Marrow Transplantation (2007) 39, 211–215 & 2007 Nature Publishing Group All rights reserved 0268-3369/07 $30.00 www.nature.com/bmt

ORIGINAL ARTICLE

N-acetyl-L-cysteine improves outcome of advanced cerebral

J Tolar1, PJ Orchard1, KJ Bjoraker2, RS Ziegler2, EG Shapiro2 and L Charnas2

1Division of Pediatric Hematology, Oncology, Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA and 2Division of Pediatric Clinical Neuroscience, Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA

Hematopoietic stem cell transplantation as a treatment and defects in cognition and motor function, depending on for childhood cerebral adrenoleukodystrophy (ALD) has localization of disease. historically only been successful in early disease. As ALD Of several ALD phenotypes that can be distinguished on is associated with oxidative damage, we reasoned that the basis of clinical onset and manifestations, the cerebral adjunctive therapy with an antioxidant agent, N-acetyl- form of ALD is the most severe, resulting in a rapid L-cysteine (NAC), may provide protection from rapid neurologic deterioration and early death.3 The prognosis neurologic decline in boys with advanced cerebral disease. for symptomatic boys with cerebral ALD with extensive We report three boys with advanced ALD, whose changes on magnetic resonance imaging neurologic status and brain radiographic findings were (MRI) is poor.4,5 The Loes score,6,7 a system of scoring stabilized by treatment including NAC 8–11 months after MRI in ALD, has been found to be predictive of survival hematopoietic stem cell transplantation. These results after hematopoietic stem cell transplantation (HSCT). contrast with previous survival data in cerebral ALD Owing to demyelination, death patients who had a similar degree of brain involvement, all occurs usually within 2–5 years of clinical onset, unless the of whom died within 1 year of stem cell infusion despite a patient receives HSCT. Overall, the 5-year survival of full donor engraftment. Thus, NAC merits investigation as patients with a Loes MRI severity score of o9 before a therapeutic strategy for patients with advanced ALD as HSCT is 92%, versus 45% for patients who have a severity an intervention that could change this lethal disease to a score of 49 and have more than one neurologic deficit.8 As condition amendable to treatment with hematopoietic a result of the increased risk of death after HSCT, stem cell transplantation. transplant is not currently recommended for individuals Bone Marrow Transplantation (2007) 39, 211–215. with advanced disease. Unfortunately, there are no other doi:10.1038/sj.bmt.1705571 current treatment alternatives. Keywords: adrenoleukodystrophy; N-acetyl-L-cysteine; Neuropsychological testing identifies the functional ; hematopoietic stem cell transplant- deficits associated with the disease process and is a sensitive ation measure of disease severity at the time of diagnosis as well as a measure of disease progression during and after treatment. The Wechsler Performance IQ (PIQ) of o80 predicts poor functional outcome after HSCT.9,10 Based on Powers’ work on oxidative stress and damage Introduction in ALD,11 we have explored the use of an agent with antioxidant and radical scavenger capabilities, N-acetyl- Adrenoleukodystrophy (ALD) is an X-linked disorder of L-cysteine (NAC), in an attempt to arrest progression of very long-chain (VLCFA) metabolism caused by demyelination in advanced ALD. NAC stimulates glu- loss of function of the peroxisomal transporter ABCD1.1,2 tathione synthesis and scavenges free radicals, which has Elevated plasma levels of VLCFA correlate with adrenal been hypothesized to provide neuroprotective capacity.12–14 and nervous system dysfunction. Initial symptoms of the Here, we show that administration of NAC before cerebral form of ALD are visual and hearing disturbances and after HSCT in three patients with advanced ALD resulted in survival of a disease process that would be expected to be lethal.

Correspondence: Dr J Tolar, Division of Pediatric Hematology, Oncology, Blood and Marrow Transplantation, Department of Pedia- trics, University of Minnesota, MMC 366, 420 Delaware Street SE, Patients and methods Minneapolis, MN 55455, USA. E-mail: [email protected] Received 5 October 2006; revised 23 November 2006; accepted 24 All boys were delivered at full-term with early develop- November 2006 mental milestones acquired within normal age level N-acetyl-L-cysteine for ALD J Tolar et al 212 expectations. The diagnosis of ALD was established in all ability to hear. The MRI indicated marked abnormalities in patients by elevation of VLCFA and characteristic changes the posterior white matter involving the occipital and by MRI. The Loes scores and PIQ data are provided in parietal lobes and extending across the splenium of the Table 1. corpus callosum and the corticospinal tracts. Before HSCT, neuropsychological testing yielded both Patient 1 auditory and visual processing impairment, severe difficul- A 10-year-old boy was admitted to the hospital with ties in fine motor speed and coordination, and poor non- headaches. An MRI was performed, demonstrating sig- verbal reasoning, in contrast to executive function and nificant abnormalities in the posterior body and splenium academic achievement which were low average. of the corpus callosum, as well as in the corticospinal tracts and middle cerebellar peduncle. Patient 3 Results of the neuropsychological testing before HSCT A 9.9-year-old boy developed an impaired ability to track indicated significant auditory and visual processing deficits, with his right eye, and difficulty walking. The whereas vocabulary, academic achievement and adaptive MRI showed bilateral parietal occipital white matter functions were low average to average. abnormalities extending into the posterior rim of the internal capsule into the front lobe, posterior temporal Patient 2 lobe and in some early involvement of descending cortical A 9.8-year-old boy developed behaviors consistent with spinal tract with gadolinium enhancement and diffusion attention deficit/hyperactivity disorder and changes in his abnormalities (Figure 1).

Table 1 Patient and transplant characteristics

Patient Age at diagnosis VLCFA C24/C22 VLCFA C26/C22 Loes score PIQ Donor Follow-up Donor chimerism (years) ratioa ratioa (HLA match) (months) (%)

1 10 1.5 0.07 16 65 MRD BM 10 100 2 9.8 1.7 0.06 14.5 53 dUCB 8 100 (4/6; 5/6) 3 9.9 1.7 0.05 14.5 68 dUCB 11 100 (both 4/6)

Abbreviations: BM ¼ bone marrow; HLA ¼ human leukocyte antigen; MRD ¼ matched related donor; PIQ ¼ performance IQ (determined by WISC-IV testing); dUCB ¼ double umbilical cord blood. aVLCFA ¼ very long-chain fatty acids (normal ranges for VLCFA C24/C22 and C26/C22 are 0.74–0.94 and 0.006–0.014, respectively).

a b ef

c d gh

Figure 1 Brain MRI scans of 10-year-old boy with advanced cerebral ALD before and after hematopoietic stem cell transplantation. Pre-transplant scan (a, b, c and d) demonstrates extensive white matter changes in periventricular, central and subcortical white matter in parietooccipital, anterior temporal and posterior frontal lobes, corpus callosum, thalamus, internal capsules, auditory and visual pathways with MRI severity score 14.5. MRI scan 90 days after transplant (e, f, g and h) shows mild generalized atrophy with loss of previously affected white matter, except in subcortical parietal area, cystic conversion in internal capsules bilaterally (f, arrow), and no extension into previously uninvolved areas.

Bone Marrow Transplantation N-acetyl-L-cysteine for ALD J Tolar et al 213 Neuropsychological results before HSCT indicated 1.0 severe visual spatial impairment, moderate coordination, auditory memory and attention problems with average verbal reasoning. 0.8 All three patients received allogeneic HSCT with NAC for severe ALD, defined as PIQ 80 and Loes score 414 o 0.6 (Table 1). Transplant protocols were approved by institu- tional review board, and informed consent was obtained in all cases before the procedure. Follow-up is reported 0.4 through October 2006. As patients with advanced ALD,

it was anticipated that they would be at very high risk Cumulative survival for dying with a standard regimen. Thus, they received 0.2 NAC 140 mg/kg/day intravenously (i.v.) followed by 70 mg/kg four times daily orally. When mucositis developed after HSCT, the NAC was administered i.v. at the same 0.0 dosing schedule (70 mg/kg four times daily). The treat- 024681012 ment with NAC was initiated following their initial Months evaluation at the University of Minnesota, with a range Figure 2 Survival after HSCT þ NAC is superior to survival after HSCT of 54–67 days before HSCT, and discontinued 114–250 alone. Survival of eight consecutive ALD patients with severe disease days after HSCT. (defined as PIQ o80 and Loes score 414) who received HSCT at this One patient received stem cell infusions derived from institution is zero at 1 year after HSCT. In contrast, all three ALD patients (with the same degree of severity) who received NAC before and after matched related donor bone marrow (patient 1). Two HSCT are alive between 8 and 11 months after HSCT. A test for the patients received an unrelated donor umbilical cord blood equality of the survival distributions using log-rank statistic was significant utilizing two cord blood grafts; the first received units (3.91, df ¼ 1, Po0.05). matched at four of six and five of six human leukocyte antigen (HLA) antigens (patient 2) and the other received two cord blood units matched at four of six HLA antigens airway edema requiring 10 days of assisted ventilation (patient 3). (patient 3). The preparative regimen consisted of busulfan 0.8 mg/kg From a functional standpoint, all boys are wheelchair i.v. four times daily for 4 consecutive days (days À9 and bound 6–9 months after transplant. Patient 1 can follow À6; total dose 12.8 mg/kg), cyclophosphamide 50 mg/kg limited commands. Patient 2 has limited expressive once daily i.v. for four consecutive days (days À4toÀ1; language and can follow commands. Patient 3 is able to total dose 200 mg/kg), horse antithymocyte globulin sit independently, can stand and take steps with support. (ATGAM) 30 mg/kg once daily i.v. for 3 consecutive days He uses a communicative device. Auditory comprehension (days À3toÀ1; total dose 90 mg/kg). To potentially reduce is good, and he follows commands in a consistent manner. brain demyelination and inflammation while waiting for He can read and his vision is similar to before transplant. transplant, patient 1 received (Campath-1 H) He is continent of bowel and bladder, and can walk with 0.3 mg/kg once daily for 3 consecutive days (days À41 to assistance from one person. À43; total dose 0.9 mg/kg). All patients received cyclospor- All three patients had stable disease as evidenced by ine and mycophenolate mofetil for graft-versus-host disease brain MRI findings 6 months after HSCT (representative prophylaxis. images are shown in Figure 1). Hematopoietic chimerism was assessed on peripheral Patients in the comparison group (defined as PIQ o80 blood leukocyte DNA by competitive polymerase chain and Loes score 414; N ¼ 8) all died within the first year reaction analysis of variable tandem repeat regions.15 after HSCT despite full donor engraftment HSCT4 A comparison group for analysis of survival consisted (Figure 2). A test for the equality of the survival of all boys transplanted at the University of Minnesota distributions using log-rank statistic was significant (3.91, with PIQ o80 and MRI Loes score 414 under the age df ¼ 1, Po0.05). of 18 years treated with HSCT before November 2005 (N ¼ 8). Discussion

Results Our findings suggest that peri-transplant administration of NAC is protective from fulminant demyelination in Patient donor graft characteristics and transplant out- advanced, symptomatic ALD, presumably because of its comes are summarized in the Table 1. The median age at antioxidant and radical scavenging properties. This ob- HSCT was 10.2 years (range, 10.1–10.3 years). Donor servation has important therapeutic implications, because engraftment (defined as donor chimerism of 95–100%) there is an urgent need for effective therapy for advanced was observed in all three patients. Significant transplant cerebral ALD patients who are not candidates for standard related morbidity consisted of the following: hemorrhagic HSCT because of disease progression. cystitis (patients 1, 2 and 3); engraftment syndrome All three boys initially presented with rapidly progressing responsive to steroids (patient 2); and spasticity and upper visual or auditory deficits, which continued to worsen

Bone Marrow Transplantation N-acetyl-L-cysteine for ALD J Tolar et al 214 during and shortly after HSCT. Currently, they appear to 4 Moser HW, Loes DJ, Melhem ER, Raymond GV, Bezman L, be stable clinically and by MRI. As ALD is associated with Cox CS et al. X-linked adrenoleukodystrophy: overview and oxidative damage, we aimed to halt rapid neurologic prognosis as a function of age and brain magnetic resonance decline observed in such patients with advanced disease imaging abnormality. A study involving 372 patients. with an adjunctive therapy with an antioxidant agent, Neuropediatrics 2000; 31: 227–239. NAC. In support of this concept, NAC has been shown to 5 Melhem ER, Loes DJ, Georgiades CS, Raymond GV, Moser HW. X-linked adrenoleukodystrophy: the role of contrast- protect neurons from apoptosis in experimental models of enhanced MR imaging in predicting disease progression. neurodegenerative conditions (e.g., , AJNR Am J Neuroradiol 2000; 21: 839–844. experimental autoimmune encephalitis, Alzheimer’s and 6 Loes DJ, Stillman AE, Hite S, Shapiro E, Lockman L, Parkinson’s diseases.16–20 Latchaw RE et al. Childhood cerebral form of adrenoleuko- NAC has been used clinically as an inhalational dystrophy: short-term effect of bone marrow transplantation mucolytic agent in bronchopulmonary illnesses including on brain MR observations. AJNR Am J Neuroradiol 1994; 15: cystic fibrosis, and as an antidote for liver injury from 1767–1771. acute acetaminophen toxicity.21 This is important, since 7 Loes DJ, Hite S, Moser H, Stillman AE, Shapiro E, Lockman several decades of toxicity data established that NAC L et al. Adrenoleukodystrophy: a scoring method for AJNR Am J Neuroradiol administration is safe (even with long-term use). Aside brain MR observations. 1994; 15: 1761–1766. from one episode of acute nausea after the first intravenous 8 Peters C, Charnas LR, Tan Y, Ziegler RS, Shapiro EG, DeFor dose and the unpleasant odor of the suspension during oral T et al. Cerebral X-linked adrenoleukodystrophy: the interna- administration, we have not observed any adverse reac- tional hematopoietic cell transplantation experience from 1982 tions related to NAC. Clearly, further studies will be to 1999. Blood 2004; 104: 881–888. needed to determine the optimal timing and dose of NAC. 9 Shapiro E, Krivit W, Lockman L, Jambaque I, Peters C, In addition, it will be important to explore to what degree Cowan M et al. Long-term effect of bone-marrow transplant- it benefits neurologic outcomes of patients with advanced ation for childhood-onset cerebral X-linked adrenoleuko- ALD (or early ALD), and whether it has any role in dystrophy. Lancet 2000; 356: 713–718. ALD outside of an adjunct to transplantation. Another 10 Shapiro EG, Balthazor M. Metabolic and neurodegenerative important issue relates to whether the protective effects disorders of childhood. In: Taylor G, Ris D, Yeates K (eds). Pediatric Neuropsychology: Research, Theory and Practice. of NAC would be important in transplantation for Guilford Press: New York, NY, USA, 2000, pp 171–205. other inherited neurological disorders such as globoid 11 Powers JM, Pei Z, Heinzer AK, Deering R, Moser AB, cell or metachromatic leukodystrophy. Moser HW et al. Adreno-leukodystrophy: oxidative stress Although the biology of these diseases is distinct, apoptosis of mice and men. 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