Aim 1: Define the dose and treatment regimen of Gal-1 necessary to increase membrane repair capacity to improve outcome measures of disease using in vivo models of LGMD2B.
We used ex vivo membrane repair assays in muscle to determine the optimal in vivo one-week rHsGal-1 dose and to define the therapeutic window in Bla/J mice. The results obtained during our one-week dose-response experiment show that there is a 6-fold therapeutic range at which rHsGal-1 was effective. The effective concentration seems to peak using a 2.7 mg/kg rHsGal-1 every seven days dosing regimen. Although the following doses did not provide benefit: 0.27 mg/kg D0, D7; 0.54 mg/kg D0, D7; and 13.5 mg/kg D0, D7, they also did not result in worsening membrane repair. This shows a safety profile of doses between 0.54–13.5 mg/kg in Bla/J mice. This 25-fold safe dosing range, along with pharmacokinetic studies, shows that the treatment of rHsGal-1 takes approximately 12 h to return to pre-dosing levels of Gal-1, indicating that this biologic may be a safe option for human patients. Using a Dab stain of psoas muscle and western blot of the gastrocnemius, we show Gal-1 is increased and that the histidine tagged Gal-1 is present in the muscle.
A one-month study using the 2.7 mg/kg rHsGal-1 treatment was used to provide additional evidence of therapeutic benefits in the form of functional, histological, and biochemical experiments. We observed increased rearing and cage movement in mice receiving treatment. A primary histological marker for LGMD2B is lipid deposition in affected muscles. Our results show a decrease in perilipin with rHsGal-1 treatment which demonstrates a decrease in fat deposition within Bla/J myofibers.
Aim 2: Define the therapeutic role of Gal-1 on chronic inflammatory response in LGMD2B models.
Decreased inflammation may be a fundamental reason for observed increased muscle integrity in treated animals, as inflammation has been shown to play a large role in the pathological symptoms of LGMD2B. Biologically, rHsGal-1 has been shown to decreases inflammation through the NF-κB pathway and upregulates cytokines with anti-inflammatory and regenerative effects in other models. We hypothesize that the decrease in fat deposition is due to decreased activation of the canonical NF-κB pathway as a result of treatment. We showed in A/J cells that rHsGal-1 does activate the canonical NF-κB pathway. Additionally, measuring inflammatory markers in Bla/J mice treated for 1 month with rHsGal-1 showed decreases in markers in NF-kB signaling such as p65, P-p65, and p-50. These results were also confirmed in patient derived cells which showed that myoblasts treated with rHsGal-1 improved membrane repair and reduces p65 and p50 levels related to NF-kB mediated inflammatory pathways.
We looked at changes in macrophage populations along with cytokine and chemokine secreted with treatment. We have preliminary data showing that conditioned media from Gal-1 treated RAW264.7 macrophages caused other RAW264.7 macrophages to convert to M2 pro-resolving phenotype more frequently than media from untreated macrophages. This suggests that Gal-1 treatment causes macrophages to secrete something into the media that promotes the conversion to M2 pro-resolving phenotype. A cytokine array showed that IL-4, CXCL-1, MCP-1 and TIMP-2 are significantly upregulated in myotubes treated for 48 hours with 0.11uM rHsGal-1 compared to PBS treated controlled cells. Additionally, quantitative western blots showed IL-4, MCP-1, and TIMP-2 were all increased in homogenized muscle samples from Bla/J mice treated for 1-month with Gal-1. CLCX-1 levels were not changed in the same samples.