The proton pump inhibitor lansoprazole improves the skeletal phenotype in dystrophin deficient mdx mice

Abstract

Background: In Duchenne muscular dystrophy (DMD), loss of the membrane stabilizing protein dystrophin results in myofiber damage. Microinjury to dystrophic myofibers also causes secondary imbalances in sarcolemmic ion permeability and resting membrane potential, which modifies excitation-contraction coupling and increases proinflammatory/apoptotic signaling cascades. Although glucocorticoids remain the standard of care for the treatment of DMD, there is a need to investigate the efficacy of other pharmacological agents targeting the involvement of imbalances in ion flux on dystrophic pathology.

Methodology/principal findings: We designed a preclinical trial to investigate the effects of lansoprazole (LANZO) administration, a proton pump inhibitor, on the dystrophic muscle phenotype in dystrophin deficient (mdx) mice. Eight to ten week-old female mice were assigned to one of four treatment groups (n = 12 per group): (1) vehicle control; (2) 5 mg/kg/day LANZO; (3) 5 mg/kg/day prednisolone; and (4) combined treatment of 5 mg/kg/day prednisolone (PRED) and 5 mg/kg/day LANZO. Treatment was administered orally 5 d/wk for 3 months. At the end of the study, behavioral (Digiscan) and functional outcomes (grip strength and Rotarod) were assessed prior to sacrifice. After sacrifice, body, tissue and organ masses, muscle histology, in vitro muscle force, and creatine kinase levels were measured. Mice in the combined treatment groups displayed significant reductions in the number of degenerating muscle fibers and number of inflammatory foci per muscle field relative to vehicle control. Additionally, mice in the combined treatment group displayed less of a decline in normalized forelimb and hindlimb grip strength and declines in in vitro EDL force after repeated eccentric contractions.

Conclusions/significance: Together our findings suggest that combined treatment of LANZO and prednisolone attenuates some components of dystrophic pathology in mdx mice. Our findings warrant future investigation of the clinical efficacy of LANZO and prednisolone combined treatment regimens in dystrophic pathology.

Figure 1. Effect of treatment on in vitro muscle force.

The maximal (A) and specific (B) EDL muscle force was compared between vehicle control (n = 8), LANZO (n = 7), prednisolone (n = 8), and combined treatment groups (n = 9). The percent differences in maximal or specific force relative to vehicle control are presented above the error bars. No statistically significant differences in maximal muscle force were detected within treatment groups. There were statistically significant within group differences in specific muscle force (p = 0.0242), although no statistically significant between group differences were observed. (C) The EDL muscles of C57Bl/10 and mdx mice in each of the four treatment groups were subjected to four lengthening contractions and the decline in maximal force was tested using a mixed-effects linear regression model. Maximal force was also shown to decrease after each subsequent contraction due to the damage incurred by the lengthening contractions. *The overall decline in maximal force was significantly lower in response to repeated lengthening contractions in the combined treatment group relative to vehicle control.

Figure 2. Effect of treatment on Rotarod latency to fall.

Latency to fall time was measured between treatment groups (n = 12 per group, with the exception of the vehicle control [n = 10]) via Rotarod testing during the last week of the trial. The data analyzed is an average of six latency to fall scores where Rotarod testing was performed two times per day for 3 consecutive days after a 24 h acclimation period. Time to fall was compared between treatment groups using a log-rank test and Kaplan-Meier estimate. Although the LANZO and combined treatment groups displayed a greater median time to fall, no statistically significant differences were detected between treatment groups.

Figure 3. Effect of treatment on histological parameters between treatment groups at 20 weeks.

H&E staining was performed on gastrocnemius muscle cross sections. Muscle histology (number of degenerating, regenerating and inflammatory foci per field) was averaged over five representative fields of non-overlapping fiber cross-sections (A–C). (A) Average number of degenerating fibers per field. (B) Average number of regenerating fibers per field. (C) Average number of inflammatory cell foci per field per group. (D–G) Representative gastrocnemius muscle cross sectional fields taken from vehicle control (D), LANZO-treated (E), prednisolone-treated (F), and combined treatment (G) groups.

Figure 4. Effect of treatment on creatine kinase.

Serum CK levels at time of sacrifice between vehicle control (n = 9), LANZO (n = 11), prednisolone (n = 12), and combined treatment (n = 11) groups. There were three mice for which a cardiac venipuncture was not obtained due to technical difficulties. All sera was obtained via cardiac venipuncture and analyzed in duplicate via enzymatic assay. Statistical analysis was performed on log-transformed data. Upon removal of two outliers in the vehicle and LANZO groups, no statistical differences remained among treatment groups (not shown).