We chose to perform a placebo-controlled study of VPA in non-ambulatory children with SMA based on encouraging preliminary observations of improved motor function during an open label study 
. Our choice to target children ages 2–8 years reflected a compromise between concerns regarding the lower limit of age where reliable assessment of motor function was possible versus concerns that improvement in older children may be constrained by other factors that would confound assessment of an SMN-enhanced treatment effect. A combined treatment regimen of VPA and carnitine was selected to avoid a potential confounding effect of carnitine depletion, because we had previously demonstrated an apparent susceptibility for more rapid depletion of carnitine with VPA treatment in this population. 
Overall, enrollment was targeted towards a more homogeneous younger cohort of subjects who theoretically might be more likely to respond to therapy.
We did not detect a statistically significant improvement after six months of treatment in our primary outcome measure - the change in overall gross motor function assessed with the MHFMS. In addition, we did not detect a significant change in secondary endpoints of strength and function, which included assessment of maximum upper and lower extremity strength via hand-held myometry and PFTs. Adverse event frequency, although not statistically different between groups, was somewhat higher in the treatment group, particularly with regard to gastrointestinal and respiratory symptoms. Although we did not observe any clinical or laboratory evidence of serious hematologic or hepatic toxicity in this study, excessive weight gain was clearly more prevalent in the active treatment group. DEXA measurements confirmed that the associated weight gain was due largely to an increase in total body fat mass in the absence of an increase in lean mass.
Given the increased fat mass in the treatment group compared to the placebo group during the phase 1 period, it is notable that we did not observe a decline in gross motor function in the treatment group as a whole. As in the open label study 
, weight gain was not uniform across the population. Non-ambulatory subjects greater than five years of age, and those having a higher BMI at baseline, were at greatest risk. Children with the greatest improvement in gross motor function as assessed by the MHFMS gained less fat mass than did those with stable or worsening MHFMS scores. The fact that the MHFMS did not decline in light of substantial weight gain in the active treatment group suggests that VPA may indeed have a modest yet measurable biologic effect that is outweighed by its confounding effect of weight gain. Increases in fat mass may not always be deleterious in this population, where both over- and under-nourished states are common. Some children may benefit from the improved nutritional intake stimulated by an effect of VPA on appetite, perhaps putting them into a more favorable anabolic state. However, in other cases, due to increased gastrointestinal symptoms related to VPA and/or L-carnitine, weight loss could be a negative confounding factor.
Although no treatment effect was detected in the primary outcome measure as assessed by the MHFMS, a post hoc analysis demonstrated a statistically significant benefit in the youngest cohort of children, those two to three years of age. In addition, individuals who received the active treatment over a full year showed modestly improved function, gaining just over 2 points on the MHFMS, although this did not reach statistical or obvious functional significance. Age proved to be a significant factor as to whether or not subjects demonstrated an increase in MHFMS scores in association with treatment. We have previously demonstrated that increased age is associated with increased severity of denervation, so it is not surprising that the youngest non-ambulatory SMA children appear more likely to demonstrate a benefit with intervention 
. Duration of treatment also appeared to be a factor and will need to be carefully considered in the design of future clinical trials in non-ambulatory SMA subjects. The ideal trial duration clearly depends on the expected mechanism of the treatment to be studied and relies heavily on the primary outcome measure chosen. This study affirms the value of the MHFMS in the multicenter pediatric clinical trial setting as a simple, reliable and easy to use outcome measure for documenting gross motor function in children as young as two years of age. However, if reinnervation is considered the most likely mechanism for a given therapeutic intervention, a trial duration of at least one year or longer may be more ideal to prove a treatment benefit using the MHFMS.
Possible disease-related biomarkers assessed in this study included quantitative SMN mRNA and maximum ulnar CMAP. Levels of flSMN and Δ7 SMN mRNA did not change with VPA. In contrast, VPA-response has been observed in SMA patient cell lines (16,17), one-third of SMA subjects (18) and in spinal cord of type III-like SMA mice (24). This result suggests that SMN expression in blood may not be a good surrogate biomarker to track VPA response. Because VPA targets are not specific, VPA response in SMA subjects may be much more complex than simply altering SMN expression. Indeed, recent data in a mild SMA model suggests that VPA may function by increasing SMN mRNA and protein, decreasing apoptosis and enhancing neuroprotection (24). Alternatively, whole blood may not be the appropriate cell type to detect changes in SMN expression, or such changes may be too small to be detected by relative quantification that relies on endogenous controls to normalize data. Finally, the assay we used has limitations, because it is not based on absolute quantification of SMN mRNA.
The strength of correlation between maximum CMAP amplitude and our primary outcome measure, the MHFMS, suggests that in non-ambulatory patients with SMA the CMAP may be a robust surrogate outcome measure for assessing treatment interventions if the putative mechanism of effect is to enhance innervated muscle mass by reinnervation or other trophic effects on nerve or muscle. However, baseline severity of denervation overall in a given cohort will profoundly impact whether or not improvement is anticipated and should be a key consideration in any trial design incorporating this as an outcome measure, as ceiling and floor effects may limit the usefulness of this assay in those of greater or lesser strength or age.
In summary, this study demonstrated no benefit from six months of treatment with VPA and L-carnitine in a young non-ambulatory cohort of subjects with SMA. Any modest biologic impact of VPA on individual outcome measures over the duration of treatment in this study was clearly outweighed by weight gain. This trial confirmed observations made in the open label study that the effects of age and weight gain with VPA are critical confounding factors that will have to be considered in the design of future clinical trials. Whether or not a subset of younger non-ambulatory SMA subjects might benefit from VPA treatment early in the course of their disease over a longer treatment period remains an outstanding question. Treatment intervention with VPA in non-ambulatory SMA children outside of a clinical study should be discouraged given the lack of clear benefit from this trial, and the potentially serious adverse events associated with VPA treatment. However, these results should be interpreted in the context of the limited treatment duration examined, an issue which should be carefully considered in the design of future clinical trials based on proposed mechanism of therapeutic action. We anxiously await the completion of ongoing studies of VPA in an ambulatory adult population (the VALIANT trial, clinicaltrials.gov ID NCT00481013) to help us further expand our knowledge about possible biologic impact of VPA in subjects with SMA. The ability to obtain strength data from the entire cohort in the VALIANT trial should help to answer outstanding questions about potential biologic effects of VPA in subjects with SMA, and whether this agent merits further study as a therapeutic agent in this disorder.