Valproate and Levocarnitine in Children With Spinal Muscular Atrophy
Valproate and Levocarnitine in Children With Spinal Muscular Atrophy
Spinal muscular atrophy (SMA), an autosomal recessive disorder, is characterized by muscle
 weakness due to degeneration of anterior horn cells in the spinal cord and brain stem nuclei.
 It has a variable incidence of 1 in 6700 to 1 in 25000 live births and prevalence of 0.12 to
 25 per 10,000 populations in different geographic areas and genetic constitution. A
 homozygous deletion/mutation involving exon 7 in SMN1 (survival motor neuron 1) is present in
 around 95% of the cases, resulting in the biochemical deficiency of the SMN protein. A
 genomic duplication at the same locus produces nearly identical SMN2 (survival motor neuron
 2) that differs from SMN1 by a nucleotide substitution that promotes exon 7 exclusion thus
 giving rise to only a fraction of the full length protein. Phenotypic variation in SMA
 correlates with the number of SMN2 gene copies and the level of SMN protein in cells.

 Several hypotheses including defective inhibition of apoptosis, glutamate excitotoxicity and
 lack of a neurotrophic factor(s) in nerve or muscle have been speculated in the pathogenesis
 of SMA.

 Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, directly increases SMN
 expression in SMA patient-derived cell lines in vitro. Till date 3 open label trials and 1
 placebo controlled RCT of VPA in human subjects have been published, all indicating a
 possible benefit in strength and/or motor function. Till date there is no effective therapy
 for SMA. Therapy is mainly supportive and palliative which can prolong lifespan and prevent
 complications to some extent without actually curing the disease.

 Children with SMA may have a reduced capacity to synthesis carnitine consequent to
 significantly diminished skeletal muscle mass. VPA independently inhibits carnitine transport
 and its metabolites deplete carnitine levels by binding to them. So along with valproate
 these patients should be supplemented with carnitine.

 With this background the investigators have planned a double blind randomized placebo
 controlled trial of Valproate and levocarnitine in 60 children (30 each in intervention and
 control arm) with Spinal Muscular Atrophy aged 2-15 years over a 2 year period with one
 baseline and four follow up visits. The study will be conducted in the Department of
 Pediatrics, AIIMS at the Myopathy clinic.
Spinal muscular atrophy is an autosomal recessive disease of varying severity that destroys
 motor neurons, resulting in atrophy and weakness of the voluntary muscles. Around 95% of the
 cases demonstrate a homozygous deletion/mutation involving exon 7 in SMN1 (survival motor
 neuron 1), resulting in the biochemical deficiency of the SMN protein.

 Although the pathogenesis of the spinal muscular atrophy remains speculative, several
 hypotheses include defective inhibition of apoptosis, glutamate excitotoxicity and lack of a
 neurotrophic factor(s) in nerve or muscle.

 Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, directly increases SMN
 expression in SMA patient-derived cell lines in vitro. It has also been demonstrated to have
 neuroprotective properties on glutamate induced excitotoxicity via up-regulation of
 alphasynuclein and increases neurite outgrowth in vitro.

 The molecular genetic basis of spinal muscular atrophy (SMA), an autosomal recessive
 neuromuscular disorder, is the loss of function of the survival motor neuron gene (SMN1). The
 SMN2 gene, a nearly identical copy of SMN1, has been detected as a promising target for SMA
 therapy. Transcriptional SMN2 activation or modulation of its splicing pattern to increase
 FL-SMN levels is believed to be clinically beneficial and therefore a crucial challenge in
 SMA research. Drugs such as valproic acid, phenylbutyrate, and sodium butyrate that mainly
 act as histone deacetylase inhibitors can mediate both: they stimulate the SMN2 gene
 transcription and/or restore the splicing pattern, thereby elevating the levels of FL-SMN2
 protein.

 Till date 3 open label trials and 1 placebo controlled RCT of VPA in human subjects have been
 published, all indicating a possible benefit in strength and/or motor function.

 Children with SMA may have a reduced capacity to synthesis carnitine consequent to
 significantly diminished skeletal muscle mass. VPA independently inhibits carnitine transport
 and its metabolites deplete carnitine levels by binding to them. So along with valproate
 these patients should be supplemented with carnitine.

 Childhood spinal muscular atrophy (SMA) is a common autosomal recessive disorder, which is
 characterized by muscle weakness due to degeneration of motor neurons in the spinal cord and
 brain stem nuclei. Progression of the disease is due to loss of anterior horn cells, thought
 to be caused by apoptosis. Positional cloning strategies have revealed several gene
 candidates including the gene for the Survival Motor Neuron(SMN) and the neuronal apoptosis
 inhibitory protein (NAIP). Both genes are duplicated on chromosome 5.

 The survival motor neuron transcript is encoded by two genes, SMNT and SMNC. The autosomal
 recessive proximal SMA that maps to 5q12 is caused by mutations in SMNT gene. SMNT exon 7 is
 not detected in approximately 95% of SMA cases because of either deletion or sequence
 conversion events.

 Recently an international collaborative effort through the European Neuromuscular SMA
 Consortium, as noted above, has established diagnostic criteria.

 1. Weakness is the central feature. The weakness should be symmetrical, proximal more than
 distal, and accompanied by hypotonia. The trunk is commonly involved. Weakness in the
 legs is characteristically greater than in the arms.

 2. Denervation demonstrated by electromyography, muscle biopsy and clinical criteria.
 Recently, the presence of the survival motor gene defect has become accepted as a
 reliable diagnostic test for spinal muscular atrophy.

 Possible therapeutic strategies include the use of motor neurotrophic agents, glutamate
 inhibitors, histone deacetylase inhibitors and embryonic grafts.

 Valproate, a short chain fatty acid histone deacetylase inhibitor has been shown to increase
 the protein level of SMN2 by 2 to 4 fold through transcription activation and restoration of
 the correct splicing of SMN 2 via increased levels of Htra2-beta1 in fibroblast cell lines
 from SMA patients and it has also been shown to raise the SMN protein level also in neuronal
 tissue.11 Till date 3 open label trials and 1 placebo controlled RCT of VPA in human subjects
 have been published, all indicating a possible benefit in strength and motor function.
 Recently the results of CARNI-VAL (valproate and carnival) trial has been published. It is a
 multicentric trial with 2 parts. The 1st one is a double blinded placebo controlled
 randomised trial of 61 SMA patients aged 2 to 8 years. It has indicated possible benefit in
 motor function and strength in younger patients with favourable and normal BMI profile. The
 2nd one is a prospective single armed trial of valproate and carnitine in 33 ambulatory
 children in SMA aged 3-17 years. No significant improvement was noted in muscle strength and
 functional status over 12 months period.

 Valproate is an FDA approved drug used for more than three decades in epilepsy treatment.
 Common side effects are dyspepsia and / or weight gain. Less common side effects are
 dysphoria, fatigue, dizziness, drowsiness, hair loss, headaches, nausea, sedation and
 tremors. It also causes hyperammonemia. It has also been reported to cause blood dyscrasias,
 impaired liver function, jaundice, thrombocytopenia and prolonged coagulation times.

 Because there has been no effective therapy for SMA, management till date consists of
 preventing or treating the complications. Complications of severe weakness include
 restrictive lung disease, poor nutrition, orthopedic deformities, immobility and psychosocial
 problems. Gene-specific therapy is not yet available. Noninvasive ventilation and
 percutaneous gastrostomy reportedly improves the quality of life with no effect on survival.
 The goal of active but nonfatiguing exercises is to maintain range of motion, increase muscle
 flexibility, and prevent contractures. Genetic counseling should be offered to all families
 of patient's with SMA.

 Sample size This has been calculated with MMT as the primary outcome using repeated measures
 design (1 pre and 4 post measurements). Assuming mean ± SD MMT score in the control group at
 enrollment as 120± 55 (based on the baseline status of patients attending our routine
 myopathy clinic) and expecting an improvement of at least 30% in the intervention arm (i.e.,
 156± 55), with correlation coefficient as 0.6, 90% power and 5% 2 sided α (i.e., 95% CI), the
 investigators require a minimum of 25 cases in each group. Considering about 20% loss to
 follow up the investigators would enroll 30 children in each of the 2 study arms, i.e., a
 total of 60 cases.

 Randomization: Block randomization with variable block size will be used to generate random
 numbers for allocation of study children in one of the two groups.

 Interim analysis It will be done after 20 patients have been enrolled to assess efficacy of
 the drug. If efficacy is clear cut further continuation of the study will not be justified.

 Patient recruitment

 All patients with diagnosis of SMA aged 2-15 years already registered in Myopathy Clinic of
 AIIMS will be recalled. Information will be sent to all Government Hospitals in Delhi that
 such a study is currently in progress and all the referrals will also be recruited. These
 patients and all those newly diagnosed at Myopathy Clinic, AIIMS will be randomized into two
 groups:

 Group I (Physiotherapy + Placebos) Group II (Physiotherapy + Valproate and Levocarnitine)
 Patients weighing less than 20 kg will receive liquid formulation of either sodium
 Valproateor placebo. Patients weighing more than 20 kg will receive oral tablets of either
 sodium Valproate/ placebo.

 Day 1-Day 10 Day 11- Day 20 Day 21 onwards < 10 Kg 1 ml BD 2 ml BD 2 ml TDS 11-15 Kg 1.5 ml
 BD 2 ml TDS 3 ml TDS 16-20 Kg 2 ml BD 3 ml TDS 4 ml TDS 21-25 Kg 100 mg (1 tab BD) 200 mg (1
 tab BD) 200 mg (1 tab TDS) 26-30 Kg 100 mg (1 tab TDS) 200 mg (1 tab TDS) 200 mg (2 -1-1)
 31-35 Kg 100 mg (1 tab TDS) 200 mg (1 tab TDS) 200 mg (2-1-2) All patients in the
 intervention arm will be supplemented with Carnitine 50mg per kg per day (maximum 1000mg) in
 2 divided doses during the entire period of valproate therapy .The other group will receive
 placebo.

 The investigators will be blinded to the code of the medicine. It will be broken only at the
 end of the study or if there are any significant side effects.

 Valproate is already being used for patients with epilepsy and has no significant side
 effects. Till date, besides conservative management no other treatment for SMA is available.
 This trial is thus justified.

 Periodic evaluation of the data will be done by a Data Safety Management Board (DSMB) which
 will be comprised by eminent experts.

 Informed consent It will be taken on a duly made consent form before starting the study. The
 patient or his parent's will have the right to withdraw from the study at any point of time
 with no disadvantage to the patient. No harmful effect is expected but in case of any
 research related injury, the best possible care at AIIMS will be provided.

 A detailed history and physical examination will be carried out on a predesigned proforma.
 All children will undergo DNA testing for exon7 deletion of SMNT gene. All will have CPK
 levels and routine hemogram and liver function tests. If the exon7 deletion is absent, EMG,
 NCV and muscle biopsy will be performed. Baseline assessment will include a detailed muscle
 charting on a 5 point scale as per the principles of manual muscle testing by a child
 physiotherapist. Other tests will include functional measure using modified Hammersmith
 Functional Motor Scale (MHFMS) and forced vital capacity (FVC) wherever possible.

 Patients will be clinically evaluated at 12, 24, 36 and 52 weeks by history, examination and
 a careful muscle charting. Other tests will include functional measure using modified
 Hammersmith Functional Motor Scale (MHFMS) and forced vital capacity (FVC) wherever possible.
 Compliance will be ensured at every visit by checking their medicine packets for leftover
 medicines. At 24 weeks and 52 weeks follow up routine hemogram, liver function tests and
 additionally at 52 weeks serum valproate levels will also be done.

 MMT (manual muscle testing) score will be expressed as total score out of 340. Similarly
 modified Hammersmith Functional Motor Scale (MHFMS) score will be expressed as total score
 out of. FVC (Forced vital capacity) will be expressed in Litres. Serum Valproate levels will
 be expressed in mg/ml. Side effect profile of valproate will be expressed as incidence of
 patients with the symptoms mentioned above. Incidence of patients with low platelet count
 (less than 1.5lacs/mm3) and elevated liver enzymes (OT and PT more than 40U/ L) will be
 recorded. Mean± 2SD change in MMT score, MHFMS score and FVC at each visit will be analysed
 using Wilcockson ranksum test and T test.
Inclusion Criteria:

 -  Children aged 2-15 years having motor weakness, hypotonia and hyporeflexia with onset
 noticed after 6 months of age.

 -  Presence of exon7 deletion of SMNT gene. OR Normal/ mildly elevated CPK with
 electrodiagnostic characteristics suggestive of neurogenic weakness, normal motor and
 sensory nerve conduction velocities and muscle biopsy showing neurogenic atrophy and
 /or evidence of reinnervation.

 Exclusion Criteria:

 1. SMA type I, onset before 6 months of age.

 2. Severely ill and unstable patients requiring life support system.

 3. Other causes like cerebral palsy, Down syndrome, connective tissue disorders,
 metabolic disorders.

 4. Pre-existing liver damage, bone marrow depression and coagulation disorders.

 5. Use of medications or supplements which interfere with valproic acid and carnitine
 metabolism within 3 months of study enrollment.

 6. Current use of either valproate or levocarnitine. If study subject is taking valproate
 and carnitine then patient must go through a washout period of 12 weeks before
 enrollment into the study