A Phase 1 Study of Oral Vitamin D3 in Boys and Young Men With X-Linked Adrenoleukodystrophy
Abstract
Background and Objectives
There are no therapies for preventing cerebral demyelination in X-linked adrenoleukodystrophy (ALD). Higher plasma vitamin D levels have been linked to lower risk of inflammatory brain lesions. We assessed the safety and pharmacokinetics of oral vitamin D dosing regimens in boys and young men with ALD.
Methods
In this open-label, multicenter, phase 1 study, we recruited boys and young men with ALD without brain lesions to a 12-month study of daily oral vitamin D3 supplementation. Our primary outcome was attainment of plasma 25-hydroxyvitamin D levels in target range (40–80 ng/mL) at 6 and 12 months. Secondary outcomes included safety and glutathione levels in the brain, measured with magnetic resonance spectroscopy, and blood, measured via mass spectrometry. Participants were initially assigned to a fixed dosing regimen starting at 2,000 IU daily, regardless of weight. After a midstudy safety assessment, we modified the dosing regimen, so all subsequent participants were assigned to a weight-stratified dosing regimen starting as low as 1,000 IU daily.
Results
Between October 2016 and June 2019, we enrolled 21 participants (n = 12, fixed-dose regimen; n = 9, weight-stratified regimen) with a median age of 6.7 years (range: 1.9–22 years) and median weight of 20 kg (range: 11.7–85.5 kg). The number of participants achieving target vitamin D levels was similar in both groups at 6 months (fixed dose: 92%; weight stratified: 78%) and 12 months (fixed dose: 67%; weight stratified: 67%). Among the 12 participants in the fixed-dose regimen, half had asymptomatic elevations in either urine calcium:creatinine or plasma 25-hydroxyvitamin D; no laboratory deviations occurred with the weight-stratified regimen. Glutathione levels in the brain, but not the blood, increased significantly between baseline and 12 months.
Discussion
Our vitamin D dosing regimens were well tolerated and achieved target 25-hydroxyvitamin D levels in most participants. Brain glutathione levels warrant further study as a biomarker for vitamin D and ALD.
Classification of Evidence
This study provides Class IV evidence that fixed or weight-stratified vitamin D supplementation achieved target levels of 25-hydroxyvitamin D in boys and young men with X-ALD without brain lesions.
X-linked adrenoleukodystrophy (ALD) is a panethnic monogenic disorder with an incidence of approximately 1 in 15,000 births.1,2 ALD is caused by pathogenic variants in a gene (ABCD1) encoding a peroxisomal surface protein that facilitates the metabolism of very long–chain fatty acids. Approximately two-thirds of male individuals with ALD develop an inflammatory cerebral demyelinating lesions (cerebral ALD) over their lifetime; female individuals are rarely affected by cerebral ALD.3,4 The risk of cerebral ALD lesion formation is highest in childhood, when almost half of male children develop cerebral ALD lesions in the first 10 years of life.3,4 Most lesions originate in the occipital or frontal white matter.5,6 Cerebral ALD lesions share histologic similarities with multiple sclerosis (MS) but do not respond to MS therapies and typically enlarge relentlessly unless hematopoietic stem cell transplantation is initiated while the lesion is still small.7,8 Because ALD is added to a growing number of newborn screening panels worldwide, a new era of surveillance for early signs of cerebral ALD lesions could facilitate clinical trials for preventive therapies.9-11
Although no preventive therapies currently exist for cerebral ALD lesions, epidemiologic evidence from MS suggest that vitamin D insufficiency predisposes to the development of brain lesions, while vitamin D supplementation may confer a reduction in brain lesions and inflammatory markers.12-18 We have identified an association between 25-hydroxyvitamin D deficiency and increased geographic latitude and cerebral ALD lesions.19 Although vitamin D's immunoregulatory effects are well established,20,21 vitamin D has more recently been linked to a range of antioxidant effects, including augmentation of glutathione, an abundant and ubiquitous antioxidant whose deficit has been implicated in ALD pathogenesis.22-28
Although vitamin D insufficiency is a common, important, and treatable childhood condition, treatment guidelines vary widely between professional societies.29,30 Moreover, there are no dosing guidelines for achieving the plasma 25-hydroxyvitamin D levels that are considered optimal for inflammatory disorders such as MS (i.e., 40–80 ng/mL),12-18 which are higher than the levels targeted by existing pediatric dosing guidelines (i.e., 20–35 ng/mL).29,30 Our primary research question was whether fixed and/or weight-based oral vitamin D dosing regimens were able to safely achieve moderately high levels (40–80 ng/mL) of 25-hydroxyvitamin D in male individuals with ALD. Secondarily, we sought to assess brain and blood glutathione as a biomarker for vitamin D in boys and young men with ALD.
Methods
Research Question
The primary objective of our study was to identify a dosing regimen for safely achieving plasma 25-hydroxyvitamin D levels between 40 and 80 ng/mL in boys and young men with ALD. We also hypothesized that oral vitamin D supplementation would increase glutathione levels in the frontal and occipital white matter and whole blood.
Standard Protocol Approvals, Registrations, and Participant Consents
The trial was registered ClinicalTrials.gov (NCT02595489) prior to participant enrollment. The protocol and consents were approved by the institutional review boards at Stanford University and Kennedy Krieger Institute. All participants provided written informed consent.
Inclusion, Exclusion, and Enrollment
Study candidates were identified for screening by the site PIs from within the clinic population at each study site, on referral from external physicians and/or on inquiry from caregiver/family. Participants were eligible for enrollment if they were male individuals with (1) a molecular diagnosis of X-linked ALD, defined as a known ABCD1 gene mutation or the presence of both an ABCD1 variant of uncertain significance and elevated very long–chain fatty acids in plasma, (2) aged between the age of 1.5 and 25 years, without evidence of gadolinium-enhancing cerebral demyelination, (3) had baseline 25-hydroxyvitamin D level less than or equal to 60 ng/mL, and (4) had normal values for serum calcium, creatinine, phosphorus, parathyroid hormone, and urinary calcium:creatinine ratio at screening. Participants were excluded if they had a history of liver or kidney disease, nephrolithiasis, hyperthyroidism, inflammatory bowel disease, medications interfering with intestinal absorption, or had contraindication to completing a brain MRI every 6 months. Participants were enrolled at 2 sites: Lucile Packard Children's Hospital and Kennedy Krieger Institute. Note that our study opened in 2016, prior to widespread adoption of newborn screening. Although we initially restricted participant age to 12 yo, after a slow start to enrollment, we expanded our upper age limit to 25 yo to facilitate enrollment.
Trial Design
This study was originally conceived as a single-arm dose escalation study designed to assess 2 fixed daily doses of vitamin D (2,000 IU vs 4,000 IU). However, an interim safety analysis suggested that younger and smaller patients were overshooting the upper target and safety threshold for 25-hydroxyvitamin D (i.e., 80 ng/mL). In response, the PI, in consultation with the study's Data Safety Monitoring Board, modified the dosing protocol to a weight-stratified protocol that stratified dosing regimens according to each participant's bodyweight during enrollment and reduced the aggressiveness of the dose titration threshold at the 6-month visit from 60 ng/mL to 40 ng/mL. The switch from a fixed-dose regimen to a weight-stratified regimen took place with the enrollment of the 12th study participant.
After enrollment of all study participants, the primary study site mailed a 3-month supply of sublingually dissolvable 1,000 IU (25 µg) vitamin D3 tablets. Participants were instructed to administer the specified number of tablets once daily. Drug was manufactured by Continental Vitamin Company. One participant on the weight-stratified regimen was provided with 1,000 IU tablets from a separate manufacturer (Renzo's Vitamins, Inc) to conform with a vegan diet.
The dosing regimens and conditional escalation parameters are summarized in Table 1. In brief, all participants in the fixed-dose group were assigned to an initial 6-month period at 2,000 IU daily. If the participant's plasma 25-hydroxyvitamin D levels remained <60 ng/mL at the 6-month study visit, the dose was increased to 4,000 IU daily. All participants in the weight-stratified dose group were assigned to either 1,000 IU, if baseline weight <20 kg, or 2,000 IU, if baseline weight >20 kg. If the participant's plasma 25-hydroxyvitamin D levels remained <40 ng/mL at the 6-month study visit, the dose was increased to either 2,000, 3,000, or 4,000 IU, depending on baseline weight. Participants who did not qualify for dose escalation remained at their starting dose. Prespecified dose reduction parameters required dose reductions in all participants with plasma 25-hydroxyvitamin D levels exceeding 80 ng/mL. To account for variations UV exposure in determining vitamin D levels, skin tone was scored on a scale of 1 (very fair) to 6 (black); sun exposure was assessed at baseline, 6 months, and 12 months, as assessed by a parent and affirmed by a study coordinator. The study protocol and statistical analysis plan are available in eSAP1.
Primary Outcome
The primary outcome was defined as the proportion of participants who achieved a plasma 25-hydroxyvitamin D level in the target range (40–80 ng/mL) at 6 months and 12 months without a triggered dose reduction. Vitamin D levels were measured by the clinical laboratories at participating institutions, both of which used tandem mass spectroscopy.
Safety Criteria and Adverse Events
Safety was assessed using clinical, laboratory, and radiologic measures. Prescription medications and over-the-counter supplement regimens were recorded at baseline and at quarterly assessments. Participants were monitored for adverse clinical events through quarterly surveys to assess for signs and symptoms associated with hypercalcemia and general adverse events. We monitored for deviations in laboratory markers associated with excessive vitamin D intake: plasma 25-hydroxyvitamin D levels and calcium were measured quarterly while urine calcium:creatinine levels were assessed every 6 months. To account for variations in UV exposure, time outdoors was assessed at each study visit by administering a survey to the parent/caregiver. Brain MRIs were completed every 6 months to screen for the appearance of cerebral ALD lesions. Prespecified events would trigger dose reduction of daily vitamin D supplementation; specified events included plasma 25-hydroxyvitamin vitamin D levels >80 ng/mL and persistently elevated urine calcium:creatinine. If urine calcium:creatinine ratio was elevated at the 6-month screening, it triggered 3 subsequent monthly measures with dose reduction implementation only if 2 of those 3 measures remained elevated (i.e., persistent elevation).
Measurement of Glutathione Levels in the Brain
Single-voxel proton magnetic resonance spectroscopy (MRS) was attempted for all participants at baseline, 6months, and 12 months. In vivo MRS data were acquired on either a GE or Philips 3T scanner using the MEGA-PRESS sequence.31,32 Occipital and frontal white matter regions were selected because they are the typical locations of cerebral ALD lesion formation.6 For each participant, data were sequentially acquired from two 2 × 6 × 2-cm voxels located centered in the occipital and frontal white matter and frontal lobes. The GSH editing pulses for the MEGA-PRESS sequence were 180° Gaussian pulses, of 20 millisecond duration, and applied at 4.56 and 7.5 ppm. A total of 256 transients were acquired for each voxel using a 5000 Hz spectral bandwidth, 2048 data points, TE/TR = 80 ms/2 s, and a total scan time of 8:40 minutes. Glutathione (GSH) levels were quantified by fitting the 2.95 ppm peak in the edited spectrum and expressed as a ratio to total creatine (GSH/tCr).33 We centralized our spectroscopy analysis to reduce intersite variability. A designated expert (M.G.) was responsible for assessing the quality of the acquired spectra. For each measure, criteria for inclusion in final analyses were as follows: (1) adequate shimming with linewidth <18 Hz, (2) flat baseline, and (3) discernible GSH peak at 2.95 ppm in the edited spectrum.
Measurement of Glutathione Levels in the Whole Blood
Blood collection, preparation, and analysis were conducted using a Sciex 4500 triple quadrupole mass spectrometer and Analyst software, as previously described by Moore et al.34 In brief, blood samples were refrigerated immediately after collection and processed within 24 hours by adding a precipitating solution of sulfosalicylic acid containing N-ethylmaleimide (NEM). Samples were then stored at −80°C. During analysis, samples were processed in a single batch using LC-MS/MS with stable isotope internal standards of GSH (GSH-13C,15N) and GSSG (GSSG-13C,15N) (Cambridge Isotope Laboratories, Inc, Tewksbury, MA) for quantitation. GSH-NEM and GSSG ions and fragments were monitored using transitions m/z 433>304 and m/z 613>355, respectively. Stable isotope internal standards were monitored as m/z 435>306 (GSH-13C, 15N-NEM) and m/z 617>359 (GSSG-13C,15N).34
Statistical Analysis
All participants who received at least 1 dose of study drug were included in the safety analysis and in the primary analysis. Appropriate summary statistics are used to present demographic and primary outcome data, overall and by dosing regimen. We used Wilcoxon signed rank sum analysis to compare change in vitamin D and glutathione levels between baseline and 6-month and 12-month time points. We used the Spearman correlation statistic to assess correlations between variables.
Data Availability
Study protocol, statistical analysis plan, and fully deidentified data related to age, weight, vitamin D dose, laboratory results, and adverse events will be shared on reasonable request from clinical and scientific investigators for the purposes of understanding the pharmacokinetic and pharmacodynamic effects of vitamin D.
Results
Study Population
Among the 22 individuals screened, 21 were enrolled (Stanford, n = 18; Kennedy Krieger, n = 3). The CONSORT Flow Diagram is presented in Figure 1. One participant was excluded because of an enhancing brain lesion on screening MRI. All participants completed all visits without loss to follow-up. The final 2 participant visits were completed remotely in March and June 2020 because of travel restrictions associated with a global coronavirus pandemic. Between October 2016 and December 2018, all participants (n = 12) were started on a fixed dose of 2,000 IU of vitamin D daily. From January 2019 through the end of recruitment in June 2019, the remaining participants (n = 9) were assigned to a weight-stratified dosing regimen with an initial 6-month dosing period of either 1,000 IU or 2,000 IU daily, as outlined in Table 1.
Demographic information and primary outcomes are summarized in Table 2. Overall, the median age (and interquartile range) was 6.7 years (9.2), median baseline weight was 20 kg (29.5), and median baseline 25-hydroxyvitamin D level was 29 ng/mL (13). Two participants were older than 18 years (19 yo and 22 yo) during enrollment. The fixed-dose group was somewhat younger (median age 6.4 vs 9 years), lighter (median weight 19.9 kg vs 33.2 kg), and had lower baseline vitamin D levels (median 28.5 vs 33.6 ng/mL) than the weight-stratified group. Two patients in each dosing group had 25-hydroxyvitamin D levels greater than 40 ng/mL at baseline with highest baseline level of 46 ng/mL. Participants were primarily White, non-Hispanic (n = 15, 71%), which was similar across groups. Skin tones were similar between dosing groups. Weekly hours spent outdoors was stable across participants in fixed-dose group (eFigure 1, links.lww.com/NXG/A586). The weight-stratified dosing group, however, was affected by a surge in enrollment such that 8 of 9 participants enrolled in the first quarter of 2019, when sun exposure and vitamin D levels are at their annual nadir (eFigure 1). This enrollment imbalance was reflected a peak in outdoor sun exposure (and to a less extent, 25-hydroxyvitamin D levels) at the midstudy point among weight-based participants.
Primary Outcome and Dose Response
At 6 months, 92% (n = 11) in the fixed-dose group and 78% (n = 7) in the weight-stratified group achieved target 25-hydroxyvitamin D levels (40–80 ng/mL) (Table 2). Half of the participants in the fixed-dose group (n = 6) qualified for dose escalation (level <60 ng/mL); 1 participant in the weight-stratified group qualified for dose escalation (level <40 ng/mL). One participant in the weight-stratified group did not complete a 6-month vitamin D level and remained on starting dose. For this participant, we used the level drawn at the 3-month time point, which was 35 ng/mL (i.e., below target), as the closest proxy for the 6-month time point.
At 12 months, 67% of both groups achieved target levels without a triggered dose reduction (n = 8 in fixed dose; n = 6 in weight stratified). Table 2 highlights the outcomes at the prespecified time points of 6 and 12 months. Figure 2 displays quarterly measures of plasma 25-hydroxyvitamin D levels during the 12-month study period. Overall, median levels were similar between groups except at months 9 and 12 when plasma 25-hydroxyvitamin D levels were higher in the fixed-dose group (eFigure 1, links.lww.com/NXG/A586). The marked decrease in 25-hydroxyvitamin D levels for 2 participants between 9 months and 12 months was due to a protocol-specified response dose reduction in response to 25-hydoxyvitamin D levels >80 ng/mL (Figure 2B). A post hoc dose-response analysis suggests that participants who received between 60 and 120 IU/kg/d tended to achieve plasma 25-hydroxyvitamin D levels in our target range after 6 months of supplementation (Figure 2C). We also found that, independent of weight, younger male individuals exhibited a less robust dose response (Figure 2D).
Compared with baseline, plasma 25-hydroxyvitamin D levels were significantly higher at all subsequent study time points (A). Most participants achieved and maintained vitamin D levels within the specified target range of 40–80 ng/mL, indicated by gray background; 4 patients exceeded the upper threshold (red dots) (B). Among all participants, daily dosing regimens between 60 and 120 IU/kg were likely to yield plasma 25-hydroxyvitamin D levels in the target (C). The dose response varied according to age, such that younger patients were less responsive to similar doses even when accounting for bodyweight (D).
Safety and Adverse Events
Adverse events (AEs) occurring over the 12-month study period are summarized in Table 3. Both predefined and general adverse events occurred more frequently among participants in the fixed-dose arm. Safety laboratory levels are summarized in Figure 2 (plasma 25-hydroxyvitamin D) and eFigure 1 (links.lww.com/NXG/A586) (serum calcium and random urinary calcium:creatinine). Among all safety laboratory measures (n = 202) of plasma 25-hydroxyvitamin D, serum calcium, and urine calcium:creatinine, we observed 8 laboratory elevations in 6 participants, all of whom were assigned to the fixed-dose regimen (eFigure 1). Four participants had 25-hydroxyvitamin D levels above predefined safety threshold of 80 ng/mL with 2 events among participants receiving a daily dose of 2,000 IU daily and 2 receiving 4,000 IU daily (Figure 2; eFigure 1). Similarly, transient elevations in urine calcium:creatinine were also observed in the fixed-dose arm (n = 2 at 2,000 IU; n = 1 at 4,000 IU) daily (eFigure 1). Serum calcium levels remained in the normal range for all participants (eFigure 1). Brain MRI and neurologic assessments also remained normal in all participants.
One participant, assigned to the fixed-dose regimen, was hospitalized for an asthma attack, which was deemed unrelated to study drug. One participant, assigned to the weight-stratified regimen, developed adrenal insufficiency, a common manifestation of ALD. Among the other AEs, participants in the fixed-dose group experienced more fever, headaches, myalgia/subjective weakness, and respiratory and gastrointestinal AEs; however, this may be due to differences in study period (overlap with flu season) and/or differences in age between the 2 dosing groups. A post hoc analysis did not show a correlation between daily dose and adverse events (eFigure 1, links.lww.com/NXG/A586). Other AEs experienced were allergic reaction, rash, insomnia, and lightheadedness.
Participant use of prescription medications and over-the-counter supplements are summarized in supplemental data. Four participants added medications or supplements during the study. Two patients added an over-the-counter nutritional supplement during the 12-month study period (vitamin C and omega-3 fatty acid supplement, n = 1; probiotic supplement, n = 1), both at the discretion of a parent. Two patients added a daily prescription medication during the study period related to a new diagnosis (prednisone for adrenal insufficiency, n = 1; inhaled steroid for asthma, n = 1).
Glutathione Levels in the Brain and Blood
Spectroscopy was acquired at 57 of 63 study time points. Six acquisitions could not be completed because of travel restrictions (n = 4) or software error (n = 2). Among the 114 acquired spectra, 2 from the occipital white matter and 13 from the frontal white matter did not meet quality threshold and were excluded. Voxels in the frontal white matter were adjacent to sinuses with poor B0 inhomogeneity.
Brain GSH/Cr levels correlated across frontal and occipital white matter regions within participants at each visit suggesting good internal validity (eFigure 2, links.lww.com/NXG/A586; Spearman r = 0.43; p = 0.005). Brain glutathione levels in both the occipital white matter (Figure 3) and frontal white matter (Figure 3) were higher at 12 months compared with those at baseline. Brain glutathione levels did not correlate with plasma vitamin D levels in either brain region (eFigure 2).
We used magnetic resonance spectroscopy to measure brain glutathione levels in the frontal and occipital white matter where cerebral ALD lesions typically begin (A). GSH/tCr ratios increased between baseline and 12 months in the occipital white matter and frontal white matter but not in the plasma (B and C). Glutathione levels did not increase significantly in the whole blood (D). In B–D, median (dot) and interquartile range (bars) are shown for all participants for whom a baseline measure was available; The Wilcoxon matched-pairs signed rank analysis was used for all comparisons between baseline and 6-month and 12-month timepoints.
Plasma samples for glutathione analysis were available from 49 time points. Glutathione levels were not significantly different between baseline and 6-month and 12-month timepoints and did not correlate with vitamin D levels (Figure 3; eFigure 2, links.lww.com/NXG/A586). The trends in brain and plasma glutathione levels were similar between fixed and weight-stratified dosing groups (eFigure 2).
Classification of Evidence
This study provides Class IV evidence that fixed or weight-based vitamin D supplementation achieved target levels in boys and young men with ALD without brain lesions.
Discussion
In this phase 1 study of oral vitamin D3 supplementation in 21 boys and young men with ALD aged 2–22 years, a weight-stratified dosing regimen achieved similar rates of 25-hydroxyvitamin D target levels as a fixed-dose regimen and had fewer safety laboratory elevations and fewer adverse events overall. No participants experienced clinical adverse events attributable to vitamin D. Glutathione/creatinine ratios in the frontal and occipital white matter increased during the 12-month study period.
Vitamin D dosing regimens in children have historically targeted levels >20 ng/mL to protect against rickets with comparatively little focus on achieving higher levels that are more commonly sought for the treatment of disorders such as MS.12-18 Our study suggests that both our tested regimens were effective at achieving our target level of 25-hydroxyvitamin D. Our dose-response curve suggests a linear relationship between the daily dose (in IU/kg) and subsequent plasma 25-hydroxyvitamin D level (see Figure 2). Due to our small sample size, our dose-response curve does not incorporate sun exposure, season, or latitude, which can also affect vitamin D level. The peak in plasma 25-hydroxyvitamin D levels at the midstudy time point in the weight-stratified group may be due to an imbalance in sun exposure that resulted from an enrollment surge in the 1st quarter of 2019. This produced a midstudy peak in sun exposure during the warmer months of summer/fall when 25-hydroxyvitamin D levels are typically highest.
Our wide age range (20 months–22 years) allowed us to assess the impact of age, independent of weight, on vitamin D dose-response. We found that younger boys required more vitamin D to achieve the same dose effect. While our findings confirm previous observations in adolescent and adult populations, we also extend this observation into a younger age group.35,36 The underlying mechanisms for this phenomenon are unknown but may be related to higher levels of physical and metabolic activity observed among children, particularly toward bone accretion.37
The fixed-dose group recorded more laboratory deviations and nonspecific adverse events than the weight-stratified group. Several participants assigned to our fixed-dose regimen achieved 25-hydroxyvitamin D level above our target threshold, although none manifested hypercalcemia or associated signs and symptoms. The higher plasma levels in the fixed-dose regimen resulted from higher supplemental dose exposures, as seen in Figure 2. The adverse events reported in both groups were nonspecific and common among children (e.g., abdominal or respiratory symptoms). Although none of these reported adverse events were considered attributable to vitamin D supplementation, it is possible that higher doses of vitamin D may predispose to mild but common and nonspecific ailments. One participant in the fixed-dose group was hospitalized for an asthma attack, which was not attributed to study drug. Although vitamin D supplementation has a demonstrated record in reducing the risk of respiratory infections and asthma attacks, this effect could be dose- dependent.38-41 Urine calcium:creatinine levels were elevated in 3 individuals receiving the fixed-dose regimen but were asymptomatic and self-resolved without intervention.
Symptoms of vitamin D toxicity, which include vomiting, weakness, and anorexia, overlap with symptoms of adrenal crisis, which is a common complication of ALD. Vitamin D toxicity is caused by hypercalcemia, which distinguishes it from adrenal insufficiency. No instances of vitamin D toxicity or adrenal crisis were reported during the study period. One participant in the weight-stratified group developed ALD-related adrenal insufficiency that was identified during routine laboratory surveillance.
Glutathione is a ubiquitous antioxidant whose deficiency predisposes to cellular injury and death in the brain and immune system.42-44 Glutathione has been previously implicated as a therapeutic biomarker for ALD. Low glutathione levels have been repeatedly observed in boys and men with ALD.27,28,45,46 Furthermore, the glutathione-boosting supplement, N-acetyl cysteine, has been linked to improved clinical outcomes in cerebral ALD.46 In this study, median levels of glutathione in the brain increased over the 12-month study period. Median levels of glutathione in whole blood were higher at 6 and 12months but were not statistically different than those at baseline. We did not observe a correlation between plasma 25-hydroxyvitamin D and plasma or brain glutathione levels, which could be explained by our small sample size and/or the nonlinear pharmacodynamics of 25-hydroxyvitamin D biology.12,38,40 The absence of a placebo control limits causal inference between vitamin D and glutathione in our sample; however, our observations are consistent with a recent and growing body of randomized placebo-controlled clinical trials linking vitamin D supplementation with increased glutathione levels in blood.22-25 Some evidence suggests vitamin D and glutathione may be synergistic, with supplementation of each boosting the other.47 Future studies seeking to boost glutathione as well as vitamin D levels might consider dietary supplementation of both vitamin D and glutathione.
Among the limitations of our study were our small sample size and lack of a placebo control. Our 12-month study was also not designed to assess the potential for long-term risks associated with high-dose vitamin D supplementation. Although previous studies suggest spectroscopy results can be reliably reproduced across clinical sites and machine vendors, technical differences may persist across sites.48 We did not account for the possibility of age-related changes in brain glutathione during childhood although significant changes over a 1-year period seem unlikely. We also did not quantify medication adherence in our study, which limits our ability to adjust for varying levels of adherence for future studies of vitamin D supplementation in this population.
There are currently no preventive therapies for cerebral ALD. The advent of newborn screening for ALD has resulted in a growing number of presymptomatic children at risk of cerebral ALD. This has increased interest and feasibility for developing preventive therapies. All current therapies for cerebral ALD are indicated only for active lesions. This includes the current standard of care that uses allogeneic hematopoietic stem cell transplant as well as candidate therapies, such as lentiviral-mediated gene-therapy using autologous stem cells, which is currently pending regulatory approval in the United States.49,50 Lorenzo oil, a mix of erucic and oleic acids, had been previously studied but was poorly tolerated and failed to demonstrate a clear clinical benefit.51,52 Although none of our participants have manifested ALD lesions during this writing, our study is insufficiently powered to offer an efficacy signal for vitamin D supplementation as a preventive therapy for ALD. However, data from our studies and others show vitamin D supplementation is well tolerated and can be safely administered. These are important criteria for any potential preventive therapy for cerebral ALD, where most of the individuals at risk will not develop brain lesions for decades, if at all.
Although vitamin D is cheap, widely available, and relatively safe, a clinical trial is required to formally determine its efficacy, and clarify its potential mechanism, as a preventive therapy for cerebral ALD. Such a study would also allow the opportunity to assess the effect of vitamin D on adrenal function in ALD. Given the stakes, many families will likely use vitamin D or sun exposure even without efficacy data. However, without a formal efficacy trial, vitamin D could, if effective, confound trials for future novel preventive therapies for ALD. Moreover, if proven effective in a trial, vitamin D could inspire new therapies for ALD. A prevention trial in ALD will present a design challenge due to the long period of follow-up required and known challenges associated with randomization in vitamin D studies, particularly when the target outcome is severe.53
Future studies of vitamin D supplementation in ALD should consider implementing a weight-stratified dosing regimen and focus on the potential efficacy of vitamin D in preventing the formation of brain lesions. Although the weight-stratified regimen underachieved regarding participants achieving the minimum target threshold of 40 ng/mL, it elicited fewer safety risks while still increasing vitamin D levels. Our proposed minimum threshold was established based on prior data suggesting a level of 40 ng/mL as sufficient to suppress PTH and reduce the future risk of developing MS in non-ALD adults.54 Whether this threshold, or any vitamin D threshold, is clinically meaningful in preventing ALD lesions remains uncertain and was beyond the scope of our study.
In summary, we find that daily oral dosing of vitamin D, particularly when stratified by weight, can safely achieve plasma 25-hydroxyvitamin D levels between 40 and 80 ng/mL in most boys and young men with ALD. Our findings also implicate brain glutathione as a candidate biomarker for vitamin D supplementation in ALD, although a causal association was not established in this study. These findings are of interest to future studies assessing the role of vitamin D in preventing brain lesions in ALD.
Glossary
- AEs
- adverse events
- MRS
- magnetic resonance spectroscopy
Acknowledgment
The authors thank the study participants and their families and Julianne Jorgensen, Sweta Patnaik, Simone Schubert, and Fe Gibbons who supported study logistics. The authors also thank the members of Data Safety Monitoring Board, including Drs Paul Fisher, Sejal Shah, and Joseph Rigdon.
Appendix Authors
Footnote
Class of Evidence: NPub.org/coe
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Information & Authors
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Published In
Neurology® Genetics
Volume 9 • Number 2 • April 2023
Copyright
Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
Publication History
Received: June 1, 2022
Accepted: January 12, 2023
Published online: March 31, 2023
Published in print: April 2023
Disclosure
K. Van Haren has received consulting fees from Bluebird bio, Minoryx, Viking Therapeutics, Poxel, and Orpheris and participated in advisory boards for Poxel, Viking, ALD Connect, and the United Leukodystrophy Foundations all outside the submitted work; G.V. Raymond has received consulting fees from Bluebird bio, Viking Therapeutics, and Minoryx for therapy development outside the submitted work; A. Fatemi has received consulting fees from Minoryx, Viking, Autobahn, Vertex, Poxel, Aevi, and Calico for scientific advising outside the submitted work, has participated in a DSMB for Bluebird Bio, and is a board member at ALD Connect. K. Cunanan, A. Avni, M. Gu, D. Pena, L.C. Chromik, M. Povazan, N.C. Rossi, J. Winterbottom, J. Goodman, V. Sundaram, T. Cowan, G.M. Enns, E. Waubant, L. Steinman, P.B. Barker, and D. Spielman have no disclosures. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NG.
Study Funding
This study was funded by NIH/NINDS K23NS087151.
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