Background
Pompe disease (glycogen storage disease type II, OMIM #232300) is a rare, autosomal recessive lysosomal storage disorder caused by deficiency of acid α-glucosidase and characterized by lysosomal glycogen storage, mainly in muscle tissue (Hirschhorn and Reuser
2001). Depending largely on how much enzyme activity is preserved, it can present at different ages, from soon after birth to late adulthood. Patients with the classic infantile form present in the first months of life with generalized muscle weakness, hypertrophic cardiomyopathy, respiratory problems, and feeding difficulties (van den Hout et al
2003). If untreated, they usually die before one year of age due to cardio-respiratory insufficiency.
Patients’ prospects were significantly improved in 2006, when enzyme-replacement therapy (ERT) with recombinant human acid α-glucosidase (Myozyme®, alglucosidase alfa) was approved. ERT prolongs lifespan, improves cardiac hypertrophy, and enables patients to reach previously unmet motor milestones (Van den Hout et al
2000,
2004; Kishnani et al
2007,
2009; Chakrapani et al
2010; Hahn et al
2015). However, response to treatment varies between patients. When treated with either 20 or 40 mg/kg every other week (eow), approximately half of patients with classic infantile Pompe disease do not survive ventilator-free beyond the age of 3 years (Kishnani et al
2009). Similarly, a substantial proportion of patients do not learn to walk, and nearly all retain residual muscle weakness (Muller et al
2009; Case et al
2012; van Gelder et al
2012). Effective clearance of glycogen from skeletal muscle is reported in only a small number of patients (Winkel et al
2003; Van den Hout et al
2004; Thurberg et al
2006; Kishnani et al
2007,
2009). Preclinical studies in mice (Bijvoet et al
1999; Raben et al
2003) and clinical studies in infantile patients (Van den Hout et al
2000,
2004; Kishnani et al
2007,
2009; McVie-Wylie et al
2008) have shown that the reduction in glycogen levels in skeletal muscle is dose-dependent. On the basis of these findings and of the published intracellular half-life of alpha-glucosidase (Van der Ploeg et al
1988,
1991; Kamphoven
2004; Maga et al
2013), we estimated that patients might benefit from a higher and more frequent dose. We therefore treated affected infants with a dose of 40 mg/kg/week, i.e., the dose previously administered to four infants treated with recombinant human acid α-glucosidase from rabbit milk (Van den Hout et al
2000,
2004). The safety and efficacy of this higher and more frequent dosing regimen was compared with that of the recommended dose of 20 mg/kg eow.
Methods
Patients
Classic infantile Pompe disease was defined as symptoms of muscle weakness within six months of birth, hypertrophic cardiomyopathy, and confirmation of total deficiency of acid α-glucosidase (GAA) activity combined with the finding of pathogenic mutations in both GAA alleles. From 2009 on we treated new patients with 40 mg/kg/week. In the current study we compared patients who started treatment with the recommended dose of 20 mg/kg eow (start before 2009) to patients who started with a dose of 40 mg/kg/week (start after 2009) and who had received the treatment for at least 3 years. Data of this ongoing investigator driven study were included until April 1 2014; or until a dose change. The study was performed independent from industry. The Medical Ethical Committee at Erasmus MC University Medical Center approved the protocols and all parents gave written informed consent.
None of the patients received immunomodulation. Only CRIM positive classic infantile patients were included, which means that the combined set of very severe mutations led to the production of at least some in-active alpha-glucosidase protein. Due to the small number of patients no comparative statistics were applied.
Clinical efficacy
Clinical efficacy was measured by assessing survival, ventilator-free survival, number of hospitalizations for respiratory infections, cardiac dimensions, and motor function. Cardiac dimensions were measured by 2D-guided M-mode echocardiographic tracings (using a Philips iE33 xMAtrix Echocardiography System, Philips Medical Systems, Andover, MA, USA), at baseline and at regular intervals thereafter. Left-ventricular mass index (LVMI) was calculated as a measure for hypertrophic cardiomyopathy (LVMI > +2z-scores (Poutanen and Jokinen
2007)) and left ventricular internal dimension (LVID) as a measure for ventricular dilatation. Motor function was examined using the Alberta Infant Motor Scale (AIMS) (Piper and Darrah
1994) and the achievement of motor milestones was examined at regular clinical assessments.
Safety
Safety assessments included the monitoring of infusion-associated reactions (IARs). Adverse events that were judged to be possibly, probably or definitely related to ERT were considered to be IARs. The severity of each IAR was indexed by clinical judgment as mild, moderate or severe (Van den Hout et al
2004).
Before enzyme infusions, blood samples were drawn at regular intervals to measure antibodies to ERT with an enzyme-linked immunosorbent assay (ELISA) (van Gelder et al
2014).
Pharmacokinetic analysis
To determine the activity of acid α-glucosidase in the blood circulation and the rate of alglucosidase alfa clearance in relation to dosing, we measured the activity in plasma during enzyme infusions with 20 mg/kg and 40 mg/kg. Blood samples were drawn before the start of the infusion, at 2 and 3 h after start, at 15 min before the end, at the end of infusion, and then 15, 30, 60, and 120 min thereafter.
To determine the percentage of the enzyme in the blood that was antibody-bound, patients’ plasma samples were incubated in the presence of Protein-A Sepharose beads to bind antibody-bound alglucosidase alfa, and in parallel in the presence of Sepharose beads only (control). After removal of the beads by centrifugation, acid α-glucosidase activity was measured in the supernatant (de Vries et al
2010). Pre-infusion serum samples were collected to determine the corresponding patients’ antibody titers by ELISA (van Gelder et al
2014).
Discussion
It is unquestionable that the introduction of enzyme replacement therapy has significantly improved the life expectancy of patients with classic infantile Pompe disease (Van den Hout et al
2000,
2004; Kishnani et al
2007,
2009; Chakrapani et al
2010). Nevertheless, nearly 50 % of the infants treated do not survive ventilator-free (Kishnani et al
2007,
2009). In this study we evaluated the efficacy and safety of a higher and more frequent dosing regimen, which we hoped would improve the patients’ clinical outcome.
Preclinical studies in mice have shown a dose dependent uptake of alglucosidase alfa in the range from 10 to 100 mg/kg (Raben et al
2003; Kamphoven
2004; McVie-Wylie et al
2008; Khanna et al
2012). In the very first clinical study in which we treated classic infantile patients with recombinant human alpha-glucosidase from rabbit milk, we observed a similar dose dependent effect in that the alpha-glucosidase activity in the skeletal muscle only normalized when the dose was increased from 15 to 20 mg/kg/week to 40 mg/kg/week (Van den Hout et al
2000,
2004, Winkel et al
2003). It is known that muscle cells are hard to treat since only a small fraction of infused enzyme actually reaches the muscle cells. Further it is by now generally accepted that treatment needs to be started before irreversible muscle damage has occurred. This combined experience was reason for us to treat patients with a dose of 40 mg/kg/week from start and not to wait until patients deteriorated. Earlier no difference in clinical response was found between infantile patients treated with either 20 mg/kg/eow and 40 mg/kg/eow (Kishnani et al
2009). This might be attributed to the lower dose and larger dose interval. Another factor that may have played a role is that there were more CRIM negative patients in the higher dose group (Kishnani et al
2009; Banugaria et al
2011, van Gelder et al
2014). CRIM negative patients tend to perform poorer. We therefore excluded CRIM negative patients from the current study.
The most notable contrast we observed between the two dose groups was the difference in overall clinical condition, which was reflected in the difference in hospital admissions for the two groups: while none of the patients treated with 40 mg/kg/week had ever had respiratory infections requiring hospitalization, 3/4 patients treated with 20 mg/kg eow required frequent readmissions. Consequently, one of these patients developed respiratory insufficiency at the age of 2.7 years. Our study results suggest that the 40 mg/kg/week dosing regimen helps to stabilize or improve the respiratory condition of affected infants better. Similarly, motor function appeared to be better in the 40 mg/kg/week dose group, all of whom learned to walk and maintained the ability to do so. Unlike 3/4 patients treated with 20 mg/kg eow learned to walk and only 2/4 could still walk at the end of the study. The loss of motor milestones in the 20 mg/kg eow dose group was preceded by infections requiring hospital admissions. Importantly walking was not regained in our patients when the dose was increased after deterioration. Recently two studies also reported minor effects of dose increase when patients perform poorly (Case et al
2015; Hahn et al
2015). It should also be noted that response to ERT varies between patients treated with the same dose. This is illustrated by 1/4 patients treated with the lower dose of 20 mg/kg eow, who performed well until the end of the follow-up at the age of 9 years.
With regard to cardiac hypertrophy, both dosing regimens worked equally well, which is explained by the fact that a lower dose is required to correct or prevent the cardiac hypertrophy compared to the skeletal muscle weakness (Bijvoet et al
1999; Van den Hout et al
2000; Raben et al
2003). For the same reason, adults with Pompe disease with residual α-glucosidase activities of up to 25 % do not generally develop hypertrophic cardiomyopathy, while they do have skeletal muscle weakness (Hirschhorn and Reuser
2001).
Although we observed no clear differences in safety parameters, the small numbers do not allow us to draw firm conclusions. While nearly all patients in each dose group experienced IARs the overall number of IARs was higher in the 40 mg/kg/week dose group. This was largely due to a single patient that had had over 50 % of the total number of IARs. A similar pattern was observed in the pivotal trials (Kishnani et al
2007,
2009). The patient with most IARs in our study had recurrent episodes of exanthema, coughing and vomiting, occasionally accompanied by saturation drops. Remarkably, the IARs started within minutes of the start of the infusion, when the infusion rate was still slow. Total IgE, serum tryptase and complement levels were within the normal range. While this patient had a relatively high sustained antibody titer, the titer was similar to that of other patients who did not develop as many IARs. At the time of writing, the patient was receiving home-based enzyme therapy without problems, and time since last IAR was over 2 years.
It is well recognized that therapeutic proteins can induce an immunological response that neutralizes the effect of ERT. Three of the four patients treated with 40 mg/kg/week and two of the four treated with 20 mg/kg eow developed a peak antibody titer of 31,250 which was estimated to be the highest titer without significant consequences for ERT at a dose of 40 mg/kg (van Gelder et al
2014). Using pharmacokinetic studies in the present study, we could not detect substantial amounts of antibody-bound alglucosidase alfa during enzyme infusion in patients whose antibody titers ranged from 1:6250 to 1:31,250. One patient receiving 40 mg/kg/week had a peak antibody titer of 1:156,250, which later declined to 1:31,250. According to earlier estimates, as much as 54 % of the administered enzyme (about 10 mg/kg) is antibody-bound at a dose of 20 mg/kg and a titer of 1:156,250 (van Gelder et al
2014). If a similar amount (10 mg/kg) were bound upon administration of 40 mg/kg, about 30 mg/kg would theoretically still be available for uptake in the target tissues.
Overall, we found no apparent correlation between the level of antibodies and the dose of ERT, although patients treated with 40 mg/kg/week tended to develop higher antibody titers than those receiving 20 mg/kg eow. This is consistent with a previous study that compared the level of antibody titers between patients treated with 20 or 40 mg/kg eow (Banugaria et al
2011). In line with previous observations (Khallaf et al
2013; van Gelder et al
2014) the patient's peak antibody titer seemed to be related to the age at start of therapy.
A further point that requires attention is that muscular problems were still observed in patients treated with 40 mg/kg/week. This may be due to insufficient glycogen clearance. As glycogen also accumulates in neural tissues, including motor neurons of the spinal cord and peripheral nerves (Gambetti et al
1971), we cannot exclude the possibility that neurological damage plays a role as well.
Our study describes a limited number of CRIM positive patients. We have chosen to report on children who received at least 3 years ERT in a dose of 40 mg/kg/week. Inclusion of more patients and longer follow-up will be needed to get the full picture. So far our group of CRIM positive children starting on 40 mg/kg/week seems to have a better outcome than those who started on 20 mg/kg eow. Our data suggest that the 40 mg/kg/week has the best effect when applied from the start.
Abbreviations
Eow, every other week; LVMI, left-ventricular mass index; IAR, infusion-associated reaction; ERT, enzyme-replacement therapy; GAA, acid α-glucosidase; AIMS, Alberta Infant Motor Scale; ELISA, enzyme-linked immunosorbent assay; MUGlc, 4-methylumbelliferyl-α-D-glucopyranoside; MU, 4-methylumbelliferon; CRIM, cross-reactive immunological material.
Acknowledgments
The authors would like to thank all patients and their parents for participating in this study. We are grateful to R. Nelisse, and J. Koemans-Schouten for material and data collection, to L. Özkan for technical assistance, and to D. Alexander for his critical reading of the manuscript. Financial support was obtained from ZonMw—Dutch Organization for Healthcare Research and Innovation of Care (Grant 152001005), ‘Prinses Beatrix Fonds (project number OP07-08)’, and the 7th Frame Program ‘EUCLYD—a European Consortium for Lysosomal Storage Diseases’ of the European Union (health F2/2008 grant agreement 201678).
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