Preclinical Studies in the mdx Mouse Model of Duchenne Muscular Dystrophy with the Histone Deacetylase Inhibitor Givinostat
Previous work has established the existence of dystrophin-nitric oxide (NO) signaling to histone deacetylases (HDACs) that is deregulated in dystrophic muscles. As such, pharmacological interventions that target HDACs (that is, HDAC inhibitors) are of potential therapeutic interest for the treatment of muscular dystrophies. In this study, we explored the effectiveness of long-term treatment with different doses of the HDAC inhibitor givinostat in mdx mice—the mouse model of Duchenne muscular dystrophy (DMD). This study identified an efficacy for recovering functional and histological parameters within a window between 5 and 10 mg/kg/d of givinostat, with evident reduction of the beneficial effects with 1 mg/kg/d dosage. The long-term (3.5 months) exposure of 1.5-month-old mdx mice to optimal concentrations of givinostat promoted the formation of muscles with increased cross-sectional area and reduced fibrotic scars and fatty infiltration, leading to an overall improvement of endurance performance in treadmill tests and increased membrane stability. Interestingly, a reduced inflammatory infiltrate was observed in muscles of mdx mice exposed to 5 and 10 mg/kg/d of givinostat. A parallel pharmacokinetic/pharmacodynamic analysis confirmed the relationship between the effective doses of givinostat and the drug distribution in muscles and blood of treated mice. These findings provide the preclinical basis for an immediate translation of givinostat into clinical studies with DMD patients.
The most common muscular dystrophy (MD) is Duchenne muscular dystrophy (DMD), a severe recessive X-linked disease that affects 1 in 3500 males and is characterized by rapid progression of muscle degeneration, eventually leading to loss of ambulation and death within the second decade of life (1,2). This disorder is caused by mutations in the dystrophin gene that result in the complete absence or, very infrequently, in the expression of a truncated, nonfunctional protein. There is currently no available therapy for children with DMD, and current treatment is based on steroids, which only marginally affect the natural history of the disease (3,4). Pharmacological strategies for the treatment of muscular dystrophies are normally designed to counter the disease progression by targeting events downstream of the genetic mutation, such as inflammation, fibrosis, fat deposition and calcium homeostasis, or by promoting endogenous regeneration (5). Because of the hurdles that still prevent the application to dystrophic patients of gene- and cell-mediated therapies, pharmacological strategies provide a unique, immediate and suitable resource for the treatment of the current generation of dystrophic patients.
We have previously demonstrated the effectiveness of histone deacetylase inhibitors (HDACi) in the treatment of muscular dystrophies, using mdx mice as models (6,7). The mdx mice are the mouse model of human DMD and therefore provide the most amenable and approachable disease model for exploratory and preclinical evaluation of experimental interventions in muscular dystrophies. We have shown that exposure to HDACi counters the disease progression in mdx mice (6). HDACi produced functional and morphological beneficial effects, such as increased cross-sectional area (CSA) of myofibers, restoration of muscle force, decreased inflammatory infiltrate and prevention of fibrotic scars, which contribute to counter the muscle loss and the functional decline that are typically observed in mdx mice (6). Interestingly, the extent to which HDACi ameliorate the mdx phenotype varies significantly among these compounds, with trichostatin A (TSA) being the most effective drug at defined concentrations (TSA 0.6 mg/kg, delivered by daily intraperitoneal injection). An interesting insight into the specific role of individual HDACs in the pathogenesis of muscular dystrophy is suggested by the comparable efficacy of MS275, which selectively inhibits class I HDACs, and pan HDACi, which inhibit both class I and II HDACs (6,7). This suggests that inhibition of class I HDACs is sufficient to exert most of the beneficial effects observed in HDACi-treated mdx mice, once again emphasizing the key contribution to DMD pathogenesis by class I HDACs.
Current studies are seeking to define the relative ability to counter DMD progression with a number of different HDACi that have long been used in clinical practice (valproic acid [VPA] and phenylbutyrate) or have recently been approved for treatment of cancer and other diseases (6,8,9). Among them, suberoylanilide hydroxamic acid (SAHA) was also effective in ameliorating the dystrophic phenotype of mdx mice. A dose-finding study was performed by Colussi et al. with SAHA, using escalating doses ranging from 0.3 to 100 mg/kg/d delivered to mdx mice for 3 months (10). This study identified efficacy in recovering functional and histological parameters within a window of doses between 0.6 and 5 mg of SAHA, with evident reduction of the beneficial effects with doses lower than 0.6 mg and higher than 5 mg. Although the reason for such a dose-dependent response of mdx mice to SAHA is still unclear, this evidence indicates that dose-finding studies should be extended to all HDACi used for the experimental treatment of muscular dystrophies. The same study revealed an interesting correlation between mdx exposure to SAHA (5 mg/kg/d) and restoration of the profile of specific plasma proteins that were differentially expressed in mdx mice versus their normal counterparts (10).
One of the hurdles that complicates the translation into clinical trials of experimental drugs that have shown a therapeutic potential in animal models of muscular dystrophy is the absence of information on critical pharmacological parameters in pediatric populations, such as DMD boys. A notable exception is represented by ITF2357 (givinostat), which is currently being tested in a phase I safety study in children affected by systemic onset juvenile arthritis (11,12). This has inspired preclinical studies addressing the effectiveness of givinostat in preventing disease progression in mdx mice after prolonged exposure.
Materials and Methods
Animals and In Vivo Treatments
C57Bl6J mdx mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Animals were used at the specified age and treated for the indicated periods with daily intraperitoneal injections of TSA (0.6 mg/kg/d) (Sigma-Aldrich, St. Louis, MO, USA) dissolved in saline solution, with gavage administration of givinostat (ITF2357) (1 mg/kg/d, 5 mg/kg/d and 10 mg/kg/d dissolved in methylcellulose 0.01%) or methylcellulose (Sigma-Aldrich) alone as control (CTR). Mice were maintained according to the standard animal facility procedures, and all experimental protocols were approved by the internal Animal Research Ethics Committee according to the Italian Ministry of Health and complied with the NIH-used Guide for the Care and Use of Laboratory Animals (13).
Exhaustion Treadmill Test
The analyses were carried out using a six-lane motorized treadmill (Exer 3/6 Treadmill; Columbus Instruments, Columbus, OH, USA) supplied with shocker plates. The first trial was performed at low intensity and for a short duration to accustom the mice to the exercise. In the protocol used, the treadmill was run at an inclination of 0° at 8 cm/s for 5 min, after which the speed was increased by 2 cm/s every 2 min up to a speed of 9 m/min. The test was stopped when the mouse remained on the shocker plate for more than 10 s without attempting to reengage the treadmill. The time to exhaustion was determined from the beginning of the test. Four tests were performed on the same animal, monthly.
Evans Blue Staining
Mice were intraperitoneally injected with 1% Evans blue dye (Sigma-Aldrich) (w/v) in phosphate-buffered saline (PBS) (pH 7.5) sterilized by passage through a Millex®-GP 0.22-µm filter (Millipore, Bedford, MA, USA). After injection, animals were returned to their cages and allowed food and water ad libitum. After 24 h the mice were killed and diaphragms were frozen (methyl butanol and N2 liquid).
Histology and Immunofluorescence
Tibialis anterior muscles were prefixed in paraformaldehyde (PFA) 0.5% and incubated at 4°C for 2 h. After fixation, to maintain good tissue morphology, the tissues were incubated in a 20% sucrose solution overnight at 4°C. The day after, the muscles were snap frozen in liquid nitrogen-cooled isopentane and then cut transversally into cryosections (10 µm) using a Leica CM 3050 S cryostat (Leica, Wetzlar, Germany).
For hematoxylin and eosin staining, cryosections were fixed in 4% PFA, washed in 1× PBS and then stained in hematoxylin for 4 min and eosin for 6 min. Then the cryosections were dried in ethanol and fixed in xylene and mounted with Eukitt mounting (O. Kindler GmbH, Freiburg, Germany).
To stain lipids, 10% formalin-fixed cells and tissues were rinsed with water and then with 60% isopropanol, stained with oil red O (Sigma-Aldrich) in 60% isopropanol and rinsed with water.
To stain fibrotic tissue Masson trichrome analysis was used. Muscle cryosections were stained in working Weigert’s iron hematoxylin solution (Sigma-Aldrich) for 5 min, washed in running tap water for 5 min and stained in Biebrich scarlet-acid fuchsin (Sigma-Aldrich) for 5 min. The cryosections were then rinsed in deionized water, placed in working phosphotungstic/phosphomolybdic acid solution for 5 min and stained in aniline blue solution for 5 min and in acid acetic 1% for 2 min. The slides were mounted with Eukitt mounting.
To stain inflammatory infiltration and in particular neutrophils and monocytes, the anti-human myeloperoxidase (MPO) monoclonal antibody (R&D Systems, Minneapolis, MN, USA) was used on tibialis anterior paraformaldehyde-fixed cryosections. The antibody was used at a concentration of 8 ug/mL after methanol (−20°C) permeabilization.
Images were acquired with a Leica confocal microscope and edited using the Photoshop software (Adobe, Seattle, WA, USA). Fields reported in the figures are representative of all examined fields.
The CSA was calculated using ImageJ software downloaded from https://doi.org/rsb.info.nih.gov/ij. Fibrotic areas were measured by selecting four representative and nonadjacent sections and photographing up to three microscopic fields. The total fibrosis was calculated from sections by evaluating image analysis algorithms for color deconvolution. ImageJ was used for image processing. The original image was segmented with three clusters, and the plugin assumes images generated by color subtraction (white represents background, blue represents collagen and magenta represents noncollagen regions). Oil red O areas were measured by using ImageJ and calculating the area of red pixels (pixel2) per field. Inflammation was quantified by counting the number of MPO-positive cells per field. Each measurement was performed by selecting four representative and non-adjacent sections and photographing up to three microscopic fields. Statistical significance was determined with the Student t test.
The Emax model (also named the Hill equation model) was applied for linking the fibrosis and MPO effects with the individual areas under the curves (AUCs). The general equation of the model is defined by four parameters: the maximum effect Emax, the shape factor m, the baseline effect E0 and the AUC50, which measures the exposure for achieving 50% of the maximum effect. On the basis of different biological and model-building criteria, different parameterizations were tested. Finally, Emax was fixed to the median value in untreated mice (Emax = 52,347 pixels for fibrosis and Emax = 17.5 for MPO), m was set to 1 and E0 = 0, which means absence of fibrosis and/or MPO as the maximum therapeutic effect. Therefore, a one-parameter Hill equation model was applied for fitting pharmacodynamic (PD) data, and the AUC50 was estimated for each PD endpoint (fibrosis and MPO). Errors (for example, differences between observed and model-predicted values), considered homoscedastic and serially uncorrelated, were described using an additive model.
The cumulative frequency function was computed for each CSA distribution curve and the 50% cumulative frequencies (MTF50) were identified and linked to the individual blood concentration AUCs.
A categorical population model was applied to describe a PD effect in terms of the running time values on treadmill.
The results of these preclinical studies demonstrate that givinostat exerts beneficial effects in the mouse model of DMD (mdx mice) that are similar or even better than those previously reported with TSA (6). The dose-response study indicates that doses higher than 1 mg/kg/d are required for a therapeutic effect of givinostat and suggests an optimal range of doses between 5–10 mg/kg/d in the mdx model. Furthermore the PK/PD analysis identified exposures of the drug expected to be beneficial. Interestingly, an AUC of approximately 600 h/nmol/L was consistently associated with a 50% effect on histological parameters, and such exposure was needed for a meaningful improvement in performance of the mdx mice on the treadmill test.
The actual reason accounting for the beneficial effect observed in mdx mice treated with doses of givinostat between 5–10 mg/kg/d is currently unknown. While these doses of givinostat could ameliorate most of the functional and histological parameters, we observed a better effect of 10 mg/kg/d in promoting increased CSA (Figure 3) and preventing fibrosis (Figure 4) and adipose infiltration (Figure 5), while treatment with 5 mg/kg/d appeared to have the best effect in restoring muscle endurance, as shown by the treadmill test (Figure 6). It is interesting to note that despite the better treadmill performance of mdx mice treated with 5 mg/kg/d of givinostat, these mice showed increased postexercise membrane permeability of diaphragms (Figure 8) as well as other muscles of the posterior legs (not shown), compared with 10 mg/kg/d concentration. This might depend on the differential impact of these doses on distinct parameters, such as muscle strength and membrane stabilization of dystrophic muscles. For instance, 5 mg/kg/d might exert maximal activity on muscle strength and endurance but have limited efficacy in stabilizing the membrane of dystrophin-deficient muscles, as suggested by the reduced effects on histological parameters in comparison with 10 mg/kg/d. The overall outcome of such effects would be paradoxical degeneration of muscles that have undergone more strenuous exercise, as shown in Figures 7 and 8.
An additional effect observed with both 5- and 10-mg/kg/d doses of givinostat was the reduction of the inflammatory infiltrate in mdx muscles (Figure 6). The relationship between reactive inflammation of dystrophic muscles and the role of local concentration of inflammatory cytokines in promoting compensatory regeneration or fibroadipogenic degeneration is still controversial (22). Although the role of chronic inflammation in the pathogenesis of DMD has been firmly established (23) and is well supported by the beneficial effects observed with steroid treatment in DMD patients (4) and antiinflammatory interventions in mdx mice (24), the inflammatory infiltrate also provides a source of environmental cues that can positively affect the activity of the cellular network that promotes muscle regeneration (22). However, over time these beneficial effects can turn into negative ones, with an exhaustion of the regeneration potential of dystrophic muscles and the compensatory repair by fibrosis and fat deposition. Because inflammation typically occurs in response to the degeneration after contraction in dystrophic muscles, the reduction of the inflammatory infiltrate observed in muscles of mdx mice treated with 5 and 10 mg/kg/d of givinostat can be the consequence of their primary beneficial effects—for example, stimulation of regeneration and inhibition of fibrosis and adipogenesis. Alternatively, the antiinflammatory action of givinostat can directly contribute to the long-term ability to reduce fibrosis and improve functional parameters.
Overall, these data demonstrate the efficacy of givinostat in a preclinical model of DMD and support the progression of givinostat into clinical studies in children affected by DMD. Importantly, the current study predicts exposures to doses that could be necessary to exert a beneficial effect, and this will help in defining the dose to be used in the clinical studies. Because pharmacological interventions with HDACi do not restore dystrophin expression (6) but instead target downstream effectors of dystrophin-NO signaling (5), they require a prolonged schedule of treatment or even life-long exposure to achieve a persistent effect in dystrophic muscles. Thus, it will be important to define the effectiveness and the potential of long-term treatment with givinostat in DMD patients in clinical trials. In this regard, an important goal of future studies will be to better understand the interactions of givinostat with other pharmacological interventions, including steroids, to optimize synergistic schedules of drug administration.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
PL Puri is an Associate Investigator of Sanford Children’s Health Research Center. This work has been supported by the following grants to PL Puri: R01AR052779 and P30 AR061303 from the National Institute of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), MDA, AFM, FILAS and EPIGEN. This work has benefited from research funding from the European Community’s Seventh Framework Programme in the project FP7-Health 2009 ENDOSTEM 241440 (Activation of vasculature associated stem cells and muscle stem cells for the repair and maintenance of muscle tissue). C Mozzetta was supported by an AFM fellowship. We thank A Sandri (Plaisant) for the excellent assistant in treating and monitoring in vivo functions of mdx mice.
- 13.Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research, Division on Earth and Life Studies, National Research Council of the National Academies. (2011) Guide for the Care and Use of Laboratory Animals. 8th edition. Washington (DC): National Academies Press. [cited 2013 Apr 23]. Available from: https://doi.org/oacu.od.nih.gov/regs/Google Scholar
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