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Abstract

Spinal muscular atrophy (SMA) is an early-onset motor neuron disease that leads to loss of muscle function. Butyrate (BA)-based compounds markedly improve the survival and motor phenotype of SMA mice. In this study, we examine the protective effects of the BA prodrug pivaloyloxymethyl butyrate (AN9) on the survival of SMNΔ7 SMA mice. Oral administration of AN9 beginning at PND04 almost doubled the average lifespan of SMNΔ7 SMA mice. AN9 treatment also increased the growth rate of SMNΔ7 SMA mice when compared to vehicle-treated SMNΔ7 SMA mice. In conclusion, BA prodrugs like AN9 have ameliorative effects on SMNΔ7 SMA mice.

Keywords

Motor neuron disease spinal muscular atrophy preclinical drug trial neonatal mouse AN9

ABBREVIATIONS

Butyrate

BA3G

Glyceryl tributyrate

CNS

Central nervous system

4PBA

4-phenylbutyrate

PND

Postnatal day

SMA

Spinal muscular atrophy

SMN

Survival motor neuron

INTRODUCTION

Spinal muscular atrophy (SMA) is an early-onset neurological disease characterized by loss of anterior horn α motor neurons in the spinal cord [1]. Limb and trunk muscles atrophy as a result of motor neuron degeneration. SMA is an autosomal recessive genetic disorder with an incidence of 1:6,000–10,0000 live births [2, 3]; in fact, it is a leading genetic cause of infant death worldwide. SMA results from the loss or mutation of the SMN1 (survival motor neuron 1) gene but retention of the near-perfectly duplicate SMN2 gene [4]. SMN2 copy number modifies the severity of SMA disease in humans as well as in transgenic mouse models for SMA (reviewed in [5]). Because of its ability to modulate disease severity, SMN2 is an ideal molecular target for the development of therapies to treat SMA.

Compounds based on the short chain fatty acid butyric acid (BA) have shown efficacy in mouse models for SMA. Continuous administration of BA via the water supply to SMA mice (SMN2;mSmn^Δ7/Δ7) results in a moderately increase in survival of these mice [6]. However, oral administration of BA directly to SMNΔ7 SMA neonates via modified gavage does not improve survival [7]. SMNΔ7 SMA mice treated with the BA analogue 4-phenylbutyrate (4PBA) or one of two BA prodrugs–glyceryl tributyrate (BA3G) or VX563–show marked improvement in survival and motor function [7]. BA is present in the forebrains of neonatal mice treated with BA3G or VX563 but not with BA [7]. The lack of efficacy of BA in SMNΔ7 SMA mice is most likely due to the poor plasma pharmacokinetics of this compound in rodents [8]. BA, therefore, can provide neuroprotective benefits in SMA mice as long as it can reach detectable levels in the central nervous system(CNS).

Pivaloyloxymethyl butyrate (AN9, Pivanex) is BA prodrug with a different chemistry from BA3G and VX563 that is metabolized by intracellular esterases to release BA along with formaldehyde and pivalic acid (2,2-dimethylpropionic acid) [9]. It possesses superior pharmacokinetics to BA and exhibits anti-tumor activity in vitro and in vivo [9 –13]. In this study, we show the protective effects of AN9 on the survival of SMNΔ7 SMA mice.

MATERIALS AND METHODS

Animals and Ethical statement

SMNΔ7 SMA mice (SMN2^+/+; SMNΔ7^+/+;mSmn^-/-) were generated from male and female carrier mice (SMN2^+/+; SMNΔ7^+/+; mSmn^+/-) [14]. The breeder mice were provided with ad libitum water and Harlan-Teklad 22/5 Rodent Diet chow [15 –17]. All experiments were conducted in accordance with the protocols described in the National Institutes of Health Guide for the Care and Use of Animals and were approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee.

Drug administration

AN9 (Pivanex) was provided by Titan Pharmaceuticals (South San Francisco, CA). Working solutions were prepared by mixing neat compound with an emulsion of 4% ethanol and Intralipid-20 (Sigma-Aldrich, St. Louis, MO) at a concentration of 0.1 mL AN9 per mL emulsion. Carrier and SMA littermate mice were dosed twice daily (b.i.d.; at 09.00 and 17.00 daily) with AN9 (200 mg/kg/d) or vehicle (4% ethanol in Intralipid-20) via oral administration as described previously [18]. Treatment began at postnatal day 4 (PND04) and continued for the lifetime of each SMA mouse. The body mass of each mouse was determined each day during treatment. The treatment cohorts were not stratified based on sex because there is no significant difference in lifespan between male and female SMNΔ7 SMA mice [19].

Butyrate measurements

Tissue levels of butyrate were measured in neonatal mice receiving a single dose of AN9 or vehicle by high-performance liquid chromatography (HPLC) as described previously [7].

Statistical analysis

Data are expressed as means±standard errors. Kaplan-Meier curves were generated from the survival and onset of body mass loss data and tested using the Mantel-Cox log rank test. All statistical analyses were performed with SPSS v.22.0.

RESULTS

SMNΔ7 SMA mice (n = 8/group) were dosed daily with either AN9 (200 mg/kg/d, b.i.d.) or vehicle (4% ethanol in Intralipid-20) beginning at PND04. Lifespan and onset of body mass loss were used as indices of the in vivo efficacy of AN9 in this mouse model [14, 19]. Oral administration of AN9 improved the mean lifespan of treated SMNΔ7 SMA mice by 84.6% (Fig. 1A; 21.0±4.9 d for AN9 vs. 11.4±0.8 d for vehicle; p = 0.039; χ² = 4.264). AN9 treatment also delayed the onset of body mass loss in SMNΔ7 SMA mice by 94.9% (Fig. 1B; 19.2±5.2 d for AN9 vs. 9.9±0.5 d for vehicle; p = 0.031; χ² = 4.641). AN9 ranked as follows when compared with other BA-based compounds tested in the SMNΔ7 SMA mouse model [7]: VX563 > AN9 > 4PBA > BA3 G > BA.

We have previously shown that BA-based drug efficacy in SMNΔ7 SMA mice is related to the levels of BA detected in the CNS [7]. Forebrain BA levels, one of the products from the metabolism of AN9 [9], were measured in neonatal mice (n = 3/group) treated with a single dose of AN9 (200 mg/kg/d) or vehicle. Tissue BA levels were measured using HPLC. In the AN9-treated mice, BA levels in the forebrain were 34.14±9.61 pmol/mg protein while vehicle-treated mice had undetectable BA levels (<12.00 mol/mg protein) in the CNS.

The body masses of SMNΔ7 SMA mice treated with AN9 (Fig. 2A; closed circles) were similar to those for vehicle-treated SMNΔ7 SMA mice (Fig. 2A; closed triangles) until PND11 after which time the drug-treated mice had higher body masses than age-matched, vehicle-treated SMNΔ7 SMA mice. After PND18, the mean body masses of AN9-treated SMNΔ7 mice were similar to drug-treated, non-SMA mice (Fig. 2A; open circles). After this timepoint, non-SMA mice treated with AN9 exhibited lower body masses than age-matched mice receiving vehicle (Fig. 2A; open triangles).

The growth rate–i.e. the change in body mass between PND14 and PND04–was diminished in SMNΔ7 SMA mice relative to non-SMA mice (Fig. 2B; p < 0.001), which is consistent with previous studies [14, 19]. AN9 increased the growth rate of SMNΔ7 SMA mice by 87% when compared against vehicle-treated SMNΔ7 SMA mice (Fig. 2B; p = 0.053). In fact, the growth rate of AN9-treated, SMNΔ7 SMA mice was similar to that for drug-treated non-SMA mice; however, the growth rate of non-SMA mice treated with AN9 was significantly diminished when compared against vehicle-treated non-SMA mice (Fig. 2B; p = 0.016). While AN9 does improve the growth rate of SMNΔ7 SMA mice, this compound may exhibit slightly toxic properties in vivo, at least in neonatal mice.

DISCUSSION

We show in this study that early administration of the BA prodrug AN9 significantly improves the survival of SMNΔ7 SMA mice. AN9 treatment also marked delays the end stage of disease as defined by the onset of body mass loss [14, 19]. Oral administration of two other BA prodrugs, BA3G and VX563, significantly ameliorate the degenerative phenotype in SMNΔ7 SMA mice [7]. AN9 is a more potent BA prodrug in these mice since its therapeutic dose is 25- to 30-fold lower than the other BA prodrugs previously tested. We can detect exogenous BA levels in the CNS of neonatal mice treated with AN9 (this study), BA3G or VX563 [7] but not with BA. BA can, therefore, provide therapeutic benefit to SMNΔ7 SMA mice if levels of the drug can be detected in the CNS.

AN9 administration also increases the growth rate, which is measured by compared the body mass of a mouse at PND14 to that at PND04, of SMNΔ7 SMA mice when compared to vehicle-treated SMNΔ7 SMA mice. At first glance, the growth rate of AN9-treated SMN Δ 7 SMA mice approaches that observed for drug-treated non-SMA mice; however, the growth rate of AN9-treated non-SMA mice is significant lower than that for vehicle-treated non-SMA mice. This latter observation would suggest that AN9 may have an off-target effect on neonatal mice. Neither BA3G nor VX563 exhibit a similar phenomenon in non-SMA mice [7]. This off-target effect could be due to the other compounds released–i.e. formaldehyde and pivalic acid [9]–from the metabolism of AN9.

How do BA-based compounds exert their protective effects on SMA mice? BA and its analogue 4PBA increase the expression of SMN in cultured fibroblasts and lymphoblasts obtained from SMA patients [6, 20]. Surprisingly, neither 4PBA nor the BA prodrug VX563 alters the levels of SMN mRNA or protein in the spinal cords of treated SMNΔ7 SMA mice [7]. BA and 4PBA are weak inhibitors of histone deacetylase (HDAC) activity [21 –24]; HDAC activity is also reduced in SMNΔ7 SMA mouse spinal cord extracts treated with 4PBA or VX563 [7]. AN9 also posseses HDAC inhibitor activity [25]. In addition to reducing HDAC activity, 4PBA and VX563 restore the phosphorylation states of Akt and one its targets, GSK3β, that are reduced in SMNΔ7 SMA mice [7]. Future studies will examine how BA-based compounds like 4PBA, VX563 and AN9 regulate the activities of HDACs and Akt signaling to protect SMA motor neurons from degeneration.

In summary, we show that the BA prodrug AN9 markedly improves the survival and growth rate of SMNΔ7 SMA mice. This study along with other work [7] demonstrate that treatment with the short chain fatty acid BA can have significantly beneficial effects on a mouse model for SMA so long as a sufficient amount of drug reaches the motor neurons in the spinal cord. BA prodrugs show promise as SMN2-independent therapeutic agents for SMA in that they protect the vulnerable motor neurons from cell death. These prodrugs could be used in concert with SMN2 inducers [26] to maximize therapeutic benefit for SMA.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

Footnotes

ACKNOWLEDGMENTS

We would like to thank Titan Pharmaceuticals for generously providing Pivanex and Dr. Arthur Burghes for providing laboratory space. This study was supported by grants from Cure SMA, the Nemours Foundation and the National Institutes of Health (P20GM103464 and P30GM114736).

References

Crawford

, Pardo

. The neurobiology of childhood spinal muscular atrophy. Neurobiol Dis. 1996;3:97–110.

Pearn

. Incidence, prevalence and gene frequency studies of chronic childhood spinal muscular atrophy. J Med Genet. 1978;15:409–13.

Cuscó

, Barceló

, Soler

, et al. Prenatal diagnosis for risk of spinal muscular atrophy. Br J Obstet Gynaecol. 2002;109:1244–9.

Lefebvre

, Bürglen

, Reboullet

, et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell. 1995;80:155–65.

Burghes

AHM

, Beattie

. Spinal muscular atrophy: Why do low levels of survival motor neuron protein make motor neurons sick? Nat Rev Neurosci. 2009;10:597–609.

Chang

, Hsieh-Li

, Jong

, et al. Treatment of spinal muscular atrophy by sodium butyrate. Proc Natl Acad Sci U S A. 2001;98:9808–13.

Butchbach

MER

, Lumpkin

, Harris

, et al. Protective effects of butyrate-based compounds on a mouse model for spinal muscular atrophy. Exp Neurol. 2016;279:13–26.

Egorin

, Yuan

, Sentz

, Plaisance

, Eiseman

. Plasma pharmacokinetics of butyrate after intravenous administration of sodium butyrate or oral administration of tributyrin or sodium butyrate to mice and rats. Cancer Chemother Pharmacol. 1999;43:445–53.

Nudelman

, Ruse

, Aviram

, et al. Novel anticancer prodrugs of butyric acid. 2. J Med Chem. 1992;35:687–94.

10.

Aviram

, Rephaeli

, Shaklai

, et al. Effect of the cytostatic butyric acid pro-drug, pivaloyloxymethyl butyrate, on the tumorigenicity of cancer cells. J Cancer Res Clin Oncol. 1997;123:267–71.

11.

Batova

, Shao

, Diccianni

, et al. The histone deacetylase inhibitor AN-9 has selective toxicity to acute leukemia and drug-resistant primary leukemia and cancer cell lines. Blood. 2002;100:3319–24.

12.

Entin-Meer

, Yang

, VandenBerg

, et al. In vivo efficacy of a novel histone deacetylase inhibitor in combination with radiation for the treatment of gliomas. Neuro-Oncology. 2007;9:82–8.

13.

Tarasenko

, Nudelman

, Tarasenko

, et al. Histone deacetylase inhibitors: The anticancer, antimetastatic and antiangiogenic activities of AN-7 are superior to those of the clinically tested AN-9 (Pivanex). Clin Exp Metastasis. 2008;25:703–16.

14.

, Pham

, Butchbach

MER

, et al. SMNΔ7, the major product of the centromeric survival motor neuron gene (SMN2), extends survival in mice with spinal muscular atrophy and associates with full-length SMN. Hum Mol Genet. 2005;14:845–57.

15.

Butchbach

MER

, Rose

Jr , Rhoades

, et al. Effect of diet on the survival and phenotype of a mouse model for spinal muscular atrophy. Biochem Biophys Res Commun. 2010;391:835–40.

16.

Narver

, Kong

, Burnett

, et al. Sustained improvement of spinal muscular atrophy mice treated with trichostatin A plus nutrition. Ann Neurol. 2008;64:465–70.

17.

Butchbach

MER

, Singh

, Gurney

, Burghes

AHM

. The effect of diet on the protective action of D156844 observed in spinal muscular atrophy mice. Exp Neurol 2014;256:1–6.

18.

Butchbach

MER

, Edwards

, Schussler

, Burghes

AHM

. A novel method for oral delivery of compounds to the neonatal SMNΔ7 model of spinal muscular atrophy. J Neurosci Methods. 2007;161:285–90.

19.

Butchbach

MER

, Edwards

, Burghes

AHM

. Abnormal motor phenotype in the SMNΔ7 mouse model of spinal muscular atrophy. Neurobiol Dis. 2007;27:207–19.

20.

Andreassi

, Angelozzi

, Tiziano

, et al. Phenylbutyrate increases SMN expression in vitro: Relevance for treatment of spinal muscular atrophy. Eur J Hum Genet. 2004;12:59–65.

21.

Boffa

, Vidali

, Mann

, Allfrey

. Suppression of histone deacetylation in vivo and in vitro by sodium butyrate. J Biol Chem. 1978;253:3364–66.

22.

Vidali

, Boffa

, Bradbury

, Allfrey

. Butyrate suppression of histone deacetylation leads to accumulation of multiacetylated forms of histones H3 and H4 and increased DNaseI sensitivity of the associated DNA sequences. Proc Natl Acad Sci U S A. 1978;75:2239–43.

23.

Candido

EPM

, Reeves

, Davie

. Sodium butyrate inhibits histone deacetylation in cultured cells. Cell. 1978;14:105–13.

24.

Davis

, Kennedy

, Chiew

, Clarke

, deFazio

. Histone deacetylase inhibitors decrease proliferation and modulate cell cycle gene expression in normal mammary epithelial cells. Clin Cancer Res. 2000;6:4334–42.

25.

Aviram

, Zimrah

, Shaklai

, Nudelman

, Rephaeli

. Comparison between the effect of butyric acid and its prodrug pivaloyloxymethylbutyrate on histones hyperacetylation in an HL-60 leukemic cell line. Int J Cancer. 1994;56:906–09.

26.

Calder

, Androphy

, Hodgetts

. Small molecules in development for the treatment of spinal muscular atrophy. J Med Chem. 2016;in press.

Effect of the Butyrate Prodrug Pivaloyloxymethyl Butyrate (AN9) on a Mouse Model for Spinal Muscular Atrophy

Abstract

Keywords

ABBREVIATIONS

INTRODUCTION

MATERIALS AND METHODS

Animals and Ethical statement

Drug administration

Butyrate measurements

Statistical analysis

RESULTS

DISCUSSION

CONFLICT OF INTEREST

Footnotes

ACKNOWLEDGMENTS

References