β2 Agonists enhance the efficacy of simultaneous enzyme replacement therapy in murine Pompe disease

Abstract

Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) has improved clinical outcomes in patients with Pompe disease; however, the response of skeletal muscle and the central nervous system to ERT has been attenuated. The poor response of skeletal muscle to ERT has been attributed to the low abundance of the cation-independent mannose-6-phosphate receptor (CI-MPR), which mediates receptor-mediated uptake of rhGAA. Hence the ability of adjunctive therapy with β2-agonists to increase CI-MPR expression in skeletal muscle was evaluated during ERT in murine Pompe disease with regard to reversal of neuromuscular involvement. Mice with Pompe disease were treated with weekly rhGAA injections (20 mg/kg) and a selective β2-agonist, either albuterol (30 mg/l in drinking water) or low-dose clenbuterol (6 mg/l in drinking water). Biochemical correction was enhanced by β2-agonist treatment in both muscle and the cerebellum, indicating that adjunctive therapy could enhance efficacy from ERT in Pompe disease with regard to neuromuscular involvement. Intriguingly, clenbuterol slightly reduced muscle glycogen content independent of CI-MPR expression, as demonstrated in CI-MPR knockout/GAA knockout mice that were otherwise resistant to ERT. Thus, adjunctive therapy with β2 agonists might improve the efficacy of ERT in Pompe disease and possibly other lysosomal storage disorders through enhancing receptor-mediated uptake of recombinant lysosomal enzymes.

Conflict of interest statement

Conflict of interest:

PSK and DDK have received research/grant support from Genzyme Corporation in the past. BT is currently employed by Genzyme Corporation.

Copyright © 2011 Elsevier Inc. All rights reserved.

Figures

Fig. 1. Muscle function and biochemical evaluatoin following low-dose clenbuterol treatment

GAA-KO mice were administered four weekly doses of rhGAA (20 mg/kg), and treated with clenbuterol (n=7), or untreated (n=6 or 3). (A) Rotarod latency at the indicated times. (B) Weight of gastrocnemius. (C) GAA enzyme levels and (D) glycogen content were evaluated in skeletal muscle, including the quadriceps, gastrocnemious (Gastroc.), tibialis anterior (Tib. Ant.), EDL, and soleus. Mean +/− standard deviation are shown. Statistically significant alterations associated with clenbuterol treatment indicated (* = p

Fig. 2. Muscle function and biochemical evaluatoin following albuterol treatment

GAA-KO mice were administered four weekly doses of rhGAA (20 mg/kg), and treated with albuterol (n=4) or untreated (n=6 or 3). (A) Rotarod latency at the indicated times. (B) Wirehang latency. (C) GAA enzyme levels and (D) glycogen content were evaluated in skeletal muscle, including the quadriceps, gastrocnemious (Gastroc.), tibialis anterior (Tib. Ant.), EDL, and soleus. Mean +/− standard deviation are shown. Statistically significant alterations associated with clenbuterol treatment indicated (* = p

Fig. 3. Decreased glycogen content in the cerebellum following β-2 agonist administration

Cerebral and cerebellar hemispheres were analyzed 5 weeks following the initiation of ERT. Groups of mice were treated with clenbuterol (n=6), albuterol (n=4) or untreated (n=6). (A) GAA enzyme levels and (B) glycogen content. Statistically significant alterations associated with clenbuterol treatment indicated (* = p

Fig. 4. Decreased glycogen accumulation in the cerebellum following β-2 agonist administration

Periodic-acid Schiff staining for glycogen in paraffin-embedded sections of the cerebellum. Original magnification 400x. (A) ERT alone. (B) ERT with concurrent albuterol treatment. (C) ERT with concurrent clenbuterol treatment. Glycogen accumulations indicated (arrows).

Fig. 5. CI-MPR expression in the cerebellum following β-2 agonist administration

(A) Western blot detection of CI-MPR and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the cerebellum of mice, treated with ERT (lanes 1–3), ERT and clenbuterol (lanes 4–6), or ERT and albuterol (lanes 7–9). Each lane represents an individual mouse. (B) Densitometry for Western blot detection of CI-MPR, normalized to GAPDH, in mice from (A).

Fig. 6. Muscle function and biochemical evaluatoin following clenbuterol treatment in DKO mice

DKO mice were administered four weekly doses of rhGAA (20 mg/kg) and treated with high-dose clenbuterol (n=7), clenbuterol alone (6), or untreated (n=7). Mean +/− standard deviation are shown. Statistically significant alterations associated with clenbuterol treatment indicated (* = p