Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is a progressively neurodegenerative and eventually life-threatening mitochondrial disorder that causes anatomohistopathological and clinical findings . An A-to-G transition mutation at nucleotide position 3243 in mitochondrial DNA (m.3243A>G) is the most common cause of this syndrome . MELAS is distinguishably characterized by the sudden, transient, and recurrent development of stroke-resembling symptoms (headache, nausea/vomiting, visual disturbance/visual field abnormalities, seizures, and impaired consciousness: ictus), with the distribution of brain lesions that are incongruent to the usual vascular territories . The primary cause for stroke-like episodes (SLE) in patients with MELAS, whether mitochondrial cytopathy, angiopathy, or both, remains controversial.
We reported that MELAS is associated with endothelial dysfunction using endothelial-dependent vasodilatation methods by a high-resolution doppler echocardiography . Because patients with MELAS have defective respiratory chain enzyme activities, a high NADH/NAD+ ratio inhibits the nitric oxide (NO) synthetase reaction to cause a decreased production of NO at the endothelial cells or smooth muscle cells in the artery. In addition, asymmetrical dimethyl-arginine (ADMA), a risk factor of ischemic heart disorders, was relatively increased in patients with MELAS , which may lead to a negative effect on the endothelial NO synthetase activity. Indeed, decreased NO metabolite concentrations during SLE and lower NO synthesis rate were observed in patients with MELAS [5–8]. If hyperactive cytochrome c oxidase (COX) may decrease the regional NO concentration, all of the above scenarios lead to the segmental vasodilatation defect especially in the segment of strongly SDH-reactive blood vessel (SSV) regions in the cerebral artery or arterioles. These findings suggest that endothelial dysfunction because of NO deficiency is involved in the pathogenesis of SLE [9▪,10,11]. Low plasma concentration of L-arginine, a potent donor of NO, observed in patients with MELAS [5,8] supports the hypothesis and indicates the therapeutic potential of L-arginine on SLE.
Using L-arginine, we evaluated the potential at acute phase treatment by L-arginine infusion to cure the symptoms associated with SLE [4,5,12], and for prevent the SLE at intrinsic phase by L-arginine oral administration to decrease the severity of SLE, as the OL-MELAS Research Study[13▪].
STUDY DESIGN OF THE OL-MELAS RESEARCH
The OL-MELAS Research is a 9-year clinical study, which integrated the pooled data from two, 2-year, phase III, prospective, multicenter, open-label clinical trials of oral and intravenous L-arginine in patients with juvenile-onset or adult-onset MELAS; the research was conducted at 10 medical institutions in Japan, in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. The protocols were approved by the institutional review board at each site. Oral L-arginine and intravenous L-arginine were purchased commercially. The clinical trials were registered at Center for Clinical Trials, Japan Medical Association (registry numbers: JMACTR-IIA00023 and JMACTR-IIA00025). We compared the results of 9-year clinical study with nationwide epidemiologic study on this patient group available in Japan, which revealed the natural course of patients with MELAS [13▪].
RESULTS OF THE CLINICAL TRIALS OF ORAL AND INTRAVENOUS L-ARGININE
In the clinical trial of oral L-arginine, before–after analysis revealed no statistically significant difference in the MELAS stroke scale, mitochondrial disease severity scores, or migraine severity scores. Namely, L-arginine therapy failed to achieve the endpoints. Nevertheless, the P value was 0.0549 in changes in the MELAS stroke scale, indicating a tendency of oral L-arginine to improve symptoms [13▪].
Regarding the efficacy of intravenous L-arginine, furthermore, the improvement rates of headache and nausea/vomiting at 2 h after completion of the initial intravenous administration were 25.0% (2/8) and 50.0% (3/6; P = 1.0000 each), respectively. Therefore, the statistical hypothesis that ‘the rate of improvement is greater than 30%’ could not be tested for these co-primary endpoints [13▪].
In the clinical trial of intravenous L-arginine study, the primary endpoints could not be achieved. We speculate that the extended window for intravenous administration from ‘3 h’ in our previous clinical study  to ‘6 h’ in the present clinical trial might have disadvantageously attenuated the pharmacologic effects of intravenous L-arginine. Indeed, L-arginine infusion during the hyperacute phase of SLE in MELAS may significantly improve the symptoms, and also completely protect the progression of neuroimaging change from transient to permanent brain atrophy  (Fig. 1).
RESULTS OF THE 7-YEAR FOLLOW-UP STUDY
At the completion of the 2-year clinical trials, both the bedriddenness and mortality rates were 0% despite the progressively neurodegenerative and eventually life-threatening nature of MELAS. At the completion of 7-year follow-up, the bedriddenness rate remained to be 0%; in contrast, the mortality rates of patients with juvenile-onset or adult-onset MELAS were 22.2 and 0.0% in patients with interictal MELAS and were 12.5 and 50.0% in patients with acute MELAS. The 9-year survival of patients who were treated with oral and intravenous L-arginine is significantly improved with those seen in the natural course of MELAS who did not get treated with L-arginine  (Fig. 2). Consequently, the ictuses developed at a plasma arginine concentration of 167 μmol/l or below are recognized in our clinical trials [13▪].
OTHER CLINICAL STUDIES OF L-ARGININE THERAPY FOR STROKE-LIKE EPISODES
In addition to our OL-MELAS research, the eligibility criteria of which included patients carrying m.3243A>G, Ganetzky et al.[14▪] conducted a retrospective analysis of the therapeutic efficacy of intravenous L-arginine for SLE in nine pediatric patients harboring mitochondrial DNA mutations or nuclear DNA mutations except for m.3243A>G. Significant therapeutic benefit of L-arginine therapy was observed in these patients as well, which suggests that treatment with L-arginine is also favorable in patients with various genetic causes. Other clinical studies of L-arginine supplement therapy for MELAS have been done in different countries [15–21], and showed the improvement of the severity of the disease progression in MELAS.
Additionally, several case studies showing the therapeutic efficacy of L-arginine on SLE have been reported. Especially, Hovsepian et al. performed magnetic resonance spectroscopy (MRS) to access the metabolic change in a SLE lesion of patients with MELAS before and after intravenous L-arginine therapy. Serial MRS showed decreased lactate peak and increased N-acetyl aspartate concentration (i.e. neuronal density) after treatment, which corresponded with clinical improvement.
PHARMACOLOGICAL EFFECTS OF L-ARGININE ON THE PATHOPHYSIOLOGICAL MECHANISM OF STROKE-LIKE EPISODES DEMONSTRATED BY USING MOLECULAR IMAGING
The recent progress of molecular imaging with positron emission tomography (PET) and magnetic resonance imaging (MRI) has shed light on the pathophysiological process of SLE and the pharmacological effects of L-arginine.
Brain MRI and single-photon emission computed tomography (SPECT) imaging usually exhibit cerebral hyperemia, vasodilatation, and vasogenic edema because of mitochondrial angiopathy in acute lesions of SLE. In addition, arterial spin labeling (ASL) perfusion MRI clearly shows serial changes in cerebral perfusion related to the disease activity of SLE. Interestingly, we discovered regional cerebral hyper perfusion on ASL imaging in the preclinical phase of SLE in some patients with MELAS  (Fig. 3). These hyperperfused areas developed into acute lesions at the clinical onset of SLE, suggesting that subclinical SLE may have already occurred several months prior to the clinical onset. Because L-arginine can accommodate endothelial dysfunction (i.e. mitochondrial angiopathy), L-arginine therapy is expected to prevent the progression to evident SLE.
In addition to a therapeutic effect on mitochondrial angiopathy as a precursor of NO, L-arginine has a facilitative effect on tricarboxylic acid (TCA) cycle metabolism (i.e. mitochondrial cytopathy) via a conversion to α-ketoglutarate. Our previous study using 11C-acetate PET showed that intravenous administration of L-arginine enhanced TCA cycle kinetics in patients with mitochondrial cardiomyopathy carrying m.3243A>G . The dual pharmacological effects of L-arginine on both mitochondrial angiopathy and cytopathy may have improved the long-term prognosis of patients with MELAS.
The OL-MELAS Research is therapeutically unique in the following achievements: a) specification of the plasma arginine concentration (trough level: 168 μmol/L) at which L-arginine can prevent an ictus that may cause irreversible brain damage, or bedriddeness in the worst case; b) presentation of ‘0%’ in the bedriddenness rate during the strict systematic administration of oral and intravenous L-arginine in the 2-year clinical trials; and c) the obvious suppression and modest retardation of disease progression at the ends of 2-year clinical trials and 7-year follow-up, respectively [13▪]. The L-arginine therapy is listed in the recent review of patient care standards for primary mitochondrial disease [25▪].
We would like to thank Dr Eisuke Inoue for his statistical analysis in the study.
Financial support and sponsorship
This study was funded by the Ministry of Health, Labour and Welfare of Japan (H14-Shouni-006) and Center for Clinical Trials, the Japan Medical Association Center (CCT-B-1803, CCT-C-2001).
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
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