Chapter 22

Development of Bile Acids as Anti-Apoptotic

and Neuroprotective Agents in Treatment

of Ocular Disease

Stephanie L. Foster, Cristina Kendall, Allia K. Lindsay, Alison C. Ziesel, Rachael S. Allen, Sheree S. Mosley, Esther S. Kim, Ross J. Molinaro, Henry F. Edelhauser, Machelle T. Pardue, John M. Nickerson,

and Jeffrey H. Boatright

Abstract  The hydrophilic bile acids ursodeoxycholic acid and tauroursodeoxycholic acid are approved by regulatory bodies of many countries for treatment of gallstones and cirrhosis. Delivery is by oral administration and side effects are minimal. This chapter reviews evidence demonstrating that systemic treatment with the two compounds is protective in models of neuronal and retinal degeneration and injury. Variability in the regulation of circulating bile acids suggests a need to explore local delivery as a treatment modality. Our initial experiments testing in vivo intraocular injections and in vitro transscleral permeability indicate that this is feasible and efficacious.

22.1  Bile Acids as Anti-Apoptotic Neuroprotectants

Ursodeoxycholic acid (UDCA) and its taurine conjugate, tauroursodeoxycholic acid (TUDCA), are hydrophilic bile acids that make up a small percentage of the bile acid pool in humans. As therapeutic compounds, they are approved by several national regulatory agencies for dissolution of gallstones (Hofmann 1994; Rubin et al. 1994; Paumgartner and Beuers 2002) and treatment of cholestatic liver disease, especially primary biliary cirrhosis (PBC) (Rubin et al. 1994; Hofmann 1999). In PBC treatment, they were originally thought to act largely through displacement of hepatotoxic, hydrophobic bile acids from the bile acid pool (Rubin et al. 1994; Hofmann 1999). However, it was subsequently determined by Steer, Rodrigues, and

J.H. Boatright (*)

Department of Ophthalmology, Emory University School of Medicine, B5511 Emory Eye Center, 1365-B Clifton Road, Atlanta, GA 30322, USA e-mail: jboatri@emory.edu

U.B. Kompella and H.F. Edelhauser (eds.), Drug Product Development for the Back of the Eye, 565 AAPS Advances in the Pharmaceutical Sciences Series 2, DOI 10.1007/978-1-4419-9920-7_22,

© American Association of Pharmaceutical Scientists, 2011

colleagues that UDCA and its conjugates are anti-apoptotic (Koga et al. 1997; Rodrigues et al. 1998, 1999), having direct effects on isolated mitochondria that prevent subsequent initiation of an apoptotic cascade (Rodrigues et al. 1998, 1999, 2003b). More recently it has been demonstrated that UDCA and TUDCA may have additional anti-apoptotic effects by activating nuclear steroid receptors (Weitzel et al. 2005; Arenas et al. 2008). Following nuclear translocation, the hydrophilic bile acids appear to modulate the E2F-1/p53/Bax pathway as part of their antiapoptotic mechanism of action (reviewed in Sola et al. 2007; Amaral et al. 2009).

The same group extended their studies in liver disease models to models of neuronal disease and injury. Using in vivo, cell culture, and in vitro approaches, they found that treatment with UDCA or TUDCA slowed cell death in several neuronal disease models, including Huntington’s disease (Rodrigues et al. 2000; Keene et al. 2001; Mangiarini et al. 1996; Davies et al. 1997), Alzheimer’s disease (Rodrigues et al. 2001; Sola et al. 2003; Joo et al. 2004; Ramalho et al. 2006; 2008a, b; Viana et al. 2009), Parkinson’s disease (Duan et al. 2002), acute hemorrhagic (Rodrigues et al. 2003a) and acute ischemic stroke (Rodrigues et al. 2002), and neuronal glutamate toxicity (Castro et al. 2004). Similar work from other laboratories shows protection neuronal damage or degeneration models. Incubation with UDCA prevents apoptosis in cisplatin-induced sensory neuropathy, possibly by suppressing p53 accumulation (Park et al. 2008). In an in vivo spinal cord injury model, rats injected systemically with TUDCA showed fewer apoptotic cord cells, less tissue injury, and better hind limb function than untreated control animals (Colak et al. 2008).

22.2  Systemic Treatment with TUDCA or UDCA

is Protective in Retinal Disease and Damage Models

Given their effects in models of neurodegeneration, it is perhaps not surprising that systemic treatment with TUDCA or UDCA is protective in both induced and genetic retinal degeneration models. Pde6brd1 (rd1) mice were injected subcutaneously or intraperitoneally with TUDCA (500 mg/kg body weight daily or every 3 days) starting at postnatal day (P)6 or P9 and continued to P21. At P21, retinal function was measured with light-adapted electroretinograms (ERG) and eyes processed for histology to assess morphology and cone survival. TUDCA-treated mice had 50% greater ERG b-wave amplitudes compared to vehicle-treated mice (Arora et al. 2009; Boatright et al. 2009a). Vehicle-treated retinas had very few outer nuclear layer (ONL) cells, but TUDCA-treated retinas had varied morphology, ranging from very little ONL to thick ONL and in some instances preservation of what appeared to be photoreceptor outer segments (Arora et al. 2009; Boatright et al. 2009a). The number of ONL cells of TUDCA-treated mice that stained for cone markers was approximately twice that in vehicle-treated mice (Arora et al. 2009; Boatright et al. 2009a). Thus, systemic treatment with TUDCA protected against loss of cone photoreceptor function and number and ONL morphology (Arora et al. 2009; Boatright et al. 2009a).

In the Pde6brd10 (rd10) mouse, a missense mutation in PDE6B causes degeneration of rods starting at about P14–16 (Chang et al. 2007; Gargini et al. 2007), about a week later than in rd1 mice (Bowes et al. 1990). ERG amplitudes are large enough to be easily measured through the first month of age, but are never normal, as would be expected in mice harboring a mutation in a visual cycle gene (Chang et al. 2002). These mice were injected subcutaneously or intraperitoneally with TUDCA similarly to the experiments with rd1 mice (500 mg/kg body weight TUDCA every 3 days) starting at P6. TUDCA treatment suppressed apoptosis and greatly slowed loss of photoreceptor number, morphology, and function (Boatright et al. 2006b; Phillips et al. 2008). In untreated rd10 mice at P18, ONL thickness and nuclei counts are about 50% of wildtype, photoreceptor outer segments are largely degenerated, and ERG a-wave and b-wave amplitudes about 50% of wildtype (Chang et al. 2007). TUDCA treatment resulted in the preservation of the number of photoreceptor cells, ONL thickness, photoreceptor outer segments, and ERG a-wave and b-wave amplitudes (Boatright et al. 2006b). TUNEL signal in P18 rd10 retina sections from mice treated with TUDCA showed was virtually absent and immunosignal for activated caspase 3 was substantially reduced, suggesting that treatment resulted in the suppression of apoptosis (Boatright et al. 2006b).

TUDCA-induced protection can extend significantly into the degeneration. By P30, the ONL of untreated rd10 mice has degenerated to about one cell layer of mainly cones, the dark-adapted a-wave is only 3% and the b-wave only 14% of wildtype mice (Chang et al. 2007; Phillips et al. 2008). TUDCA-treated retinas had dark-adapted a-waves that were maintained to 30% of wildtype and lightand darkadapted b-waves maintained to 45% of wildtype, indicating preservation of both rod and cone function (Phillips et al. 2008). The number of photoreceptor nuclei was fivefold greater in TUDCA-treated mice than in vehicle-treated mice. Similar to the effect on rod photoreceptors at P18, treatment preserved cone outer segment morphology in the P30 retina (Phillips et al. 2008). Overall, TUDCA treatment delayed morphological and functional loss by 12 days over the course of the degeneration to P30 (Phillips et al. 2008).

TUDCA treatment also protects against light-induced retinal degeneration (LIRD) in mice and rats, an environmental model of blindness (Reme et al. 1998; Chen et al. 2003). Adult albino Balb/C mice were subcutaneously injected with TUDCA (500 mg/kg body weight) or vehicle, dark-adapted for 18 h, injected again, then exposed to 7 h of bright (10,000 lux) or dim (200 lux) light (Chen et al. 2003), then returned to regular rearing lighting conditions. ERGs, retinal morphology, and apoptosis markers were assessed at various times post-exposure. TUDCA treatment nearly completely prevented the massive disruption of photoreceptor cells, extreme disorganization, and apoptosis signal throughout the ONL typically seen within 24 h of damaging light exposure. Such protection was observed to 21 days post-exposure, the longest post-exposure duration of these assessments in our experiments (Boatright et al. 2006b and unpublished observations). Further, ERG amplitudes were maintained in TUDCA-treated mice exposed to bright light, even up to 7 weeks post-exposure (Yang et al. 2008), suggesting that protection is fairly long-term.

Oveson et al. (2011) recently demonstrated the effectiveness of systemic TUDCA treatment in rd10 and LIRD mouse models of retinal degeneration. They extended previous work by demonstrating that, in addition to protecting retinal function and morphology as described above and elsewhere (Chang et al. 2007; Phillips et al. 2008; Boatright et al. 2006b; Yang et al. 2008), TUDCA treatment suppressed superoxide radical formation in the LIRD mouse and provided significant protection against loss of cone photoreceptor number and function in the rd10 mouse out to P50 (Oveson et al. 2011), significantly longer than we or others previously reported.

Other genetic retinal degeneration models respond to systemic TUDCA treatment. TUDCA treatment slows retinal degeneration in s334ter-3 and P23H-3 rats, rat lines that were genetically engineered to have rhodopsin mutation identical to ones common in autosomal dominant retinitis pigmentosa (ADRP) patients (Steinberg et al. 1996). s334ter-3 rats were systemically injected daily from birth with TUDCA (Mulhern et al. 2008). Retinal sections from P5 and P10 rats showed that TUDCA treatment significantly decreased markers for reactive oxygen species, endoplasmic reticulum (ER) stress, and apoptosis. Retinal degeneration as assessed by morphology was also delayed in TUDCA-treated rats (Mulhern et al. 2008). TUDCA treatment also slows retinal degeneration in P23H-3 rats (Fernandez-Sanchez et al. 2008, 2009). Rats were injected intraperitoneally (500 mg/kg body weight) once per week from P20 through 4 months old. Photoreceptor inner and outer segments, ONL nuclei counts, and the capillary retinal network were preserved in TUDCA-treated compared to vehicle-treated rats and TUNEL signal was lower in TUDCA-treated rats compared to controls (Fernandez-Sanchez et al. 2008, 2009).

In addition to these models of ADRP, the hydrophilic bile acids prevent disease progression in a model of age-related macular degeneration (AMD). Systemic treatment with UDCA or TUDCA suppresses choroidal neovascularization (CNV) in a laser-treated rat model of wet AMD (Woo et al. 2010). Rats were injected intraperitoneally the day before ocular argon laser photocoagulation and daily thereafter for 14 days with UDCA (500 mg/kg) or TUDCA (100 mg/kg). TUDCA treatment suppressed laser-induced increases in vascular endothelial growth factor (VEGF) levels in the retina. Either UDCA or TUDCA treatment reduced CNV lesion dimensions and clinically significant fluorescein leakage (Woo et al. 2010). As with the responses in other models of ocular disease, systemic treatment with UDCA or TUDCA has effects in this posterior ocular disease model.

These several examples and others reviewed previously (Boatright et al. 2009a) demonstrate that TUDCA or UDCA delivered systemically in animal models of retinal degeneration and neurodegeneration is protective. Further, we and others have demonstrated that TUDCA prevents apoptosis and cell death in general in various cell culture models, including retinoblastoma cell lines (Do et al. 2003; German Moring et al. 2003). This suggests that TUDCA can have direct effects on cells and it allows for speculation that systemically delivered bile acids result in elevated levels of bile acids at posterior ocular cellular targets. This is further supported by a recent clinical trial with ALS patients in which orally delivered UDCA resulted in elevated UDCA levels in cerebral spinal fluid (CSF) that correlated with dosage concentration (Parry et al. 2010).