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. 2014 Sep 2;22(11):2004–2012. doi: 10.1038/mt.2014.138

A Chaperone Enhances Blood α-Glucosidase Activity in Pompe Disease Patients Treated With Enzyme Replacement Therapy

Giancarlo Parenti 1,2, Simona Fecarotta 1, Giancarlo la Marca 3, Barbara Rossi 2, Serena Ascione 1, Maria Alice Donati 4, Lucia Ovidia Morandi 5, Sabrina Ravaglia 6, Anna Pichiecchio 6, Daniela Ombrone 3, Michele Sacchini 4, Maria Barbara Pasanisi 5, Paola De Filippi 7, Cesare Danesino 7, Roberto Della Casa 1, Alfonso Romano 1, Carmine Mollica 8, Margherita Rosa 1, Teresa Agovino 1, Edoardo Nusco 2, Caterina Porto 1,2, Generoso Andria 1,*
PMCID: PMC4429731  PMID: 25052852

Abstract

Enzyme replacement therapy is currently the only approved treatment for Pompe disease, due to acid α-glucosidase deficiency. Clinical efficacy of this approach is variable, and more effective therapies are needed. We showed in preclinical studies that chaperones stabilize the recombinant enzyme used for enzyme replacement therapy. Here, we evaluated the effects of a combination of enzyme therapy and a chaperone on α-glucosidase activity in Pompe disease patients. α-Glucosidase activity was analyzed by tandem-mass spectrometry in dried blood spots from patients treated with enzyme replacement therapy, either alone or in combination with the chaperone N-butyldeoxynojirimycin given at the time of the enzyme infusion. Thirteen patients with different presentations (3 infantile-onset, 10 late-onset) were enrolled. In 11 patients, the combination treatment resulted in α-glucosidase activities greater than 1.85-fold the activities with enzyme replacement therapy alone. In the whole patient population, α-glucosidase activity was significantly increased at 12 hours (2.19-fold, P = 0.002), 24 hours (6.07-fold, P = 0.001), and 36 hours (3.95-fold, P = 0.003). The areas under the curve were also significantly increased (6.78-fold, P = 0.002). These results suggest improved stability of recombinant α-glucosidase in blood in the presence of the chaperone.

Introduction

Pompe disease (OMIM 232300, ORPHA365) is a metabolic myopathy caused by mutations of the GAA gene and by functional deficiency of the acid α-glucosidase (GAA, acid maltase, E.C.3.2.1.20), a glycoside hydrolase involved in the lysosomal breakdown of glycogen.1 The lack of functional GAA results in the typical pathological hallmarks of the disease, extensive glycogen storage in multiple tissues, and massive accumulation of autophagic vesicles and autophagic debris in muscles.2

Cardiac and skeletal muscles are particularly vulnerable to these cellular abnormalities, and are at the same time the main sites of pathology in Pompe disease and the major targets of therapy, although recent studies indicate that neural deficits contribute to muscle weakness in Pompe disease.3,4 The severity of cardiac and muscular manifestations varies broadly in Pompe patients. Infantile-onset patients may present within the first months of life with the “classic” form of the disease, characterized by hypotonia, hypertrophic cardiomyopathy, respiratory infections, and, if untreated, die within the first year of life. In “non-classic” infantile-onset patients, heart involvement is usually absent. In late-onset patients, clinical manifestations are mostly related to skeletal muscle involvement. Although characterized by an attenuated course, late-onset phenotypes are highly debilitating and cause progressive motor impairment, respiratory failure, and premature death.

The only approved treatment for Pompe disease, enzyme replacement therapy (ERT) with human recombinant GAA5 (rhGAA, alglucosidase), has been characterized by both extraordinary successes and unexpected failures. ERT was effective in reversing cardiac involvement and prolonging survival in classic infantile-onset patients.6,7 In late-onset patients, ERT resulted in improvements of neuromuscular deficits and stabilization of respiratory function.8,9 On the other hand, some manifestations related to the involvement of specific muscles and fiber types apparently remain refractory to treatment. Selective resistance of specific muscles or fibre types to ERT has been reported.10 The relative insensitivity of skeletal muscles to ERT has been ascribed to a number of factors, such as disturbed autophagic pathway, the age at start of therapy,11 and the cross-reactive material status of patients.12 These observations suggest that ERT may not be sufficient to treat all the aspects of Pompe disease and that alternative approaches would be needed.

An emerging strategy to treat diseases due to protein misfolding, including lysosomal storage disorders, is based on the use of small-molecule “chaperones,” that, by interacting with mutant misfolded proteins, enhance their correct conformation, and prevent their degradation by the endoplasmic reticulum-associated degradation system. This approach was proposed also for Pompe disease.13,14 The most extensively studied GAA chaperone molecules are two glucose analogues, the imino sugars 1-deoxynojirimycin (DNJ, AT2220) and N-butyldeoxynojirimycin (NB-DNJ, miglustat).

Although pharmacological chaperone therapy was initially designed to prevent degradation of misfolded mutant proteins, we showed that chaperones are able to enhance the efficacy of the wild-type recombinant enzyme used for ERT both in a study performed in Pompe fibroblasts and in the animal model of the disease.15 Coadministration of NB-DNJ and rhGAA in Pompe disease fibroblasts resulted in increased intracellular activity, with facilitated lysosomal trafficking, maturation and stability of rhGAA in cells. Preclinical in vitro and in vivo studies15,16 highlighted the potential of treatment protocols based on the combination of different therapeutic strategies and opened the way to translation into a clinical trial.

Here, we describe the results of the first clinical trial on the combination of ERT with one of the imino sugar chaperones, NB-DNJ, already investigated in preclinical studies. This drug has already been approved for clinical use for different indications, as an inhibitor of substrate synthesis in Gaucher disease17 and in Niemann-Pick disease type C,18 and its pharmacokinetics and safety profile have been well characterized. We chose to utilize a biochemical measure, the profile of GAA activity in dried blood spots (DBS) after ERT, as endpoint of the study, because evaluation of this parameter did not require invasive procedures or hospitalization of patients.

Results

This open, intra-patient interventional study was designed to evaluate whether ERT with rhGAA (Myozyme, Genzyme, Cambridge, MA; 20–40 mg/kg/infusion) in combination with the chaperone NB-DNJ (miglustat, Zavesca, Actelion, Basel, Switzerland; 80 mg/m2 × 4 doses) resulted in significantly higher GAA activity in DBS, when compared with the activities obtained with ERT alone.

Thirteen patients, followed at four Italian centers, were enrolled in the study (Table 1).

Table 1. Demographics, molecular and clinical data of patients enrolled in the study.

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The duration of the study was 16 months. After a baseline assessment for 2 months with rhGAA alone (Figure 1a, protocol ERT1), the patients were treated for 12 months with the combination of rhGAA and NB-DNJ (protocol ERT+CHAP), and subsequently were re-evaluated for 2 months with rhGAA alone (protocol ERT2).

Figure 1.

Figure 1

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Protocol of ERT and NB-DNJ administration and DBS sampling. (a) The overall duration of the study was 16 months. The patients were subjected to a baseline assessment for two months with rhGAA alone (ERT1), then were treated for 12 months with the combination of rhGAA and NB-DNJ (ERT-CHAP), and subsequently were re-evaluated for two months with rhGAA alone (ERT2). GAA profiling in DBS was performed three times at baseline (ERT1) and three times in the first ERT cycles of ERT+CHAP, and again three times at the end of ERT+CHAP, and three times in ERT2. (b) Patients were treated with rhGAA at standard doses of 20 or 40 mg/kg/infusion (day 0). rhGAA was reconstituted according to the manufacturer's instructions and given intravenously, in 5–7 hours. NB-DNJ was given in four doses of 80 mg/m2 each, one on the evening before ERT (day -1), and three on the day of the infusion (day 0). The sampling for GAA activity in DBS was performed before each ERT infusion (day −1), and on days 1, 3, 5, 7, 9, and 11, (DBS sampling 1) at the beginning of the study (protocol ERT1, months 1–2, and first round of sampling in ERT-CHAP, months 3–4). To better evaluate the GAA profile in the first 72 hours following ERT, at the end of the ERT+CHAP period (months 13–14) and during ERT2 (months 15–16), GAA activities were assayed in DBS every 12 hours for three days (DBS sampling 2).

The primary endpoint of the study was to obtain increased GAA activity in DBS with the combination protocol ERT+CHAP. The timing of DBS sampling and of clinical and biochemical evaluations, are indicated in Figure 1b and in Table 2.

Table 2. Clinical and biochemical procedures.

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The experimental protocol was well tolerated by patients. Only minor adverse events related to the combination therapy were reported (Supplementary Table S1).

DBS profiles after ERT

In all patients included in the study, the GAA activity profiles in DBS were characterized by a rapid peaking within the first 24 hours after the start of ERT, and by a decline of GAA activity within 2–3 days to levels that were close to baseline. The GAA activity profile and the peak levels obtained in each patient studied were consistent and reproducible in the different cycles of therapy. On the other hand, although most patients were treated with equivalent doses of recombinant enzyme, the average peak activities varied considerably among different patients. Plasma anti-GAA antibody titers were studied in 10 patients and did not correlate with GAA peak levels (Supplementary Material).

DBS profiles with and without chaperone

When the rhGAA was given in combination with NB-DNJ in 11 patients we observed increases of the areas under the curve of GAA activity greater than 1.85-fold, when compared with areas obtained with ERT alone (Figure 2a).

Figure 2.

Figure 2

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GAA activity profiles in DBS. (a) The combination of ERT and NB-DNJ (protocol ERT+CHAP) resulted in increases of GAA activity peak areas greater than 1.85-fold, compared to ERT alone in patients 0101, 0103, 0201,0202, 0203, 0204, 0301, 0302, 0303, 0401, 0402). (b) In the whole population, the average GAA activities were significantly increased compared to ERT alone at 12 hours (P = 0.002), 24 hours (P = 0.001), 36 hours (P = 0.003), and remained significantly higher up to 72 hours (inset). The increase in the areas under the curve ranged between 1.90 and 3.42-fold, with an average increase of 6.78, and was also highly statistically significant (P = 0.002).

When the data of the whole population were pooled there was a 2.19-fold increase (range 1.10–14.53) of GAA activity at 12 hours with the combined treatment, compared with the activities obtained with ERT alone (Figure 2b and Table 3). The increase was statistically significant (P = 0.002). At 24 hours the average increase in activity with the combination of ERT and NB-DNJ was 6.07-fold (range 1.65–29.19, P = 0.001) (Table 3). At 36 hours, the average increase in activity with the combination of ERT and NB-DNJ was 3.95-fold (range 2.33–5.84, P = 0.003) (Table 3). The increase in the areas under the curve ranged between 1.31- and 7.88-fold, with an average increase of 2.59, and was also statistically significant (P = 0.002) (Table 3). Significant differences in GAA activity persisted at 48 (P = 0.002), 60 (P = 0.004), and 72 hours (P = 0.002) (Figure 2b, inset).

Table 3. GAA activities and statistics in the whole population.

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In the 10 patients tested, the combination protocol was not associated with substantial and consistent changes in plasma anti-GAA antibody titers (Supplementary Material).

Other outcome measures

None of the additional parameters evaluated in the patients revealed significant changes. Plasma CK levels showed substantial intra and inter-patient variations. A trend towards a decrease of CK levels with the combination therapy (ERT+CHAP) was observed in patient 0102. In all other patients, no variations of CK values were observed (Supplementary Figure S1).

Urinary GLC4 was measured by tandem mass spectrometry in all patients (Supplementary Figure S2). Four of them (0101, 0102, 0103, 0202), presenting with infantile-onset and more severe phenotypes (see Table 1), showed elevated levels of GLC4 excretion, in some cases with substantial fluctuations among the different samples. In all other patients (with milder phenotypes), the GLC4 levels remained within normal range throughout the study. None of the patients showed significant changes in GLC4 excretion with the ERT+CHAP protocol, compared with samples obtained during the baseline assessment (ERT1).

At baseline (V0), in patients younger than 18 years (0101, 0102, 0103, 0201, 0203, 0204), the MRI signs of muscle adipose substitution were overall very mild and scattered, without any detectable pattern of selective muscle involvement. Adult patients (0301, 0302, 0303, 0401, 0402) showed selective muscle involvement in the lower limbs, with a pattern similar to that previously reported in Pompe patients.19 Paraspinal and psoas muscles were also involved. In the shoulder girdle there was a prominent involvement of subscapularis muscles in all adult patients.

At the end of the study (V5), MRI was performed in 10 patients (excluding patient 0301). The MR images showed an unchanged qualitative pattern of distribution of intramuscular fatty replacement in all patients; patient 0303 showed a modest reduction of trophism at the vastus lateralis.

Nine patients were evaluated by the six-minute walk test (6MW) (Supplementary Figure S3). For six of them, historical data were available, while in three, we only have the data collected during the trial. In five patients (0101, 0201, 0204, 0205, 0303), the distance walked was increased (more than 50 meters) at the end of the study, compared to baseline. In three (0203, 0302, 0402), the distance remained unchanged. Patient 0401, who had already experienced a progression of motor impairment during ERT alone and was not able to walk more than 70 meters, showed a minor decrease in the distance walked. When the data from all nine patients were pooled an increase in the average distance walked (+58 m ± 61) was observed at V6 (12 months) (Supplementary Figure S3, bottom). However, this increase was not statistically significant.

The levels of dyspnoea at the 6MW test (Supplementary Figure S4a), the expected FVC (Supplementary Figure S4b) and the MMT/MRC test (Supplementary Figure S4c) remained substantially stable in the patients studied.

GAA activities in DBS and tissues from the Pompe disease mouse model

We replicated the studies done in patients in the Pompe disease murine model. This allowed us to analyze, in addition to DBS, the most clinically relevant tissues, namely skeletal muscles, in which increased therapeutic efficacy of ERT would be required, and liver, the organ that clears up to 85% of the recombinant enzyme provided by ERT.

GAA activity was assayed in DBS and tissues every day for 3 days. In DBS, the GAA activity in mice treated with ERT alone was increased 24 hours after the injection (Figure 3a, dotted line), and showed a rapid decrease in the following time-points. A parallel increase in GAA activity was detectable in liver, gastrocnemius and quadriceps, where the GAA activity persisted up to 72 hours (Figure 3b–d, dotted lines). As expected, the greatest increase was observed in liver.

Figure 3.

Figure 3

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GAA activities in the Pompe diseasemouse. Pompe disease mice received a single retro-orbital injection of rhGAA with or without an oral administration of NB-DNJ by gavage, and GAA activity was measured in DBS and tissues (liver, quadriceps and gastrocnemius) at 24, 48, and 72 hours. For each time point the average and SD of the activities in three to five animals is shown. The DBS GAA profile in mice showed a maximum peak at 24 hours after the injection and a rapid decrease of activity over the following days (dotted line). A parallel increase in GAA activity was detectable in liver, gastrocnemius and quadriceps, where the GAA activity persisted up to 72 hours (dotted lines). In the animals treated with the combination of ERT and NB-DNJ a significantly higher peak (approximately fivefold compared to ERT alone; P = 0.01 at 24 and 48 hours) was observed in DBS (continuous line). At 48 hours, a statistically significant increase in GAA activity with the combination protocol was observed in liver (P = 0.02), and in quadriceps (P = 0.04). The activities obtained in tissues from the Pompe disease mice treated with ERT alone or with the combination protocol were compared using the Student's t-test.

In the animals treated with the combination of ERT and NB-DNJ GAA activity in DBS showed a fivefold increase compared to ERT alone (Figure 3a, continuous line). We observed a greater increase in activity at 48 hours, that was statistically significant in liver (P < 0.02) and quadriceps (P = 0.04) (Figure 3b–d, continuous lines). The average fold increase at 48 hours in tissues was lower compared to that seen in DBS (liver 1.89, quadriceps 2.22, and gastrocnemius 1.34). In the animals treated with the combination therapy, only traces of NB-DNJ were found in liver within the first 24–48 hours, while the drug was undetectable in muscles (not shown).

Discussion

Over the past years, different lines of research provided rationale and proof of principle to use a combination of ERT and pharmacological chaperone therapy. Some studies suggested that the recombinant enzymes used for ERT are relatively unstable after injection and during their route to reach the lysosomes, where their activities are required. In the animal model of Gaucher disease, a lysosomal disorder due to β-glucocerebrosidase deficiency, only a fraction of the recombinant β-glucocerebrosidase (imiglucerase) injected in the animals could be recovered from tissues,20 and the half-life of imiglucerase in tissues appeared to be shorter than expected. rhGAA was shown to be unstable at neutral pH21 and in blood.22 Other studies, performed both in cultured fibroblasts and in the animal models of Pompe and Fabry disease patients, demonstrated that the physical stability of recombinant enzymes could be enhanced by the presence of enzyme-specific chaperones, with an increase in the activity and in the amounts of protein assessed by western blot analysis.15,21,22 Studies performed in cultured fibroblasts of Pompe and Fabry disease incubated with the respective recombinant enzymes showed that the enhancing effect was only seen when the proper chaperone was used, indicating that the stabilizing effect of chaperones is specific, and is not mediated by other activities.23

Further support to the use of the combination of chaperones and recombinant enzymes derived from biochemical and computational analyses that highlighted the structural changes of wild-type lysosomal enzymes induced by the binding of chaperones.21,24

In this study, we translated this preclinical evidence into the first clinical trial based on the combined use of ERT and a chaperone. As a first-line measure to document the effects of such a combination, we chose the least invasive procedure, GAA activity in DBS, in order to cause minimal discomfort to patients. Other similar trials, that are in progress for Pompe25 and Fabry disease,26 are based on more invasive protocols, that may be distressing, especially in young patients. The earliest time point chosen for DBS sampling was 12 hours after the ERT infusion. Earlier time points (when GAA activity is expected to be even higher) were excluded in this study, being not relevant for the increased stability of rhGAA in the presence of the chaperone. In fact, previous studies performed in wild-type rats infused with rhGAA demonstrated that the effect of the chaperone AT2220 on the stability of the recombinant enzyme in vivo became most evident several hours (4–8) after an ERT infusion.16 The heterogeneity of the patient population was not considered a relevant issue for the purpose of the study, being a biochemical measure the main endpoint. In fact, our results show that the enhancing effect of the chaperones is independent of the clinical presentation and is observed in patients with different phenotypes and genotypes.

We found increased and prolonged GAA activity in DBS with the combination of chaperones and rhGAA, compared to the activities obtained with ERT alone. The results were consistent and reproducible in each individual patient, even after one year. The effect of NB-DNJ on GAA activity in DBS was evident in 11 patients. Overall, when the average data from all patients were pooled, this result was highly significant at all time points up to 72 hours. This may reflect an increased stability of the enzyme in plasma, as shown in the animal models of Pompe and Fabry disease,16,22 or in blood cells similarly to what observed in Pompe fibroblasts.15 This might allow a better bioavailability of the enzyme for the target tissues, where greater therapeutic efficacy is needed. Variations in GAA peak activities in DBS among different patients cannot be clearly explained and may reflect individual variations in kinetics and uptake of rhGAA. In vitro studies suggested that the efficacy of rhGAA may vary in cells from patients with different phenotypes, and that the integrity (or the disruption) of the mannose-6-phosphate pathway impacts the correction of enzyme activity by rhGAA.27 Although these studies have been done in fibroblasts (not the best cellular model to study a myopathy, like Pompe disease), they may reflect a more general variability in the handling of the recombinant enzyme by other cell types, including muscle fibers, in patients with different phenotypes.

In the parallel study that we conducted in the animal model of the disease, we confirmed that coadministration of chaperones increased the stability of rhGAA in DBS. In liver and muscles, we observed significant increase in GAA activity at 48 hours.

We also evaluated some biochemical and clinical measures that in previous work had been monitored as indicators of disease severity. Although in few patients we observed an improved performance at the 6MW test, none of the markers studied was significantly changed with the combination protocol. However, this is not surprising if we consider the design of our intra-patient study, performed in subjects that had already been treated for long periods with ERT and were switched to a different therapeutic regimen for a relatively short period, and the limitations of commonly used clinical outcome measures.28 In other ERT trials in Pompe disease,8,9 these markers were compared in large cohorts of treated versus untreated patients, thus allowing statistical significance of the results. Overall, the clinical measures evaluated in our study suggested stability of the disease course that, in principle, may indicate a favorable response to the combination treatment. In this respect, it must be pointed out that a retrospective study on the Pompe patients treated in the UK29 showed that, after an initial improvement in the first 1–2 years, the clinical course of Pompe disease patients on ERT was characterized by a plateau and subsequent resumed disease progression.

In conclusion, this is the first study that reports on the results of a clinical trial based on the combination of ERT and chaperone therapy with NB-DNJ in a lysosomal storage disease. We showed that the combination protocol leads to higher GAA levels in blood, compared to those obtained with ERT alone. A combined treatment is only expected to result in a very limited increase in the costs of therapies, since the chaperone is administered only at the time of ERT. For an adult patient a single Miglustat box would be sufficient for almost a year. Compared to the cost of a single rhGAA infusion this cost is negligible. It is possible to speculate, based on preclinical studies obtained in our laboratory and by other groups, that similar imino sugar chaperones, such as AT2220, may show the same enhancing effect on ERT. Also, it may be reasonable to expect that ERT and chaperone coformulation for intravenous administration may be as effective. In principle, the enhancing effect of chaperones on ERT may also apply to second-generation recombinant enzymes that are presently under evaluation,30,31 or in combination with strategies aimed at improving muscle targeting of recombinant GAA.32,33 This study did not address the question as to whether this effect of chaperones on ERT results in greater therapeutic efficacy of ERT. Clinical efficacy of the combination protocol should be tested in future trials in larger cohorts of patients, preferably with homogeneous phenotypes, and for longer observational periods.

Materials and Methods

Study design and participants. The study protocol (MIGLU-3, EudraCT n 2010-024647-32) was approved by the Ethical Committees of the centers involved in the study. All patients, or their legal guardians, signed an informed consent before enrollment in the study.

Thirteen Pompe disease patients were recruited for the study. The patients were followed at four Italian centers (Department of Translational Medicine, Federico II University, Naples, first two digits of patients' code 01; Meyer's Hospital, University of Florence, Florence, codes 02; Istituto C. Besta, Milan, codes 03; Istituto C. Mondino, Pavia, patients' codes 04). All these Institutions are referral centers for the diagnosis, care and follow-up of inborn errors of metabolism.

In all patients, the clinical and enzymatic diagnosis was confirmed by the molecular analysis of the GAA gene. The common juvenile c.32-13T>C mutation was found in 10 patients. Patient 0102 carries this mutation in cis with the mutation c.1655T>C, and the mutation c.1856G>A on the second allele. Patient 0205 is a compound for the complex mutation c.1833_1839 del; c.1846G>T; c.1847_1848 insT and the c.32-13T>C mutation. The mutation c.1655T>C was detected in three patients.

At enrollment all patients had been on ERT with rhGAA at standard doses (20–40 mg/kg/every 2 weeks) for variable periods (1–8 years). One patient (0103) was affected by the classic infantile-onset (IC) form, two patients (0101, 0102) had the non-classic infantile-onset (INC) form; the others were affected by late-onset (LO) Pompe disease. Four patients had severe motor impairment (0102, 0103, 0202 and 0401), two required non-invasive ventilatory support (0102, 0302), and one required invasive-mechanical ventilation through tracheostomy.

After a baseline assessment for 2 months with rhGAA alone (Figure 1a, protocol ERT1), the patients were treated for 12 months with the combination of rhGAA and NB-DNJ (protocol ERT+CHAP), and subsequently were re-evaluated for 2 months with rhGAA alone (protocol ERT2). One patient (0301) exited the study at month 2 of the ERT+CHAP protocol.

The rhGAA doses were 40 (patient 0103) or 20 (all other patients) mg/kg/infusion. NB-DNJ was given in four doses of 80 mg/m2 each, one on the evening before ERT, and three on the day of the infusion (Figure 1b).

The timing of clinical and biochemical evaluations of the patients enrolled is indicated in Table 2.

DBS sampling. The sampling for GAA activity in DBS was performed three times with ERT alone and three times with the combination of ERT and NB-DNJ at the beginning of the study (protocols ERT1 and first round of sampling in ERT+CHAP) (Figure 1b). In this part of the study, DBS samples were taken before each ERT infusion, after 24 hours and then every other day for the following 13 days until the next infusion. In the second part of the study (ERT+CHAP and ERT2), to better evaluate the GAA profile in the first 72 hours after ERT (that appeared to be more critical for the GAA activity profiling) DBS were obtained before the infusion and every 12 hours for the first 3 days after ERT (Figure 1b). Also, in this part of the study, DBS were obtained three times with the combination of rhGAA and NB-DNJ and three times with rhGAA alone.

At the end of the study, for each patient and each time-point the number of samples collected and suitable for analysis varied between three and six samples with ERT alone and between three and six with the combination of ERT and the chaperone. For some patients, occasional samples collected at home were unsuitable for analysis, causing deviation from the planned number of six samples for each time-point/patient.

DBS samples were collected on the day of infusion by the medical team, or at home (other time-points) by patients or caregivers according to a standard operating procedure, for which patients and caregivers had been adequately instructed and trained.

DBS were prepared by spotting blood drops corresponding to approximately 25 μl of whole blood on filter paper. Blood spots were dried for a period of at least 2 hours at room temperature and were stored at −18 °C until analysis in sealed plastic bags containing desiccant and a humidity indicator card.

The samples were shipped in dry ice by the centers in Naples, Milan, and Pavia to the laboratory of the Meyer's Hospital in Florence, where the analysis of GAA activity in DBS was performed.

GAA activity assay. The activity of GAA was measured in blood spots according to previous studies.34 Spots of 3.2 mm (containing about 2.8−3.0 μl blood) were punched from each DBS sample into a 96-well plate. Twenty μl of substrate were added to each well. The plates, covered with lids and aluminum foil, were placed in a water bath set at 37 °C for 20−24 hours. After incubation, the reaction mixtures were quenched with 60 μl of methanol containing 0.1% formic acid, centrifuged (ALC PK 120 R, DJB Labcare, Newport Pagnell, UK) to remove any suspended paper particles (2,500 rpm for 3 minutes). Blank reactions in duplicate were performed for each enzyme assay by using 3.2 mm diameter disks from blank filter paper. GAA activitiy was calculated by subtracting the blank contribution.

TMS analysis was performed on an API 3200 (Applied Biosystems, Foster City, CA) equipped with the ESI ionization probe fitted on the Turbo-V source. For the online trapping and-cleanup/liquid chromatography configuration, an Agilent 1100 LC-binary pump and an Agilent 1100 thermostated autosampler were used. In order to operate the Agilent 1100 binary pump as two independent pumping heads, internal plumbing has been modified by removing the mixing-T and leaving the dumper on the channel A. The trapping and cleanup procedure was centered on a Perfusion column POROS R1/20 2 × 30 mm (Applied Biosystems). Separation chromatography was performed through a Metachem Polaris C18 3 μm, 2 × 50 mm (Metachem, Lake Forest, CA). The amount of product generated by the enzyme assay was calculated from the ion abundance ratio between the enzymatic reaction product (P) and the associated internal standard (P/IS), multiplied by the amount of added IS, and divided by the response factor ratio of P and IS. The enzyme activity expressed in units of “μmol/hour/l whole blood” was then calculated from the amount of product further divided by the used amount of blood and by the incubation time.

Samples in which GAA activity exceeded 150 μmol/hour/l were diluted to avoid the saturation of the mass spec detector.

Statistical analysis. All data were analyzed using the SPSS software (SPSS, Chicago, IL). Data were analyzed using the Wilcoxon signed rank test, a nonparametric test to compare two dependent variables. The mean of GAA levels at different time-points and areas under the curve were calculated for each patient. The area under the curve was calculated from the concentration-time data by the linear trapezoid method. We set up discrete blocks under the curve. Each rectangle represents an approximation of the area for that interval. The sum of the rectangles is equivalent to a trapezoid which extends linearly between concentration (C) and observation time (t). The area of each trapezoid was calculated according to the equation: area = 1/2 (C1 + C2) (t2 − t1). The difference between the calculated means obtained with ERT alone were compared to the means obtained with the combination protocol in the whole population studied. The α was set at 0.05 for all dependent variables in the study. A significant result would indicate that plasma GAA levels or areas under the curve changed significantly when comparing the data with ERT alone or with the combination treatment. Intra-patient comparison of GAA levels and areas was performed using descriptive statistics.

Additional procedures

Clinical evaluations. Plasma creatine kinase (CK), and routine blood biochemistry were performed according to standard procedure in the laboratories of each participating centre.

Urinary glucose tetrasaccharide (GLC4) was analyzed according to An et al.35

Muscular strength and function were evaluated according to Gross Motor Function Measure GMFM (Version 1.0, Mac Keith Press, 2002)36 in patients younger than four years and Manual muscle testing by Medical Research Council MMT/MRC37 in patients older than 4 years. The 6 minutes walking test was performed according to the statements of American Thoracic Society.38 The subjective assessment of dyspnoea and muscular fatigue was performed using the Borg Scale.39

Muscle imaging. Muscle MRI was performed in 11 patients (0101, 0102, 0103, 0201, 0203, 0204, 0301, 0302, 0303, 0401, 0402) evaluating every single muscle bulk of the following muscle districts: inferior limb, pelvic girdle, paraspinal muscles, and shoulder girdle at baseline (V0) and at the end of the study (V5).

Muscle MRI was done according to previously described methods.19 Muscle images were obtained with a Philips Gyroscan Intera 1.5 (Milan, Florence, Pavia) or 3T (Naples), using axial T1-weighted spin-echo sequences (TR, 300 ms; TE, 10 ms; two averages; thickness, 10 mm; slice gap, 1 mm; RFOV, 80%; FOV, 400 mm) in order to study the inferior limb, the pelvic girdle, the paraspinal muscles, and the scapular shoulder girdle. The total imaging time was about 50 minutes. The scans were examined by two independent observers, blinded to clinical data, looking for normal and abnormal muscle bulk (atrophy) and for normal and abnormal signal intensity within the different muscles. Each muscle group was graded according to the degree of fatty degeneration following the scale proposed by Mercuri et al.40 as follows: Stage 0: normal appearance. Stage 1: early moth-eaten appearance, with scattered small areas of increased signal. Stage 2a: late moth-eaten appearance, with numerous discrete areas of increased signal with beginning confluence, comprising less than 30% of the volume of the individual muscle. Stage 2b: late moth-eaten appearance, with numerous discrete areas of increased signal with beginning confluence, comprising 30–60% of the volume of the individual muscle. Stage 3: washed-out appearance, fuzzy appearance due to confluent areas of increased signal. Stage 4: end-stage appearance, muscle replaced increased density connective tissue and fat, with only a rim of fascia and neurovascular structures distinguishable.

Animal studies. Animal studies were performed according to the EU Directive 86/609, regarding the protection of animals used for experimental purposes. Every procedure on the mice was performed with the aim of ensuring that discomfort, distress, pain, and injury would be minimal. Mice were euthanized following avertin anesthesia by cervical dislocation.

A KO Pompe disease mouse model obtained by insertion of neo into the Gaa gene exon 6 (ref. 41) was purchased from Charles River Laboratories (Wilmington, MA) and maintained at the Cardarelli Hospital's Animal Facility (Naples, Italy).

Six month-old mice received NB-DNJ (6 mg/kg), dissolved in 0.5 ml saline, administered daily by gavage for 2 days, or with 0.5 ml saline. On the second day, the animals were injected into the retro-orbital vein with rhGAA at a dose of 40 mg/kg. Both NB-DNJ and rhGAA doses corresponded to those used for the clinical study.

DBS spots were obtained 24, 48, and 72 hours after the injection of rhGAA. GAA assay in dried blood spots was performed as indicated above.

The animals were sacrificed at different times (24, 48, and 72 hours) after ERT, and liver, gastrocnemii and quadriceps were harvested, homogenized, and stored at −80 °C until they were used for the assay of GAA activity. For each time point (both DBS and activity in tissues), we tested three to five animals.

GAA activity in tissues was measured as indicated in previous studies,15 using the fluorogenic artificial substrate 4-methylumbelliferyl (4MU)-alfa-D-glucopyranoside (Sigma-Aldrich, St Louis, MO).

SUPPLEMENTARY MATERIAL Figure S1. Plasma CK levels in the PD patients. Figure S2. Urinary GLC4 measured by tandem mass spectrometry. Figure S3. Six-minute walk test (6MW). Figure S4. Clinical measures in the patients included in the trial. Table S1. Adverse events in patients treated with the combination of ERT and NB-DNJ. Material.

Acknowledgments

This work was supported by the Telethon Foundation, Rome, Italy (grant GUP09017 to G.A. and grant TGPMT4TELD to G.P.). We thank Graciana Diez-Roux (Telethon Institute of Genetics and Medicine) for critically reviewing the manuscript and for helpful discussion and suggestions. We thank Antonio Correra, Maria Teresa Carbone, Daniela Concolino, Demetrio Costantino, Giuseppina Mansi, Paolo Buonpensiero, and Rosaria Tuzzi that collaborated in the treatment and monitoring of patients. We are also very grateful to the patients and their families for participating in this study. G.A. and G.P. designed the study, analyzed the results, and wrote the manuscript; S.F. collected and analyzed the data, and collaborated in the writing; G.l.M. analyzed the dried blood spot samples; B.R., K.P., and E.N. performed the animal studies; S.A. collaborated in the collection and interpretation of data and performed the statistical analysis; M.A.D., M.S., L.O.M., M.B.P., C.D., S.R., P.D.F., R.D.C., and A.R. treated and monitored the patients; A.P. and C.M. examined the muscle MRIs of patients; D.O. analyzed the dried blood spot samples; M.R. and T.A. collaborated in the collection and interpretation of data. The authors declare no conflict of interest.

Supplementary Material

Supplementary Figure S1

Plasma CK levels in the PD patients.

Click here for additional data file. (130.2KB, pdf)
Supplementary Figure S2

Urinary GLC4 measured by tandem mass spectrometry.

Click here for additional data file. (134.8KB, pdf)
Supplementary Figure S3

Six-minute walk test (6MW).

Click here for additional data file. (647.9KB, pdf)
Supplementary Figure S4

Clinical measures in the patients included in the trial.

Click here for additional data file. (1.1MB, pdf)
Supplementary Table S1

Adverse events in patients treated with the combination of ERT and NB-DNJ.

Click here for additional data file. (66.8KB, pdf)
Material
Click here for additional data file. (1.1MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure S1

Plasma CK levels in the PD patients.

Click here for additional data file. (130.2KB, pdf)
Supplementary Figure S2

Urinary GLC4 measured by tandem mass spectrometry.

Click here for additional data file. (134.8KB, pdf)
Supplementary Figure S3

Six-minute walk test (6MW).

Click here for additional data file. (647.9KB, pdf)
Supplementary Figure S4

Clinical measures in the patients included in the trial.

Click here for additional data file. (1.1MB, pdf)
Supplementary Table S1

Adverse events in patients treated with the combination of ERT and NB-DNJ.

Click here for additional data file. (66.8KB, pdf)
Material
Click here for additional data file. (1.1MB, pdf)

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