Carlo Viscomi, Alberto B Burlina,   Imad Dweikat, Mario Savoiardo,    Costanza Lamperti, Tatjana Hildebrandt,Valeria Tirantitiranti@istituto-besta.it& Massimo

 

Ethylmalonic encephalopathy is caused by mutations in ETHE1, a mitochondrial matrix sulfur dioxygenase, leading to failure to detoxify sulfide, a product of intestinal anaerobes and, in trace amounts, tissues. Metronidazole, a bactericide, or N-acetylcysteine, a precursor of sulfide-buffering glutathione, substantially prolonged the lifespan of Ethe1-deficient mice, with the combined treatment being additive. The same dual treatment caused marked clinical improvement in five affected children, with hardly any adverse or side effects.

 

Main

Accumulated sulfide (H2S) due to ETHE1 impairment (Supplementary Fig. 1) inhibits cytochrome c oxidase, short-chain acyl CoA dehydrogenase (COX, also known as SCAD)1 and possibly other enzymatic activities as well, damages intestinal mucosa and endothelia and alters the vessel tone2, resulting in hemorrhagic diarrhea, episodes of petechial purpura with edematous acrocyanosis and progressive neurological failure, which are the hallmarks of ethylmalonic encephalopathy. H2S is mainly released by bacterial anaerobes of the large intestine, but endogenous production in trace amounts occurs in virtually all organs2, making H2S a physiological 'gasotransmitter'3.

We used an Ethe1−/− mouse model1 to test a therapeutic strategy aiming at decreasing bacterial production of H2S and neutralizing toxic H2S. Metronidazole4 is a bactericidal nitroimidazole effective against anaerobic bacteria. We administered the recommended daily dosage for children, 30 mg per kg body weight, by two intraperitoneal injections in six Ethe1−/− mice from post-partum day 18 (P18). The median survival shifted from 27 to 54.5 d (Fig. 1a).

Figure 1: Clinical, morphological and biochemical features of Ethe1−/− mice and human subjects.

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(a)   Kaplan-Meyer survival probability curves of untreated versus treated Ethe1−/− mice. The P values refer to the results of the log rank test. NAC, N-acetylcysteine. (b) COX histochemical reaction in the large intestine of P30 mice. In the untreated Ethe1−/− specimen (left) COX is absent in mucosal cells facing the lumen and reduced in cryptal cells, whereas a comparable reaction is present in treated Ethe1−/− (middle) and control (right) Ethe1+/+ specimens. (c) Neuroradiological and cutaneous features in patients before and during metronidazole and N-acetylcysteine combined treatment. Top left, fluid attenuated inversion recovery MRI of a transverse section of subject 1's brain at 28 months of age, one month before starting the treatment; top right, the same sequence as at left performed at 36 months of age, 7 months after starting the treatment. Compared to the image at left, the right one shows reduction of brain atrophy (see the difference in size of the sylvian fissure, indicated by an arrow, and lateral ventricles), regression of white matter changes (see the encircled area of the centrum semiovale) and increased thickness of the genu of the corpus callosum (bracket), but increased abnormalities in the neostriatum (asterisk). Middle left, severe plantar acrocyanosis and swelling in subject 2 before treatment (age 30 months); middle right, the same frame shot as at left of subject 2 after treatment (age 36 months) shows the resolution of both acrocyanosis and edema. Bottom left, petechial rash in subject 3 (age 17 months); bottom right, the rash resolved after treatment (age 23 months). (d) Plasma EMA before and after treatment. Left, serial measurements of plasma EMA in subject 1 before (mean value = 10.5 ± 1.5 μM) and after (mean value = 4.2 ± 0.3 μM) the start (arrow) of combined metronidazole and N-acetylcysteine treatment (unpaired Student's t test P = 3.65 × 10−6). Right, plasma EMA in subjects 1–5 before and after the start of metronidazole and N-acetylcysteine treatment. **P = 0.003 (paired, two-tailed Student's t test). Informed consent was obtained from the human subjects' parents for the use of biological material from all human subjects. Mouse studies were approved by the Ethics Committee of the Foundation 'Carlo Besta' Neurological Institute, in accordance with guidelines of the Italian Ministry of Health. The use and care of mice followed the Italian Law D.L. 116/1992 and the EU directive 86/609/CEE. Standard food and water were given ad libitum.

(b)   Mitochondrial glutathione (GSH)5 can accept the sulfur atom of H2S through the action of sulfide-CoQ reductase6 (Supplementary Fig. 1), forming nontoxic GSSH persulfide (which can, in turn, become a substrate of ETHE1 (refs. 1,6)). We added N-acetylcysteine7, 8, 9, a cell-permeable precursor of GSH10, to drinking water (1%, pH 7.0) in a second group of six Ethe1−/− mice starting on P18, resulting in a daily intake of 5 g per kg body weight. The median survival shifted to 49 d (Fig. 1a). Combined treatment led the median survival of a third group of six Ethe1−/− mice to shift to 71.5 d, indicating an additive effect (Fig. 1a). Although the serum concentration of thiosulfate, a stable and readily measurable index of H2S, was still much higher than normal, it significantly (P < 0.0015, two-tailed, unpaired Student's t test) decreased in treated Ethe1−/− mice at P30 compared to untreated Ethe1−/− mice (Supplementary Fig. 2), suggesting that the combined therapy acted specifically against accumulation of H2S and H2S-related compounds. Quantitative motor tests performed at P30 showed significantly (P < 0.016, two-tailed, unpaired Student's t test) lower activity of untreated Ethe1−/− mice as compared to control littermates, whereas values in treated Ethe1−/− mice overlapped those of the controls (Supplementary Fig. 3). At P45, treated Ethe1−/− mice actually showed significantly higher motor activity than control littermates (P < 0.016, two-tailed, unpaired Student's t test) indicating a possible hyperactive state (Supplementary Fig. 3). In sharp contrast with untreated Ethe1−/− mice, treated Ethe1−/− mice had an increase in body weight similar to, albeit less than, control littermates (Supplementary Fig. 4). Although the histochemical reaction to COX was equally low in muscle and brain of treated and untreated Ethe1−/− mice at P30 (Supplementary Fig. 5) and remained low in a treated Ethe1−/− mouse at P45 (data not shown), the severe COX depletion in colonocytes of untreated Ethe1−/− mice at P30 disappeared completely and persistently in the treated Ethe1−/− mice (Fig. 1b). This result suggests that recovery of COX proficiency varies widely between rapidly proliferating cells such as colonocytes and postmitotic cells such as muscle fibers and neurons, possibly because of the combined effect of H2S-mediated inhibition and structural degradation of COX1 and slow turnover of mitochondrial respiratory chain complexes in mouse muscle11 and neurons12. COX deficiency in essential tissues may ultimately contribute to early death of treated mice, in spite of the marked delay associated with therapy.

(c)   Given the results in the mouse model, we treated an Italian patient (subject 1), carrying a homozygous mutation in ETHE1 (ref. 13) encoding an L55P substitution. He had manifested the clinical and biochemical signs typical of ethylmalonic encephalopathy since 6 months of age (Fig. 1c and Supplementary Fig. 6). Despite dietary manipulations consisting of reducing intake of monosaccharides, which have a stronger osmotic effect, in favor of polysaccharides and consuming only hydrolyzed proteins, as well as coenzyme Q10 supplementation (50 mg per day), progression of his main clinical features occurred in the following months. A magnetic resonance imaging (MRI) scan at 28 months showed brain atrophy, leukodystrophy and patchy lesions of the basal nuclei (Fig. 1c). Diffuse petechiae of the trunk and limbs and acrocyanosis were worsened, as observed by the patient's physician.

(d)   At 29 months of age, we added a daily therapy consisting of oral metronidazole (30 mg per kg body weight) and N-acetylcysteine (105 mg per kg body weight); the latter was administered orally but with the intravenous formulation to avoid collateral osmotic effects due to the sugar additive of the granular preparation. During the next 8 months, we observed an increase in body weight (+1.5 kg), a marked reduction and then virtual disappearance of diarrhea, petechial showers and acrocyanosis (Table 1 and Supplementary Fig. 6) and marked neurological improvement, with a lower frequency of seizure attacks from several per day to less than one a week. The EEG showed the disappearance of slow-voltage waves. Truncal hypotonia became less severe, and spontaneous motor activity ensued at the lower limbs. A brain MRI at 36 months of age showed a reversion of brain atrophy and a reduction in leukodystrophy, although the lesions in the neostriatum had become more evident (Fig. 1c). Ethylmalonic acid (EMA) in urine and plasma dropped significantly and persistently (Fig. 1d and Table 1), and so did the concentrations of C4 acylcarnitines (Table 1). Occasional gastroesophageal reflux after ingestion of N-acetylcysteine responded well to ranitidine.

(e)   Table 1: Clinical and laboratory features of individuals with ethylmalonic encephalopathy

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We were encouraged enough by the long-lasting improvement of subject 1 to extend the same therapy to four Palestinian patients, a 32-month-old boy (subject 2), a 19-month-old boy (subject 3), a 12-month-old girl (subject 4) and a 10-month-old boy (subject 5), all formally unrelated but sharing the same homozygous 505+1G>T splice-site mutation in ETHE1 (ref. 14). After >3 months of therapy, these patients showed clinical, biochemical and MRI improvements similar to those of subject 1 (Table 1, Fig. 1c and Supplementary Fig. 7). In all five subjects, plasma EMA and serum thiosulfate (Fig. 1d and Table 1) concentrations consistently decreased during treatment, indicating a specific and considerable effect of the combined therapy on the metabolic abnormality associated with ethylmalonic encephalopathy.

The noteworthy prolongation of lifespan in Ethe1−/− mice and the evidence-based clinical improvement in patients make the combined treatment with metronidazole and N-acetylcysteine the most effective therapy available for ethylmalonic encephalopathy. Prenatal screening of the ETHE1 gene in at-risk fetuses, and of urinary EMA in neonates, could prompt pediatricians to apply this treatment in a timely manner to prevent or reduce irreversible brain damage.

 

supplementary

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其他關於acetylcysteine的文章可以點這邊:http://mulicia.pixnet.net/blog/search/acetylcystein/1
 

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