Neurol Sci DOI 10.1007/s10072-014-1683-y
ORIGINAL ARTICLE
Results from a pilot study on amiodarone administration in monogenic frontotemporal dementia with granulin mutation A. Alberici • S. Archetti • A. Pilotto • E. Premi • M. Cosseddu A. Bianchetti • F. Semeraro • M. Salvetti • M. L. Muiesan • A. Padovani • B. Borroni
•
Received: 1 December 2013 / Accepted: 11 February 2014 Ó Springer-Verlag Italia 2014
Abstract Frontotemporal dementia (FTD) is one of the most important neurodegenerative conditions and Granulin (GRN) is one of the major genes associated to the disease. FTD-GRN patients are still orphan for any evidence-based target-therapy approach. Interestingly, it has been recently found that alkalizing agents rescued haploinsufficiency in cellular models expressing FTD-GRN mutations. We set up a pilot phase II clinical trial in five FTD patients with GRN Thr272s(g.1977_1980delCACT) mutation, to determine if amiodarone (200 mg/day) may (1) reverse progranulin deficiency and (2) delay disease progression. Each patient was scheduled for 7 study visits over 12 months period. We assessed GRN levels at baseline and after amiodarone administration during the treatment course. Somatic and neurologic examinations, along with cognitive and behavioral assessment were recorded as well. No significant
A. Alberici (&) A. Pilotto E. Premi M. Cosseddu A. Padovani B. Borroni Department of Medical Sciences, Clinic of Neurology, Spedali Civili, University of Brescia, Piazzale Spedali Civili 1, 25100 Brescia, Italy e-mail: [email protected] S. Archetti Third Laoratory, Spedali Civili, Piazzale Spedali Civili 1, 25100 Brescia, Italy A. Bianchetti Medicine Unit, Sant’Anna Hospital, 25100 Brescia, Italy F. Semeraro Oftalmology Unit, Spedali Civili, University of Brescia, Piazzale Spedali Civili 1, 25100 Brescia, Italy M. Salvetti M. L. Muiesan Department of Internal Medicine, Spedali Civili, University of Brescia, Piazzale Spedali Civili 1, 25100 Brescia, Italy
effect on peripheral GRN levels was observed. In treated FTD, disease course did not differ when compared with a group of untreated FTD-GRN patients. This is the first trial targeting progranulin rescue in FTD-GRN patients using amiodarone. Despite the negative findings, it may be interesting to extend this attempt to a larger sample of subjects and to other alkalizing agents to restore granulin haploinsufficiency. Keywords Frontotemporal dementia Progranulin Granulin Amiodarone Treatment
Introduction Frontotemporal dementia (FTD) is a common cause of dementia in presenium, also recognized affecting aged population. Presenting features are characterized by social misconduct, language and executive deficits, or motor impairment, therefore encompassing different clinical syndromes: the behavioral variant FTD (bv-FTD), the agrammatic (avPPA) and semantic (svPPA) variant of primary progressive aphasia [1–3]. The broad clinical spectrum is the result of the predominant atrophy of frontal and temporal lobes, in which at autopsy examination the distinctive hallmarks are mainly represented by microtubule-associated protein tau (MAPT) depositions, TAR DNA-binding protein 43 (TDP-43) inclusions, or fused in sarcoma (FUS) [4]. The strong genetic background, with almost 40 % of patients showing a positive family history, prompted the identification of a number of genes responsible for the disease. In particular, mutations in granulin (GRN), MAPT, and more recently in the hexanucleotide repeat expansion in C9orf72, are considered the most frequent causes of genetic FTD [4].
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Despite the great effort to find genes associated with the disease, FTD is a condition still orphan for any therapeutic approach. The wide clinical, pathological and genetic variability, along with the small number of patients, have been so far prevented the development of clinical trials, although it has been established a methodology for outcome measures [5]. Potential strategies to cure FTD might consider treating symptoms or intervening on the pathological determinants. However, in clinical practice, the drugs already approved for Alzheimer’s disease are frequently administered to FTD patients as an off-label prescription, and in spite to any evidence-based medicine [6]. Only a handful of clinical trials on pharmacological interventions are currently ongoing, or have been established in the last few years (http://www.clinicaltrial.gov) (http://www.clinicaltrialsregister.eu). Interventions on pathological determinants of FTD included a bulk of studies on agents interfering with MAPT or TDP-43 deposits [7]. The evidence that mutations within GRN gene, encoding for progranulin, reduce or almost abolish the concentrations of peripheral circulating protein, constituted a particular appealing start point to develop drug targeting and to restore progranulin levels. One of the most promising studies in this direction has been proposed by Capell and colleagues [8], who showed how alkalizing reagents, and vacuolar ATPase inhibitors may significantly increase progranulin levels in in vitro cellular models. On the basis of these in vitro results, although with the limitation that the drug has not been tested in specific animal models (i.e. GRN KO mice), we set up a pilot II phase, open-label clinical trial to administer amiodarone in five patients with FTD bearing the pathogenic mutation Thr272fs(g.1977_1980delCACT) in GRN gene.
All patients had a reliable caregiver who could accompany them for all the study visits. Exclusion criteria were represented by: (1) any medical condition that may interfere with the study, (2) FTD-CDR [ 10 [5, 9]. No patient was taking memantine or an acetylcholinesterase inhibitor. Antidepressant or typical antipsychotic medications (promazine or haloperidol) were admitted for symptomatic treatment of behavioral disorders. Written informed consent was obtained from the patients and their caregivers. The study was approved by the Local Ethical Committee, and it was registered at the European Agency for Clinical trials (http://www.clinicaltrialsregister.eu) as ReProIn-FTD 2011-004571-37, EUDRACT: 2011-00457137. Procedures Each patient was scheduled for 7 study visits over 12 months period. After the screening visit, the amiodarone medication was dispensed with an initial dosage of 1,200 mg daily for 1 week, followed by 200 mg daily for 1 year. Patients returned at weeks 1, 4, 12, 24, 54, and stopped the treatment after 1 year. A further evaluation 6 months after stopping the treatment was scheduled. The visits included somatic and neurologic examination, along with cognitive and behavioral assessment, and blood test for detecting progranulin serum levels. In addition, ECG, ophthalmologic evaluation, thyroid function and serum electrolytes were determined at weeks 1, 4, 24, 54, to check possible adverse events related to amiodarone therapy. Compliance was assessed by counting amiodarone tablets remaining in the blister pack. Adverse events were planned to be recorded in accordance to local Ethical Committee recommendations. Serious adverse events were defined as those requiring hospital admission or death.
Patients and methods Study design and participants
Genetic screening and serum progranulin level determinations
We set up an open label, phase II, trial to administer amiodarone for 12 months to 5 patients suffering from FTD and bearing the pathogenic mutation Thr272fs (g.1977_1980delCACT) in GRN gene. Patients had to be aged more than 18, to have mild disease stage, i.e. FTDmodified Clinical Dementia Rating Scale (FTD-CDR) score of 5–10, and a positive genetic screening for a known GRN pathogenic mutation, causing progranulin deficiency [5, 9]. The patients were enrolled at the Centre of Neurodegenerative Disorders; University of Brescia, Italy. An extensive cognitive and behavioral assessment was applied to each patient, as previously reported as well as brain neuroimaging scan [10].
Genomic DNA was extracted from peripheral blood using a standard procedure. All the 12 exons plus exon 0 of GRN, and at least 30 base pairs (bp) of their flanking introns were evaluated by Polymerase Chain Reaction (PCR) and subsequent sequencing. GRN Thr272(g.1977_1980delCACT) was tested as previously described [9]. Venous blood samples were drained at fast, to collect serum. Progranulin determinations were performed in duplicate in the same ELISA batch according to manufacturer (Adipogen, USA), before treatment (baseline, T = 0), after 1 week (T = 1), and at weeks 4 (T = 2), 12 (T = 3), 24 (T = 4), 54 (T = 5). On the basis of our data from 11 subjects bearing GRN pathogenic mutations (5 affected; 6 unaffected, Age-
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Neurol Sci Table 1 Main clinical characteristics of FTD patients bearing GRN T272SfsX10 mutation included in the study Patient ID code
Gender
Education (years)
Family history
Age at evaluation (years)
Age at disease onset (years)
Clinical phenotype
MMSE
FTD-CDR T0
FTD-CDR T5
1
F
5
Positive
57
54
bvFTD
15.9
10
16
2
F
5
Positive
65
64
bvFTD
18.9
10
12.5
3 4
M F
5 5
Positive Positive
70 65
68 63
bvFTD bvFTD
23.3 25.9
6 6
16 21
5
F
8
Positive
65
60
avPPA
20
6.5
10
FTD frontotemporal dementia, F female, M male, bvFTD behavioral variant of FTD, avPPA agrammatic variant of primary progressive aphasia, MMSE mini-mental state examination, FTD-CDR FTD-modified clinical dementia rating scale, T0 baseline evaluation, T5 evaluation after 54 weeks
Progranulin levels (ng/ml)
120 100 80 Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
60 40 20 0 To
T1
T2
T3
T4
T5
Time
Fig. 1 Serum progranulin levels at different time evaluation in patients treated with amiodarone
years = 64.6 ± 9.2; 40.0 ± 18.6, Gender-Female % = 60.0; 33.3) as compared to 25 Alzheimer Disease patients (Ageyears = 74.3 ± 8.9, Gender-Female % = 68.0), and to 36 healthy controls (Age-years = 57.5 ± 14.8, Gender-Female % = 61.1), we established the serum GRN cutoff level of 125 ng/mL, to distinguish GRN null mutations carriers, with a specificity of 97.4 % and a sensitivity of 81.8 % (unpublished data). Outcomes Primary endpoint was the dosage of progranulin levels over the time, determined in serum at T = 0–5; the secondary endpoint was the clinical course at 1 year, as compared to natural disease course in untreated GRN mutation carriers.
10, a disease duration less than 5 years). Almost all included patients had a bvFTD, one had avPPA; main clinical features are reported in Table 1. Serum progranulin levels were determined at baseline (T = 0), before amiodarone administration, and after 1-week, 4, 12, 24, 54 weeks of treatment. As shown in Fig. 1, patient 1 presented a higher serum GRN level to what reported as optimal plasma GRN cutoff value for predicting null mutations, but under the normal GRN determination, as based on our unpublished data [11]. Indeed, for patients who have a positive family history, and a younger disease onset, with a borderline plasma or serum GRN levels, the complete sequence of the gene should be recommended. No significant increase was observed after amiodarone therapy at different times, as reported in Fig. 1. Two patients dropped out after the third month treatment for the onset of behavioral disturbances characterized by agitation (patient 3), and jealousy delusions (patient 1). The three other patients completed the study, in the absence of any adverse event, with no significant increase of progranulin levels (Fig. 1). Patient 4 was institutionalized at the end of the study because of the severity of the disease. Disease stage after 1-year treatment is reported in Table 1 (i.e. FTD-CDR). We compared this group of patients treated with amiodarone to other 13 FTD with the same mutations (mean age = 57.61 ± 6.75; 46.2 % male), who were followed in the previous years and did not enter into the present study. These two subgroups of FTD-GRN patients were comparable in terms of disease severity (i.e., FTD-CDR; patients treated with amiodarone: 7.70 ± 2.10 vs. untreated: 8.19 ± 2.71, p = 0.703). During 1-year follow-up, the mean increase of FTD-CDR did not differ between the treated (mean value 7.40 ± 5.14 points) and untreated (mean value 4.50 ± 2.19 points) FTD patients (p = 0.289).
Results We enrolled five FTD patients with GRN Thr272fs (g.1977_1980delCACT) mutation (mean age = 64.4 ± 4.7; 20 % male), all with a positive family for dementia, in a relatively early stage of their disease (FTD-CDR less than
Discussion FTD is a devastating disease for patients who are bereaved from their personality or their ability to communicate with
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language, with an enormous impact on their relatives and high costs for the society. Previous studies attempted to verify the efficacy of different symptomatic therapies on the disease course, although with poor results. In a trial with galantamine administered to behavioral and aphasic variants of FTD, no effect on behavior or language was found, excluding a minor trend of efficacy in the aphasic patients [12]. A significant improvement in scores on the Neuropsychiatric Inventory and reduced recognition of angry facial expressions was observed in patients with bvFTD who received a single dose of oxytocin [12]. Citalopram was demonstrated to be effective in relieving from behavioral disturbances in a small number of patients [13]. More recently, the findings of a multicentre, randomized, double-blind, placebo-controlled trial of memantine showed no treatment benefit, in analogy to what reported in a previous French trial [14–16]. At our best knowledge, the present study represents a first pilot trial targeting pathological determinants associated to the disease in FTD, in particular in patients bearing GRN mutation. The molecular pathways that lead from reduced progranulin levels to TDP43 inclusions and therefore to neurodegeneration are still to be clarified, but the possibility to rescue progranulin levels offers an opportunity for strategies of intervention. The paper by Capell and colleagues [8] demonstrated an increase of progranulin level in lymphoblastoid derived from loss-of-function carrier treatment with different alkalizing agents. Using a different approach, Cenik and colleagues [17] identified suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor as an enhancer of GRN expression. Among the different agents proposed in the paper by Capell [9], amiodarone is largely used in cardiologic application, and therefore was sought to be firstly assayed. Unfortunately, our results, although limited to a small number of patients, are not promising in this sense, but may help in future to identify other alkalizing agents, including chloroquine, or different approaches to be pursued. We acknowledge that the present study has several limitations, in first instance the drug has not been tested on specific animal models, in particular in available GRN KO mice [18–20]. The rationale to administer amiodarone directly in patients, by-passing a first step in animal models, was based on the broad use of amiodarone in cardiologic settings, since a long time. However, amiodarone final dosage was limited to what commonly used for atrial fibrillation. Finally, the number of recruited subjects is very small, and the study lacks of plasma amiodarone levels determinations, along with the lack of cerebrospinal fluid progranulin determination. Nevertheless, we provided a first hint for future multicentre trial aiming to contrast progranulin deficiency in FTD.
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Acknowledgments The authors wish to acknowledge the helpful and generous collaboration of patients and their family members, which was essential for this study. The authors are indebted with Dr. Francesca Ferrari for technical assistance. Conflict of interest All co-authors agreed with the contents of the manuscript and there is no financial interest to report.
References 1. Neary D, Snowden JS, Gustafson L, Passant U, Stuss D, Black S, Freedman M, Kertesz A, Robert PH, Albert M, Boone K, Miller BL, Cummings J, Benson DF (1998) Frontotemporal lobar degeneration: a consensus of clinical diagnostic criteria. Neurology 51(6):1546–1554 2. Rascovsky K, Hodges JR, Knopman D, Mendez MF, Kramer JH, Neuhaus J, van Swieten JC, Seelaar H, Dopper EG, Onyike CU, Hillis AE, Josephs KA, Boeve BF, Kertesz A, Seeley WW, Rankin KP, Johnson JK, Gorno-Tempini ML, Rosen H, PrioleauLatham CE, Lee A, Kipps CM, Lillo P, Piguet O, Rohrer JD, Rossor MN, Warren JD, Fox NC, Galasko D, Salmon DP, Black SE, Mesulam M, Weintraub S, Dickerson BC, Diehl-Schmid J, Pasquier F, Deramecourt V, Lebert F, Pijnenburg Y, Chow TW, Manes F, Grafman J, Cappa SF, Freedman M, Grossman M, Miller BL (2011) Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia. Brain 134:2456–2477 3. Gorno-Tempini ML, Hillis AE, Weintraub S, Kertesz A, Mendez M, Cappa SF, Ogar JM, Rohrer JD, Black S, Boeve BF, Manes F, Dronkers NF, Vandenberghe R, Rascovsky K, Patterson K, Miller BL, Knopman DS, Hodges JR, Mesulam MM, Grossman M (2011) Classification of primary progressive aphasia and its variants. Neurology 76:1006–1014 4. Sieben A, Van Langenhove T, Engelborghs S, Martin JJ, Boon P, Cras P, Cras P, De Deyn PP, Santens P, Van Broeckhoven C, Cruts M (2012) The genetics and neuropathology of frontotemporal lobar degeneration. Acta Neuropathol 124:353–372 5. Knopman DS, Kramer JH, Boeve BF, Caselli RJ, Graff-Radford NR, Mendez MF, Mendez MF, Miller BL, Mercaldo N (2008) Development of methodology for conducting clinical trials in frontotemporal lobar degeneration. Brain 13:2957– 2968 6. Lo´pez-Pousa S, Calvo´-Perxas L, Lejarreta S, Cullell M, Mele´ndez R, Herna´ndez E, Bisbe J, Perkal H, Manzano A, Roig AM, Turro´-Garriga O, Vilalta-Franch J, Garre-Olmo J, Registry of Dementias of Girona Study Group (ReDeGi Study Group) (2012) Use of antidementia drugs in frontotemporal lobar degeneration. Am J Alzheimers Dis Demen 27:260–266 7. Bigni B, Premi E, Pilotto A, Padovani A, Borroni B (2012) Disease-modifying therapies in frontotemporal lobar degeneration. Curr Med Chem 19:1008–1020 8. Capell A, Liebscher S, Fellerer K, Brouwers N, Willem M, Lammich S, Lammich S, Gijselinck I, Bittner T, Carlson AM, Sasse F, Kunze B, Steinmetz H, Jansen R, Dormann D, Sleegers K, Cruts M, Herms J, Van Broeckhoven C, Haass C (2011) Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase. J Neurosci 31:1885–1894 9. Borroni B, Agosti C, Premi E, Cerini C, Cosseddu M, Paghera B, Bellelli G, Padovani A (2010) The FTLD-modified Clinical Dementia Rating scale is a reliable tool for defining disease severity in frontotemporal lobar degeneration: evidence from a brain SPECT study. Eur J Neurol 17(5):703–707. doi:10.1111/j. 1468-1331.2009.02911.x (Epub 2009 Dec 29)
Neurol Sci 10. Borroni B, Grassi M, Agosti C, Premi E, Archetti S, Alberici A, Bellelli G, Caimi L, Di Luca M, Padovani A (2010) Establishing short-term prognosis in Frontotemporal Lobar Degeneration spectrum: role of genetic background and clinical phenotype. Neurobiol Aging 31:270–279 11. Ghidoni R, Stoppani E, Rossi G, Piccoli E, Albertini V, Paterlini A, Glionna M, Pegoiani E, Agnati LF, Fenoglio C, Scarpini E, Galimberti D, Morbin M, Tagliavini F, Binetti G, Benussi L (2012) Optimal plasma progranulin cutoff value for predicting null progranulin mutations in neurodegenerative diseases: a multicenter Italian study. Neurodegener Dis 9(3):121–127. doi:10.1159/000333132 (Epub 2011 Nov 26) 12. Kertesz A, Morlog D, Light M, Blair M, Davidson W, Jesso S, Brashear R (2008) Galantamine in frontotemporal dementia and primary progressive aphasia. Dement Geriatr Cogn Disord 25:178–185 13. Jesso S, Morlog D, Ross S, Pell MD, Pasternak SH, Mitchell DG, Kertesz A, Finger EC (2011) The effects of oxytocin on social cognition and behaviour in frontotemporal dementia. Brain 134:2493–2501 14. Herrmann N, Black SE, Chow T, Cappell J, Tang-Wai DF, Lanctoˆt KL (2012) Serotonergic function and treatment of behavioral and psychological symptoms of frontotemporal dementia. Am J Geriatr Psychiatry 20:789–797 15. Boxer AL, Knopman DS, Kaufer DI, Grossman M, Onyike C, Graf-Radford N, Mendez M, Kerwin D, Lerner A, Wu CK, Koestler M, Shapira J, Sullivan K, Klepac K, Lipowski K, Ullah J, Fields S, Kramer JH, Merrilees J, Neuhaus J, Mesulam MM, Miller BL (2013) Memantine in patients with frontotemporal
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ORIGINAL ARTICLE
Results from a pilot study on amiodarone administration in monogenic frontotemporal dementia with granulin mutation A. Alberici • S. Archetti • A. Pilotto • E. Premi • M. Cosseddu A. Bianchetti • F. Semeraro • M. Salvetti • M. L. Muiesan • A. Padovani • B. Borroni
•
Received: 1 December 2013 / Accepted: 11 February 2014 Ó Springer-Verlag Italia 2014
Abstract Frontotemporal dementia (FTD) is one of the most important neurodegenerative conditions and Granulin (GRN) is one of the major genes associated to the disease. FTD-GRN patients are still orphan for any evidence-based target-therapy approach. Interestingly, it has been recently found that alkalizing agents rescued haploinsufficiency in cellular models expressing FTD-GRN mutations. We set up a pilot phase II clinical trial in five FTD patients with GRN Thr272s(g.1977_1980delCACT) mutation, to determine if amiodarone (200 mg/day) may (1) reverse progranulin deficiency and (2) delay disease progression. Each patient was scheduled for 7 study visits over 12 months period. We assessed GRN levels at baseline and after amiodarone administration during the treatment course. Somatic and neurologic examinations, along with cognitive and behavioral assessment were recorded as well. No significant
A. Alberici (&) A. Pilotto E. Premi M. Cosseddu A. Padovani B. Borroni Department of Medical Sciences, Clinic of Neurology, Spedali Civili, University of Brescia, Piazzale Spedali Civili 1, 25100 Brescia, Italy e-mail: [email protected] S. Archetti Third Laoratory, Spedali Civili, Piazzale Spedali Civili 1, 25100 Brescia, Italy A. Bianchetti Medicine Unit, Sant’Anna Hospital, 25100 Brescia, Italy F. Semeraro Oftalmology Unit, Spedali Civili, University of Brescia, Piazzale Spedali Civili 1, 25100 Brescia, Italy M. Salvetti M. L. Muiesan Department of Internal Medicine, Spedali Civili, University of Brescia, Piazzale Spedali Civili 1, 25100 Brescia, Italy
effect on peripheral GRN levels was observed. In treated FTD, disease course did not differ when compared with a group of untreated FTD-GRN patients. This is the first trial targeting progranulin rescue in FTD-GRN patients using amiodarone. Despite the negative findings, it may be interesting to extend this attempt to a larger sample of subjects and to other alkalizing agents to restore granulin haploinsufficiency. Keywords Frontotemporal dementia Progranulin Granulin Amiodarone Treatment
Introduction Frontotemporal dementia (FTD) is a common cause of dementia in presenium, also recognized affecting aged population. Presenting features are characterized by social misconduct, language and executive deficits, or motor impairment, therefore encompassing different clinical syndromes: the behavioral variant FTD (bv-FTD), the agrammatic (avPPA) and semantic (svPPA) variant of primary progressive aphasia [1–3]. The broad clinical spectrum is the result of the predominant atrophy of frontal and temporal lobes, in which at autopsy examination the distinctive hallmarks are mainly represented by microtubule-associated protein tau (MAPT) depositions, TAR DNA-binding protein 43 (TDP-43) inclusions, or fused in sarcoma (FUS) [4]. The strong genetic background, with almost 40 % of patients showing a positive family history, prompted the identification of a number of genes responsible for the disease. In particular, mutations in granulin (GRN), MAPT, and more recently in the hexanucleotide repeat expansion in C9orf72, are considered the most frequent causes of genetic FTD [4].
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Neurol Sci
Despite the great effort to find genes associated with the disease, FTD is a condition still orphan for any therapeutic approach. The wide clinical, pathological and genetic variability, along with the small number of patients, have been so far prevented the development of clinical trials, although it has been established a methodology for outcome measures [5]. Potential strategies to cure FTD might consider treating symptoms or intervening on the pathological determinants. However, in clinical practice, the drugs already approved for Alzheimer’s disease are frequently administered to FTD patients as an off-label prescription, and in spite to any evidence-based medicine [6]. Only a handful of clinical trials on pharmacological interventions are currently ongoing, or have been established in the last few years (http://www.clinicaltrial.gov) (http://www.clinicaltrialsregister.eu). Interventions on pathological determinants of FTD included a bulk of studies on agents interfering with MAPT or TDP-43 deposits [7]. The evidence that mutations within GRN gene, encoding for progranulin, reduce or almost abolish the concentrations of peripheral circulating protein, constituted a particular appealing start point to develop drug targeting and to restore progranulin levels. One of the most promising studies in this direction has been proposed by Capell and colleagues [8], who showed how alkalizing reagents, and vacuolar ATPase inhibitors may significantly increase progranulin levels in in vitro cellular models. On the basis of these in vitro results, although with the limitation that the drug has not been tested in specific animal models (i.e. GRN KO mice), we set up a pilot II phase, open-label clinical trial to administer amiodarone in five patients with FTD bearing the pathogenic mutation Thr272fs(g.1977_1980delCACT) in GRN gene.
All patients had a reliable caregiver who could accompany them for all the study visits. Exclusion criteria were represented by: (1) any medical condition that may interfere with the study, (2) FTD-CDR [ 10 [5, 9]. No patient was taking memantine or an acetylcholinesterase inhibitor. Antidepressant or typical antipsychotic medications (promazine or haloperidol) were admitted for symptomatic treatment of behavioral disorders. Written informed consent was obtained from the patients and their caregivers. The study was approved by the Local Ethical Committee, and it was registered at the European Agency for Clinical trials (http://www.clinicaltrialsregister.eu) as ReProIn-FTD 2011-004571-37, EUDRACT: 2011-00457137. Procedures Each patient was scheduled for 7 study visits over 12 months period. After the screening visit, the amiodarone medication was dispensed with an initial dosage of 1,200 mg daily for 1 week, followed by 200 mg daily for 1 year. Patients returned at weeks 1, 4, 12, 24, 54, and stopped the treatment after 1 year. A further evaluation 6 months after stopping the treatment was scheduled. The visits included somatic and neurologic examination, along with cognitive and behavioral assessment, and blood test for detecting progranulin serum levels. In addition, ECG, ophthalmologic evaluation, thyroid function and serum electrolytes were determined at weeks 1, 4, 24, 54, to check possible adverse events related to amiodarone therapy. Compliance was assessed by counting amiodarone tablets remaining in the blister pack. Adverse events were planned to be recorded in accordance to local Ethical Committee recommendations. Serious adverse events were defined as those requiring hospital admission or death.
Patients and methods Study design and participants
Genetic screening and serum progranulin level determinations
We set up an open label, phase II, trial to administer amiodarone for 12 months to 5 patients suffering from FTD and bearing the pathogenic mutation Thr272fs (g.1977_1980delCACT) in GRN gene. Patients had to be aged more than 18, to have mild disease stage, i.e. FTDmodified Clinical Dementia Rating Scale (FTD-CDR) score of 5–10, and a positive genetic screening for a known GRN pathogenic mutation, causing progranulin deficiency [5, 9]. The patients were enrolled at the Centre of Neurodegenerative Disorders; University of Brescia, Italy. An extensive cognitive and behavioral assessment was applied to each patient, as previously reported as well as brain neuroimaging scan [10].
Genomic DNA was extracted from peripheral blood using a standard procedure. All the 12 exons plus exon 0 of GRN, and at least 30 base pairs (bp) of their flanking introns were evaluated by Polymerase Chain Reaction (PCR) and subsequent sequencing. GRN Thr272(g.1977_1980delCACT) was tested as previously described [9]. Venous blood samples were drained at fast, to collect serum. Progranulin determinations were performed in duplicate in the same ELISA batch according to manufacturer (Adipogen, USA), before treatment (baseline, T = 0), after 1 week (T = 1), and at weeks 4 (T = 2), 12 (T = 3), 24 (T = 4), 54 (T = 5). On the basis of our data from 11 subjects bearing GRN pathogenic mutations (5 affected; 6 unaffected, Age-
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Neurol Sci Table 1 Main clinical characteristics of FTD patients bearing GRN T272SfsX10 mutation included in the study Patient ID code
Gender
Education (years)
Family history
Age at evaluation (years)
Age at disease onset (years)
Clinical phenotype
MMSE
FTD-CDR T0
FTD-CDR T5
1
F
5
Positive
57
54
bvFTD
15.9
10
16
2
F
5
Positive
65
64
bvFTD
18.9
10
12.5
3 4
M F
5 5
Positive Positive
70 65
68 63
bvFTD bvFTD
23.3 25.9
6 6
16 21
5
F
8
Positive
65
60
avPPA
20
6.5
10
FTD frontotemporal dementia, F female, M male, bvFTD behavioral variant of FTD, avPPA agrammatic variant of primary progressive aphasia, MMSE mini-mental state examination, FTD-CDR FTD-modified clinical dementia rating scale, T0 baseline evaluation, T5 evaluation after 54 weeks
Progranulin levels (ng/ml)
120 100 80 Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
60 40 20 0 To
T1
T2
T3
T4
T5
Time
Fig. 1 Serum progranulin levels at different time evaluation in patients treated with amiodarone
years = 64.6 ± 9.2; 40.0 ± 18.6, Gender-Female % = 60.0; 33.3) as compared to 25 Alzheimer Disease patients (Ageyears = 74.3 ± 8.9, Gender-Female % = 68.0), and to 36 healthy controls (Age-years = 57.5 ± 14.8, Gender-Female % = 61.1), we established the serum GRN cutoff level of 125 ng/mL, to distinguish GRN null mutations carriers, with a specificity of 97.4 % and a sensitivity of 81.8 % (unpublished data). Outcomes Primary endpoint was the dosage of progranulin levels over the time, determined in serum at T = 0–5; the secondary endpoint was the clinical course at 1 year, as compared to natural disease course in untreated GRN mutation carriers.
10, a disease duration less than 5 years). Almost all included patients had a bvFTD, one had avPPA; main clinical features are reported in Table 1. Serum progranulin levels were determined at baseline (T = 0), before amiodarone administration, and after 1-week, 4, 12, 24, 54 weeks of treatment. As shown in Fig. 1, patient 1 presented a higher serum GRN level to what reported as optimal plasma GRN cutoff value for predicting null mutations, but under the normal GRN determination, as based on our unpublished data [11]. Indeed, for patients who have a positive family history, and a younger disease onset, with a borderline plasma or serum GRN levels, the complete sequence of the gene should be recommended. No significant increase was observed after amiodarone therapy at different times, as reported in Fig. 1. Two patients dropped out after the third month treatment for the onset of behavioral disturbances characterized by agitation (patient 3), and jealousy delusions (patient 1). The three other patients completed the study, in the absence of any adverse event, with no significant increase of progranulin levels (Fig. 1). Patient 4 was institutionalized at the end of the study because of the severity of the disease. Disease stage after 1-year treatment is reported in Table 1 (i.e. FTD-CDR). We compared this group of patients treated with amiodarone to other 13 FTD with the same mutations (mean age = 57.61 ± 6.75; 46.2 % male), who were followed in the previous years and did not enter into the present study. These two subgroups of FTD-GRN patients were comparable in terms of disease severity (i.e., FTD-CDR; patients treated with amiodarone: 7.70 ± 2.10 vs. untreated: 8.19 ± 2.71, p = 0.703). During 1-year follow-up, the mean increase of FTD-CDR did not differ between the treated (mean value 7.40 ± 5.14 points) and untreated (mean value 4.50 ± 2.19 points) FTD patients (p = 0.289).
Results We enrolled five FTD patients with GRN Thr272fs (g.1977_1980delCACT) mutation (mean age = 64.4 ± 4.7; 20 % male), all with a positive family for dementia, in a relatively early stage of their disease (FTD-CDR less than
Discussion FTD is a devastating disease for patients who are bereaved from their personality or their ability to communicate with
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language, with an enormous impact on their relatives and high costs for the society. Previous studies attempted to verify the efficacy of different symptomatic therapies on the disease course, although with poor results. In a trial with galantamine administered to behavioral and aphasic variants of FTD, no effect on behavior or language was found, excluding a minor trend of efficacy in the aphasic patients [12]. A significant improvement in scores on the Neuropsychiatric Inventory and reduced recognition of angry facial expressions was observed in patients with bvFTD who received a single dose of oxytocin [12]. Citalopram was demonstrated to be effective in relieving from behavioral disturbances in a small number of patients [13]. More recently, the findings of a multicentre, randomized, double-blind, placebo-controlled trial of memantine showed no treatment benefit, in analogy to what reported in a previous French trial [14–16]. At our best knowledge, the present study represents a first pilot trial targeting pathological determinants associated to the disease in FTD, in particular in patients bearing GRN mutation. The molecular pathways that lead from reduced progranulin levels to TDP43 inclusions and therefore to neurodegeneration are still to be clarified, but the possibility to rescue progranulin levels offers an opportunity for strategies of intervention. The paper by Capell and colleagues [8] demonstrated an increase of progranulin level in lymphoblastoid derived from loss-of-function carrier treatment with different alkalizing agents. Using a different approach, Cenik and colleagues [17] identified suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor as an enhancer of GRN expression. Among the different agents proposed in the paper by Capell [9], amiodarone is largely used in cardiologic application, and therefore was sought to be firstly assayed. Unfortunately, our results, although limited to a small number of patients, are not promising in this sense, but may help in future to identify other alkalizing agents, including chloroquine, or different approaches to be pursued. We acknowledge that the present study has several limitations, in first instance the drug has not been tested on specific animal models, in particular in available GRN KO mice [18–20]. The rationale to administer amiodarone directly in patients, by-passing a first step in animal models, was based on the broad use of amiodarone in cardiologic settings, since a long time. However, amiodarone final dosage was limited to what commonly used for atrial fibrillation. Finally, the number of recruited subjects is very small, and the study lacks of plasma amiodarone levels determinations, along with the lack of cerebrospinal fluid progranulin determination. Nevertheless, we provided a first hint for future multicentre trial aiming to contrast progranulin deficiency in FTD.
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Acknowledgments The authors wish to acknowledge the helpful and generous collaboration of patients and their family members, which was essential for this study. The authors are indebted with Dr. Francesca Ferrari for technical assistance. Conflict of interest All co-authors agreed with the contents of the manuscript and there is no financial interest to report.
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