Martin K. Childers

Assessment: Botulinum neurotoxin in the treatment of autonomic disorders and pain (an evidence-based review): report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology.

Objective: To perform an evidence-based review of the safety and efficacy of botulinum neurotoxin (BoNT) in the treatment of autonomic and urologic disorders and low back and head pain. Methods: A literature search was performed including MEDLINE and Current Contents for therapeutic articles relevant to BoNT and the selected indications. Authors reviewed, abstracted, and classified articles based on the quality of the study (Class I–IV). Conclusions and recommendations were developed based on the highest level of evidence and put into current clinical context. Results: The highest quality literature available for the respective indications was as follows: axillary hyperhidrosis (two Class I studies); palmar hyperhidrosis (two Class II studies); drooling (four Class II studies); gustatory sweating (five Class III studies); neurogenic detrusor overactivity (two Class I studies); sphincter detrusor dyssynergia in spinal cord injury (two Class II studies); chronic low back pain (one Class II study); episodic migraine (two Class I and two Class II studies); chronic daily headache (four Class II studies); and chronic tension-type headache (two Class I studies). Recommendations: Botulinum neurotoxin (BoNT) should be offered as a treatment option for the treatment of axillary hyperhidrosis and detrusor overactivity (Level A), should be considered for palmar hyperhidrosis, drooling, and detrusor sphincter dyssynergia after spinal cord injury (Level B), and may be considered for gustatory sweating and low back pain (Level C). BoNT is probably ineffective in episodic migraine and chronic tension-type headache (Level B). There is presently no consistent or strong evidence to permit drawing conclusions on the efficacy of BoNT in chronic daily headache (mainly transformed migraine) (Level U). While clinicians’ practice may suggest stronger recommendations in some of these indications, evidence-based conclusions are limited by the availability of data.

Common patterns of clinical motor dysfunction.

An upper motor neuron syndrome often leads to the development of stereotypical patterns of deformity secondary to agonist muscle weakness, antagonist muscle spasticity and changes in the rheologic (stiffness) properties of spastic muscles. Indentification of the spastic muscles that contribute to deformity across a joint allows therapeutic denervation to be implemented with the maximum likelihood of success. Identifying responsible muscles can be complex, since many muscles may cross the joint involved, and not all muscles with the potential to cause deformity will be spastic. Strategies including polyelectromyography and diagnositc blocks with local anesthetics can be used to test hypoteses regarding the deformity, providing information for more long‐term denervation. In this review, we discuss frequently observed patterns of deformity associated with problematic spasticity, paresis, contracture, and impaired voluntary motor control

Canine Models of Duchenne Muscular Dystrophy and Their Use in Therapeutic Strategies

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder in which the loss of dystrophin causes progressive degeneration of skeletal and cardiac muscle. Potential therapies that carry substantial risk, such as gene- and cell-based approaches, must first be tested in animal models, notably the mdx mouse and several dystrophin-deficient breeds of dogs, including golden retriever muscular dystrophy (GRMD). Affected dogs have a more severe phenotype, in keeping with that of DMD, so may better predict disease pathogenesis and treatment efficacy. Various phenotypic tests have been developed to characterize disease progression in the GRMD model. These biomarkers range from measures of strength and joint contractures to magnetic resonance imaging. Some of these tests are routinely used in clinical veterinary practice, while others require specialized equipment and expertise. By comparing serial measurements from treated and untreated groups, one can document improvement or delayed progression of disease. Potential treatments for DMD may be broadly categorized as molecular, cellular, or pharmacologic. The GRMD model has increasingly been used to assess efficacy of a range of these therapies. A number of these studies have provided largely general proof-of-concept for the treatment under study. Others have demonstrated efficacy using the biomarkers discussed. Importantly, just as symptoms in DMD vary among patients, GRMD dogs display remarkable phenotypic variation. Though confounding statistical analysis in preclinical trials, this variation offers insight regarding the role that modifier genes play in disease pathogenesis. By correlating functional and mRNA profiling results, gene targets for therapy development can be identified.

MTM1 mutation associated with X-linked myotubular myopathy in Labrador Retrievers.

Mutations in the MTM1 gene encoding myotubularin cause X-linked myotubular myopathy (XLMTM), a well-defined subtype of human centronuclear myopathy. Seven male Labrador Retrievers, age 14–26 wk, were clinically evaluated for generalized weakness and muscle atrophy. Muscle biopsies showed variability in fiber size, centrally placed nuclei resembling fetal myotubes, and subsarcolemmal ringed and central dense areas highlighted with mitochondrial specific reactions. Ultrastructural studies confirmed the centrally located nuclei, abnormal perinuclear structure, and mitochondrial accumulations. Wild-type triads were infrequent, with most exhibiting an abnormal orientation of T tubules. MTM1 gene sequencing revealed a unique exon 7 variant in all seven affected males, causing a nonconservative missense change, p.N155K, which haplotype data suggest derives from a recent founder in the local population. Analysis of a worldwide panel of 237 unaffected Labrador Retrievers and 59 additional control dogs from 25 other breeds failed to identify this variant, supporting it as the pathogenic mutation. Myotubularin protein levels and localization were abnormal in muscles from affected dogs, and expression of GFP-MTM1 p.N155K in COS-1 cells showed that the mutant protein was sequestered in proteasomes, where it was presumably misfolded and prematurely degraded. These data demonstrate that XLMTM in Labrador Retrievers is a faithful genetic model of the human condition.

Gene Therapy Prolongs Survival and Restores Function in Murine and Canine Models of Myotubular Myopathy

Intravenous injection of an adeno-associated viral vector expressing the myotubularin (MTM1) gene improves survival and rescues skeletal muscle function in mice and dogs affected by myotubular myopathy. Restoring Skeletal Muscle Function X-linked myotubular myopathy is a fatal disease of skeletal muscle that affects about 1 in 50,000 male births. Patients harbor mutations in the MTM1 gene and are typically born floppy, with severely weak limb and respiratory muscles. Survival requires intensive support, often including tube feeding and mechanical ventilation, but effective therapy is not available for patients. Gene replacement therapy using adeno-associated viral (AAV) vectors has potential for the treatment of inherited diseases like myotubular myopathy. Therefore, Childers et al. tested the effects of a recombinant AAV vector expressing myotubularin in two animal models of myotubularin deficiency: Mtm1 knockout mice and dogs carrying a naturally occurring MTM1 gene mutation. Results in both mice and dogs showed that a single intravascular injection of AAV strengthened severely weak muscles, corrected muscle pathology, and prolonged survival. No toxicity or immune response was observed in dogs. These results demonstrate the efficacy of gene replacement therapy for myotubular myopathy in animal models and pave the way to a clinical trial in patients. Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype 8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathology, and prolonged survival throughout a 6-month study. Similarly, single-dose intravascular delivery of a canine AAV8-MTM1 vector in XLMTM dogs markedly improved severe muscle weakness and respiratory impairment, and prolonged life span to more than 1 year in the absence of toxicity or a humoral or cell-mediated immune response. These results demonstrate the therapeutic efficacy of AAV-mediated gene therapy for myotubular myopathy in small- and large-animal models, and provide proof of concept for future clinical trials in XLMTM patients.

Evaluating motor end-plate-targeted injections of botulinum toxin type A in a canine model.

Tarsal joint forces were measured in dogs over 70 days following botulinum toxin type A (BTX‐A) injections. Three dogs were injected at motor end‐plates located by electromyography (EMG), while 3 dogs were similarly injected, but without EMG guidance. Extension forces were significantly (P < 0.05) smaller in limbs injected at motor end‐plates than in corresponding limbs on days 14 and 35. There were no significant differences at other times. Using these techniques, EMG end‐plate targeting potentiates effects of BTX‐A.

Contraction force generated by tarsal joint flexion and extension in dogs with golden retriever muscular dystrophy

Force generated due to torque caused by tarsal joint flexion and extension was measured noninvasively at 3, 4.5, 6, and 12 months of age in dogs with the Duchenne homologue, golden retriever muscular dystrophy (GRMD). Absolute and body-weight-corrected GRMD twitch and tetanic force values were lower than normal at all ages (P<0.01 for most). Tarsal flexion and extension were differentially affected. Flexion values were especially low at 3 months, whereas extension was affected more at later ages. Several other GRMD findings differed from normal. The twitch/tetany ratio was generally lower; post-tetanic potentiation for flexion values was less marked; and extension relaxation and contraction times were longer. The consistency of GRMD values was studied to determine which measurements will be most useful in evaluating treatment outcome. Standard deviation was proportionally greater for GRMD versus normal recordings. More consistent values were seen for tetany versus twitch and for flexion versus extension. Left and right limb tetanic flexion values did not differ in GRMD; extension values were more variable. These results suggest that measurement of tarsal tetanic force should be most useful to document therapeutic benefit in GRMD dogs.

Comparison of two injection techniques using botulinum toxin in spastic hemiplegia

This study sought to test the hypothesis that injections of botulinum toxin type A (BTX-A) at the mid belly of the gastrocnemius muscle in spastic hemiplegic adults produce superior clinical results to proximal injections directed toward the muscular origin. We designed a randomized, double-blind, placebo-controlled intervention study at a university tertiary care setting. Seventeen subjects with chronic spastic hemiplegic gait were enrolled from a volunteer community sample; time range from acute neurologic insult was 0.75 to 31 yr; age range was 19 to 71 yr; gender consisted of 11 men and 4 women; diagnoses were 12 patients with stroke, 2 with traumatic brain injuries, and 1 with a brain tumor. Two subjects were withdrawn from the study because of (1) acute vascular occlusion before intervention and (2) noncompliance with follow-up visits. After baseline measurements, subjects were injected with 50 units of BTX-A (volume, 0.5 cc) into the medial or lateral gastrocnemius: (1) proximally at one site near the muscular origin; (2) distally at three sites along the mid belly. We measured outcome using the Fugl-Meyer score, Ashworth scale, ankle range of motion, and a timed 50-ft fastest walk. No outcome measures showed a significant effect attributable to site of injections. Confounding variables included physical therapy and varying duration of illness in the study cohort. We conclude that the results failed to support the hypothesis that BTX-A injections at the mid belly of the gastrocnemius produced superior functional improvements to injections located near the muscular origin using localization techniques described. Additional research comparing more precise localization methods for BTX-A injections might further establish the importance of electromyographic guidance using BTX-A in management of spasticity.

Dystrophin-deficient cardiomyocytes derived from human urine: New biologic reagents for drug discovery

The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patients skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patients dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.

Enzyme replacement therapy rescues weakness and improves muscle pathology in mice with X-linked myotubular myopathy

No effective treatment exists for patients with X-linked myotubular myopathy (XLMTM), a fatal congenital muscle disease caused by deficiency of the lipid phosphatase, myotubularin. The Mtm1δ4 and Mtm1 p.R69C mice model severely and moderately symptomatic XLMTM, respectively, due to differences in the degree of myotubularin deficiency. Contractile function of intact extensor digitorum longus (EDL) and soleus muscles from Mtm1δ4 mice, which produce no myotubularin, is markedly impaired. Contractile forces generated by chemically skinned single fiber preparations from Mtm1δ4 muscle were largely preserved, indicating that weakness was largely due to impaired excitation contraction coupling. Mtm1 p.R69C mice, which produce small amounts of myotubularin, showed impaired contractile function only in EDL muscles. Short-term replacement of myotubularin with a prototypical targeted protein replacement agent (3E10Fv-MTM1) in Mtm1δ4 mice improved contractile function and muscle pathology. These promising findings suggest that even low levels of myotubularin protein replacement can improve the muscle weakness and reverse the pathology that characterizes XLMTM.