Introduction
X-Adrenoleukodystrophy (X-ALD) is the most frequently inherited leukodystrophy, with a minimum incidence of 1 in 14,700 live births [
1]. The gene mutated in the disease (
ABCD1) encodes the ALD protein (ALDP), a peroxisomal transporter that imports very long-chain fatty acids (VLCFA) into the peroxisome for degradation [
2,
3]. Elevated plasma VLCFA is a pathognomonic biomarker for this disorder, although it lacks predictive value for disease severity or progression [
4]. Different phenotypes have been described in X-ALD. Virtually all patients who reach adulthood develop the most frequent phenotype, adrenomyeloneuropathy (AMN), in their third and fourth decades of life. The initial symptoms are limited to the spinal cord and peripheral nerves. Patients develop progressive spastic paraparesis, sensory ataxia with impaired vibration sense, sphincter dysfunction, pain in the legs, and impotence [
5]. Neurophysiological findings reveal axonal neuropathy and disturbances in evoked potentials [
6]. Brain magnetic resonance imaging (MRI) results are often abnormal, mainly affecting the corticospinal tract [
7]. Approximately 80% of patients suffer from adrenocortical insufficiency [
5]. Women carriers often present mild myelopathy after the fourth decade of life [
8]. The most severe disease phenotype, cerebral childhood adrenoleukodystrophy (cALD), presents rapidly progressive inflammatory brain demyelination with a lethal outcome unless diagnosed early and treated with hematopoietic bone marrow transplant [
9] or the new available hematopoietic stem cell therapy [
10].
At present, there is no satisfactory treatment for patients with AMN [
11]. Oxidative stress is a major factor driving X-ALD pathogenesis [
12‐
17] and appears very early in life [
12]. At the origin of this, redox imbalance is a combination of increased production of mitochondrial radical oxygen species (ROS) caused by the excess of VLCFA [
18], together with a deficient endogenous antioxidant response [
13,
19]. The combination of antioxidants used herein, α-tocopherol (vit E), N-acetylcysteine (NAC), and α-lipoic acid (LA), has been shown to be synergistic
in vitro, halting the clinical progression and axonal damage in a murine model of AMN [
20]. Moreover, this combination ameliorates key metabolic pathways contributing to the pathogenetic cascade, such as energy production [
21], mitochondrial biogenesis and respiration [
22], proteostasis [
23,
24], and endoplasmic reticulum stress [
25]. These findings indicate that oxidative damage is a very early driver of pathogenesis, whereas providing a strong rationale for clinical translation. To date, no biomarkers of disease progression have been identified in X-ALD, as the VLCFA levels in plasma do not correlate with the onset or severity of symptoms. The present work contributes to filling this knowledge gap.
Discussion
This open-label trial was primarily envisaged to translate the positive preclinical results obtained on a mouse model of adrenomyeloneuropathy treated with the same combination of antioxidants, by identifying a safe, well-tolerated dose that would achieve biological efficacy in patients. The design also aimed to identify and validate meaningful biomarkers to monitor biological efficacy or target engagement to guide larger, randomized placebo-controlled studies. We believe this objective was successfully achieved and provide herein a panel of quantitative oxidative lesion markers and lipid inflammatory mediators in the plasma, cytokines in the plasma and PBMCs, neopterin in the CSF, and 8-oxo-dG in the urine, which may be useful for monitoring future treatments and disease progression. Most importantly, we report the normalization of several inflammation markers upon antioxidant treatment, in addition to markers of oxidative damage, indicating that redox and inflammatory homeostasis are deeply intertwined in this disease.
Indeed, the pro-inflammatory derivatives of arachidonic acid (TBX2, 12S-HETE, and 15S-HETE) and several pro-inflammatory cytokines and chemokines (IL-8, TNF,
IFNA2, IL-4, IL-36A, and
CCR3) exhibited greatly decreased or normalized values after the antioxidant treatment. Of note, the treatment increased the levels of the protective cytokine adiponectin, which was previously reported to be significantly diminished in a small cohort of subjects with AMN [
27] and a mouse model [
44]. Furthermore, antioxidant treatment increased the already-elevated levels of the anti-inflammatory cytokine
IL-10, suggesting a protective effect of the antioxidants in this series. Although AMN has long been considered to be a non-inflammatory disorder—as opposed to cerebral childhood ALD or adult-onset cerebral AMN—mounting evidence indicates that pro-inflammatory cascades are significant although controlled under tight regulatory cues [
27]. On this pro-inflammatory background, which may represent the “default form” of the disease, additional genetic or environmental hits, such as brain trauma, may provoke conversion to the rapidly progressive and lethal cerebral ALD or AMN forms [
45].
In this trial, we have learned that redox homeostasis is directly linked to inflammatory status in this disease, so that antioxidants may help to ameliorate the chronic, low-grade pro-inflammatory state that characterizes AMN and, as a consequence, modify or slow disease progression. It is tempting to speculate that our results may strengthen the rationale for the improved outcomes of hematopoietic stem cell transplantation in the presence of NAC [
46] and argue for the use of antioxidants as a companion treatment also for the most severe cerebral inflammatory forms of the disease, cALD. In addition, using penalized regression methods, we discovered markers with predictive value for the response to treatment. Indeed, the levels of MCP1 negatively affected the percentage of improvement in the walked distance in the 6MWT, indicating that several subjects showed a correlation between an improvement in walking distance and a decrease in MCP1 levels following treatment. The reverse was also true for patient 7, who showed increased MCP1 levels and a poorer 6MWT result in the final visit. Thus, the response of MCP1 to the antioxidant treatment (by decreasing its levels in comparison with baseline) may be considered a candidate predictor of disease progression in this cohort. Of note, MCP1 has been shown to be increased in the CSF of children with cALD [
47], underscoring the interest in this biomarker across discordant disease phenotypes. This chemokine is the most potent inducer of the signal transduction pathways leading to monocyte transmigration [
48,
49], and accumulating evidence suggests that MCP1 and its receptor, CCR2, may be prime targets to combat neuroinflammation [
50]. The same conclusion applies to 15S-HETE, a lipid mediator of inflammation and potent agonist of PPARbeta/delta receptors [
51,
52], which showed an inverse correlation with an improvement in walking distance in this series. Thus, confirmation of the predictive value of MCP1 and 15S-HETE in additional, larger cohorts is warranted.
With all the natural, obliged reservation that emanates from a small open trial, it is intriguing that the treatment appeared to confer a statistically significant clinical benefit in several parameters. We detected a significant improvement in 6MWT, particularly in subjects with higher EDSS scores. The 6MWT appeared to be sufficiently sensitive to discern any possible differences in the distances walked and to be a good clinical outcome measure for the follow-up of moderately or severely affected subjects during the treatment.
Although a placebo effect cannot be excluded, the robust improvements in several biological outcome measures, as well as some mild but statistically significant amelioration in independent clinical and neurophysiological outcomes, may suggest an intriguing positive signal. Furthermore, this work provides a series of markers to monitor the biological effects of prospective trials, some of which, such as MCP1, have the potential to correlate with disease progression and clinical efficacy. Likewise, the 6MWT may be meaningful to monitor the clinical treatment efficacy. The observed tolerability, safety, and signs of clinical benefit of this pilot study may warrant the long-term use of these or other next-generation antioxidants [
53] in a phase III, double-blind, randomized, placebo-controlled clinical trial with a larger number of subjects. The safety and tolerability of the doses used may facilitate extension of similar antioxidant combinations to other diseases that share both axonal degeneration as a significant component of clinical progression and redox imbalance as a primary or early contributing pathogenic factor [
54,
55].
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