinum toxin-induced focal paresis in mice. METHODS: We combined an automated wheel-running paradigm with low-dose botulinum toxin injections into the calf muscles of wild-type mice. Half of the mice were injected either before the nightly running or before the daily resting period. RESULTS: After botulinum toxin injections, running distance and maximum velocity decreased dose-dependently. The degree and duration of decrease between the respective groups with regard to the time-points of injection were identical. CONCLUSIONS: This in vivo paradigm quantifies the degree of otherwise clinically inapparent botulinum toxin-induced focal calf muscle paresis. Increased muscle activity after low-dose injections does not influence the efficacy of botulinum toxin in normal muscles.

Validation of the Parkinson's Disease Caregiver Burden Questionnaire in Progressive Supranuclear Palsy.

Progressive supranuclear palsy (PSP) is an atypical Parkinson syndrome with axial akinetic-rigid symptoms, early postural instability, and ocular motor impairments. Patients experience a rapid loss of autonomy and care dependency; thus, caregivers must assist in the activities of daily living early in the course of the disease. Caregiver burden is an extremely important factor in disease management. However, there are no specific questionnaires for assessment of caregiver burden in PSP. This study aims to validate the Parkinson's disease caregiver burden questionnaire (PDCB) as a specific measure of caregiver burden in PSP. PSP patients were assessed by the PSP rating scale, PSP quality-of-life questionnaire (PSP-QoL), Montreal cognitive assessment test (MoCA), and geriatric depression scale (GDS-15). Caregivers filled out the short form 36-health survey, GDS-15, PDCB, and the caregiver burden inventory (CBI). 22 patient caregiver pairs completed the study. PDCB showed a highly significant correlation with the CBI (r 0.911; p < 0.001). Internal reliability of the PDCB measured by Cronbach's alpha was favourable at 0.803. These data support the specificity of the PDCB in PSP caregivers. Future studies with larger sample sizes of PSP patients and caregivers and a multicentric longitudinal design should be performed to gain further insight of caregiver burden in PSP.

Regulation of dual leucine zipper kinase activity through its interaction with calcineurin.

Hyperglycemia enhancing the intracellular levels of reactive oxygen species (ROS) contributes to dysfunction and progressive loss of beta cells and thereby to diabetes mellitus. The oxidation sensitive calcium/calmodulin dependent phosphatase calcineurin promotes pancreatic beta cell function and survival whereas the dual leucine zipper kinase (DLK) induces apoptosis. Therefore, it was studied whether calcineurin interferes with DLK action. In a beta cell line similar concentrations of H2O2 decreased calcineurin activity and activated DLK. DLK interacted via its φLxVP motif (aa 362-365) with the interface of the calcineurin subunits A and B. Mutation of the Val prevented this protein protein interaction, hinting at a distinct φLxVP motif. Indeed, mutational analysis revealed an ordered structure of DLK's φLxVP motif whereby Val mediates the interaction with calcineurin and Leu maintains an enzymatically active conformation. Overexpression of DLK wild-type but not the DLK mutant unable to bind calcineurin diminished calcineurin-induced nuclear localisation of the nuclear factor of activated T-cells (NFAT), suggesting that both, DLK and NFAT compete for the substrate binding site of calcineurin. The calcineurin binding-deficient DLK mutant exhibited increased DLK activity measured as phosphorylation of the downstream c-Jun N-terminal kinase, inhibition of CRE-dependent gene transcription and induction of apoptosis. These findings show that calcineurin interacts with DLK; and inhibition of calcineurin increases DLK activity. Hence, this study demonstrates a novel mechanism regulating DLK action. These findings suggest that ROS through inhibition of calcineurin enhance DLK activity and thereby lead to beta cell dysfunction and loss and ultimately diabetes mellitus.

Reduced Expression of GABA A Receptor Alpha2 Subunit Is Associated With Disinhibition of DYT-THAP1 Dystonia Patient-Derived Striatal Medium Spiny Neurons.

DYT-THAP1 dystonia (formerly DYT6) is an adolescent-onset dystonia characterized by involuntary muscle contractions usually involving the upper body. It is caused by mutations in the gene THAP1 encoding for the transcription factor Thanatos-associated protein (THAP) domain containing apoptosis-associated protein 1 and inherited in an autosomal-dominant manner with reduced penetrance. Alterations in the development of striatal neuronal projections and synaptic function are known from transgenic mice models. To investigate pathogenetic mechanisms, human induced pluripotent stem cell (iPSC)-derived medium spiny neurons (MSNs) from two patients and one family member with reduced penetrance carrying a mutation in the gene THAP1 (c.474delA and c.38G > A) were functionally characterized in comparison to healthy controls. Calcium imaging and quantitative PCR analysis revealed significantly lower Ca2+ amplitudes upon GABA applications and a marked downregulation of the gene encoding the GABA A receptor alpha2 subunit in THAP1 MSNs indicating a decreased GABAergic transmission. Whole-cell patch-clamp recordings showed a significantly lower frequency of miniature postsynaptic currents (mPSCs), whereas the frequency of spontaneous action potentials (APs) was elevated in THAP1 MSNs suggesting that decreased synaptic activity might have resulted in enhanced generation of APs. Our molecular and functional data indicate that a reduced expression of GABA A receptor alpha2 subunit could eventually lead to limited GABAergic synaptic transmission, neuronal disinhibition, and hyperexcitability of THAP1 MSNs. These data give pathophysiological insight and may contribute to the development of novel treatment strategies for DYT-THAP1 dystonia.

Meningioma causing gabapentin-responsive secondary SUNCT syndrome.

Short-lasting unilateral neuralgiform headache with conjunctival injection and tearing (SUNCT) is one of the rarest and most serious headache disorders. Cases of symptomatic SUNCT syndromes are reported, which demonstrate that brain imaging is very important for diagnosis. In this study, we describe the first case of secondary SUNCT syndrome caused by a meningioma. So far, a clearly effective therapy for SUNCT syndrome has not been known. In this case, however, SUNCT was completely responsive to gabapentin. This underlines that this drug is worthy of being considered as a potential therapeutic option in the treatment of SUNCT syndrome.

Therapeutic effects of Tetanus neurotoxin in spinal cord injury: a case series on four dogs.

STUDY DESIGN: Case series on four dogs. OBJECTIVES: To determine the alleviation of motor symptoms in spinal cord injury (SCI) by tetanus neurotoxin (TeNT). SETTING: Different Berlin veterinary clinics, Germany. METHODS: We report on the effect of intramuscular injections of low-dose TeNT into paretic hind limb muscles 2-157 weeks after SCI due to lumbar disc herniation in a clinical case series on four dogs. All dogs underwent unsuccessful or incomplete surgical decompression prior to TeNT treatment. TeNT was injected on a compassionate basis. Stance, gait ability and the diameter of the rectus femoris muscle were assessed as parameters. RESULTS: All four dogs improved their stance and three of these dogs improved in gait at 4 and 6 weeks after TeNT injections without evidence of side effects or spreading of TeNT effects. At the same time, the size of the rectus femoris muscle diameter increased considerably as compared with baseline (baseline: 100%; 4 weeks: 148.7% ± 10.9%; 6 weeks: 137.1% ± 7.9%). CONCLUSIONS: Facilitation of α-motor neurons by TeNT injections into paretic hind limb muscles of four dogs improved standing and/or gait abilities and partly reversed muscle atrophy after SCI. The absence of generalized or painful muscle spasms supports the safety of low-dose TeNT. Therefore, TeNT might evolve as a promising therapeutic option for muscle paresis of central origin, e.g. in individuals with SCI, stroke or multiple sclerosis.

The role of human serum albumin and neurotoxin associated proteins in the formulation of BoNT/A products.

Botulinum neurotoxin (BoNT) is synthesized as a progenitor toxin complex (PTC) by Clostridium botulinum. This PTC comprises, in addition to the neurotoxin itself, neurotoxin associated proteins (NAPs) which are composed of three hemagglutinins and one non-toxic, non-hemagglutinin protein. After oral ingestion, these NAPs protect the neurotoxin from the low pH and proteases in the gastrointestinal tract and play a role in the entry via the intestinal barrier. Two of the three therapeutically used botulinum neurotoxin serotype A (BoNT/A) products (onabotulinumtoxinA and abobotulinumtoxinA) contain different amounts of NAPs, while incobotulinumtoxinA, lacks these proteins. In addition, human serum albumin (HSA) that is supposed to stabilize BoNT/A is added at different concentrations. Up to now, the function of the NAPs and HSA after parenteral therapeutic application is not completely understood. To investigate the influence of NAPs and HSA on potency of BoNT/A, we used the ex vivo mouse phrenic nerve hemidiaphragm assay. Increasing doses of HSA resulted dose-dependently in a more pronounced effect of BoNT/A. Though, a plateau was reached with concentrations of 0.8 mg/ml HSA and higher, the accessory addition of NAPs in a relevant amount (4 ng/ml) did not further enhance the effect of BoNT/A. In conclusion, in our ex vivo assay an adequate concentration of HSA prevented BoNT/A from loss of effect and supplementary NAPs did not alter this effect. A confirmation of these data in an in vivo assay is still lacking. However, it might be supposed that even in clinically applied BoNT/A products an increase of HSA accompanied by the avoidance of NAPs could potentially reduce the injected dose and, thus, the risk of unwanted side effects, the treatment costs as well as the risk of a secondary therapy failure due to BoNT/A neutralizing antibodies.

Botulinum neurotoxin serotype D - A potential treatment alternative for BoNT/A and B non-responding patients.

OBJECTIVES: Botulinum neurotoxin serotypes A and B (BoNT/A & B) are highly effective medicines to treat hyperactive cholinergic neurons. Due to neutralizing antibody formation, some patients may become non-responders. In these cases, the serotypes BoNT/C-G might become treatment alternatives. BoNT/D is genetically least related to BoNT/A & B and thereby circumventing neutralisation in A/B non-responders. We produced BoNT/D and compared its pharmacology with BoNT/A ex vivo in mice tissue and in vivo in human volunteers. METHODS: BoNT/D was expressed recombinantly in E. coli, isolated by chromatography and its ex vivo potency was determined at mouse phrenic nerve hemidiaphragm preparations. Different doses of BoNT/D or incobotulinumtoxinA were injected into the extensor digitorum brevis (EDB) muscles (n = 30) of human volunteers. Their compound muscle action potentials were measured 11 times by electroneurography within 220 days. RESULTS: Despite a 3.7-fold lower ex vivo potency in mice, a 110-fold higher dosage of BoNT/D achieved the same clinical effect as incobotulinumtoxinA while showing a 50% shortened duration of action. CONCLUSIONS: BoNT/D blocks dose-dependently acetylcholine release in human motoneurons upon intramuscular administration, but its potency and duration of action is inferior to approved BoNT/A based drugs. SIGNIFICANCE: BoNT/D constitutes a potential treatment alternative for BoNT/A & B non-responders.

BoNT/AB hybrid maintains similar duration of paresis as BoNT/A wild-type in murine running wheel assay.

The highly potent Botulinum neurotoxins (BoNT) are successful drugs to treat neuromuscular disorders. Efforts are being made to further reduce the injected BoNT dose and to lengthen the interval between treatments. Detailed knowledge of the BoNT structure-activity relationship (SAR) allows combining the best features of the different BoNT serotypes. Of all seven BoNT serotypes A-G, BoNT/A displays the highest potency despite low neuronal binding affinity, while BoNT/B exhibits much higher affinity. Recently, a new BoNT/AB hybrid (AABB) was constructed comprising the catalytic and translocation domain of BoNT/A and the 50kDa cell binding domain of BoNT/B. Here, we compared BoNT/A wild-type (AAAA) and AABB with regard to ex vivo potency and in vivo potency, efficacy and duration of action using the mouse phrenic nerve hemidiaphragm assay and the murine running wheel assay, respectively. The ex vivo potency of AABB was found to be 8.4-fold higher than that of AAAA. For the latter, two and 5 pg each of AAAA and AABB, respectively, were bilaterally injected into the calf muscles and mouse running wheel performance was automatically monitored during the following weeks to determine potency, efficacy and duration. Mice displayed a dose-dependent impairment of running performance. AABB showed potency, efficacy and duration equal to AAAA demonstrating successful exchange of the cell binding domain. AABB might combine the higher potency and longer duration of BoNT/A with the target specificity for the autonomic nervous system of BoNT/B. AABB might therefore constitute an improved treatment option for acetylcholine-mediated autonomic disorders such as hypersalivation or hyperhidrosis.

In-vivo comparison of the neurotoxic potencies of incobotulinumtoxinA, onabotulinumtoxinA, and abobotulinumtoxinA.

Three botulinum neurotoxin type A (BoNT/A) products, incobotulinumtoxinA, onabotulinumtoxinA, and abobotulinumtoxinA, all manufactured by different methods, are employed in clinical practice. Comparing the three BoNT/A products is difficult because their concentrations and volumes differ and the precise dose equivalence ratio is not known. We aimed to compare the neurotoxic potencies by a systematic analysis of injected volume and dose. The potency of BoNT in inducing hind limb paresis was assessed by analyzing the wheel-running performance of mice. To standardize the volume, the effect of an identical dose of incobotulinumtoxinA dissolved in different volumes of saline (15, 10, 5, and 2µl) was studied in four groups of mice (n=13-15). The potencies of the BoNT products were then compared by injecting identical volumes (10µl) containing different doses into both hind leg muscles. Mice injected with incobotulinumtoxinA showed a volume-dependent reduction in wheel-running, with larger volumes inducing more intense paresis. A standardized volume containing the same number of mouse units of the BoNT/A products produced different degrees of paresis. The conversion ratio of incobotulinumtoxinA and onabotulinumtoxinA is estimated to be between 1:0.75 and 1:0.5. OnabotulinumtoxinA displayed a two-fold greater potency than abobotulinumtoxinA. Doses of onabotulinumtoxinA and abobotulinumtoxinA that produce an identical severity of pareses even result in the same duration of pareses. This wheel-running assay allows one to compare the neurotoxic potency of different volumes and doses of the BoNT products in vivo. Our results argue against common clinical practice because incobotulinumtoxinA and onabotulinumtoxinA are not readily interchangeable and a two-fold dose of abobotulinumtoxinA is needed to induce an effect identical to onabotulinumtoxinA. In addition, this emphasizes that the duration of BoNT-induced effect is the same as long as equipotent doses of BoNT are injected.

Enhanced cardiac TBC1D10C expression lowers heart rate and enhances exercise capacity and survival.

TBC1D10C is a protein previously demonstrated to bind and inhibit Ras and Calcineurin. In cardiomyocytes, also CaMKII is inhibited and all three targeted enzymes are known to promote maladaptive cardiomyocyte hypertrophy. Here, in accordance with lack of Calcineurin inhibition in vivo, we did not observe a relevant anti-hypertrophic effect despite inhibition of Ras and CaMKII. However, cardiomyocyte-specific TBC1D10C overexpressing transgenic mice exhibited enhanced longevity. Ejection fraction and exercise capacity were enhanced in transgenic mice, but shortening of isolated cardiomyocytes was not increased. This suggests longevity resulted from enhanced cardiac performance but independent of cardiomyocyte contractile force. In further search for mechanisms, a transcriptome-wide analysis revealed expressional changes in several genes pertinent to control of heart rate (HR) including Hcn4, Scn10a, Sema3a and Cacna2d2. Indeed, telemetric holter recordings demonstrated slower atrial conduction and significantly lower HR. Pharmacological reduction of HR was previously demonstrated to enhance survival in mice. Thus, in addition to inhibition of stress signaling, TBC1D10C economizes generation of cardiac output via HR reduction, enhancing exercise capacity and survival. TBC1D10C may be a new target for HR reduction and longevity.

Accurate quantification of tetanus neurotoxin-induced focal spasticity in mice using complex running wheels.

Tetanus neurotoxin (TeNT) enhances activity of motoneurons by blocking spinal inhibitory interneurons. Based on this pathomechanism, we propose that low-dosage intramuscular injections of TeNT could serve as a specific treatment for central paretic muscles. However in vivo TeNT research is restricted because of the fear of triggering widespread muscle spasms. In addition, no reliable test to measure the in vivo toxicity of low-dosage TeNT is available. We introduce a novel wheel running-based paradigm with mice to quantify functional effects and thus the toxicity of low-dosage TeNT in vivo. We accustomed three groups of wildtype mice (n=14) to using a complex running wheel with irregularly spaced crossbars. Each group received an injection with a different low-dosage of TeNT (0.15 ng, 0.1 ng or 0.05 ng TeNT) into both tibialis anterior muscles. The maximum running velocity and accumulative running time of the groups were recorded during the following weeks. Three days after TeNT injections, the mice exhibited an increase in muscle tone of the injected tibialis anterior muscles but no generalized symptoms. However, we found that normal running in the complex wheel set-up was disturbed such that the maximum running velocity and running time of the mice decreased with the size of the dose. This effect peaked on the fifth and sixth nights after injection and returned to baseline level again within the next two weeks. With this novel in vivo automated paradigm we can accurately and objectively quantify the duration and degree of TeNT-induced focal increase in muscle tone.

Transcranial direct current stimulation induces polarity-specific changes of cortical blood perfusion in the rat.

OBJECTIVE: Transcranial direct current stimulation (tDCS) induces changes in cortical excitability and improves hand-motor function in chronic stroke. These effects depend on polarity, duration of stimulation and current intensity applied. Towards evaluating the therapeutic potential of tDCS in acute stroke, we investigated tDCS-effects on cerebral blood flow (CBF) in a tDCS rat model adapted for this purpose. METHODS: In a randomised crossover design eight Sprague-Dawley rats received three single cathodal and anodal tDCS for 15 min every other day. At each polarity, current intensities of 25, 50 and 100 µA were applied. CBF was measured prior and after tDCS for at least 30 min with laser Doppler flowmetry (LDF). RESULTS: At higher intensities (50 and 100 µA) anodal tDCS increased CBF up to 30 min. At 100 µA CBF was increased by about 25%, at 50 µA by about 18%. In contrast, cathodal tDCS led to a decrease of CBF, likewise depending on the current intensity applied. At 100 µA the effects were about 25% of baseline levels and persisted for at least 30 min. At 25 and 50 µA, baseline-levels were mostly re-established within 30 min. CONCLUSIONS: tDCS modulates CBF in a polarity specific way, the extent of modulation depending on the stimulation parameters applied. Because of its polarity-specificity, we assume that CBF-alterations are causally related to tDCS-induced alterations in cortical excitability via neuro-vascular coupling. tDCS may constitute a therapeutic option in acute stroke patients or in patients at risk for vasospasm-induced ischemia after subarachnoid hemorrhage.