22.3  Potential Need for Local Delivery of Bile Acids as Neuroprotectants

It thus appears that systemic routes as a delivery modality may be sufficient in TUDCA or UDCA treatment of posterior ocular disease. Oral delivery of either leads to few and minimal side effects (Parry et al. 2010; Nakagawa et al. 1990; Crosignani et al. 1996; Setchell et al. 1996; Invernizzi et al. 1999), results in elevated serum and plasma levels of UDCA and its conjugates (Batta et al. 1989, 1993; Setchell et al. 1996; Invernizzi et al. 1999; Parry et al. 2010), and clearly slows or prevents retinal damage or degeneration in a number of animal models. However, systemic delivery may not be sufficient due to possible individual variability in response to systemic dosing.

Though TUDCA and UDCA have been approved for treatment liver and gall bladder afflictions for decades, relatively little is known about the pharmacokinetics of these bile acids in normal, diseased, or dosed states (Invernizzi et al. 1999). Studies that do report bile acid levels in blood and non-hepato-biliary tissues generally do not focus on UDCA and its conjugates as these bile acids do not make up a substantial proportion of the bile acid pool in humans. The studies that do report circulating levels of UDCA and its conjugates indicate great variability in serum, plasma, or CSF levels among individuals (Invernizzi et al. 1999; Parry et al. 2010).

There are significant differences in UDCA and TUDCA pharmacokinetics following oral administration. TUDCA administration leads to greater biliary UDCA enrichment than UDCA administration, probably because hepatic extraction of taurine-conjugated bile acids is more efficient than that of their unconjugated forms (Invernizzi et al. 1999). This may result in better clinical efficacy for treatment of hepato-biliary disease. Of added importance in regards to these and other potential therapeutic uses is that TUDCA undergoes much less biotransformation to lithocholic acid than does UDCA (Invernizzi et al. 1999). Lithocholic acid is cytotoxic and there are concerns that a harmful side effect of long-term UDCA treatment can be liver damage (Invernizzi et al. 1999).

Of more direct importance to neuroprotection uses, oral dosing with either UDCA or TUDCA produces high serum concentrations of UDCA conjugates. Oral administration of UDCA results in UDCA and its conjugates becoming the dominant bile acids in biliary bile and absolute concentrations in blood increase over tenfold (Fedorowski et al. 1977; Parquet et al. 1985; Oka et al. 1990; Stiehl et al. 1990; Batta et al. 1993; Rubin et al. 1994). About half of an UDCA dose is absorbed from the portal blood into liver via first pass extraction, where it is conjugated with glycine, forming glycoursodeoxycholic acid (GUDCA), or taurine, forming TUDCA (Nakagawa et al. 1990; Hofmann 1994; Rubin et al. 1994; Paumgartner and Beuers 2002). The percentage absorbed decreases with increasing dose such that absolute and proportional enrichment of the biliary bile with UDCA and conjugates plateaus at an as-yet undefined dose due to epimerization of UDCA to chenodeoxycholic acid (CDCA) and endogenous bile acid synthesis (Tint et al. 1982; Parquet et al. 1985; Walker et al. 1992; Hofmann 1994). UDCA and conjugates are excreted from

the biliary tree and resorbed through the enterohepatic circulation or metabolized to insoluble salts and excreted in the feces (Rubin et al. 1994). Oral TUDCA produces similar changes in bile acid composition and concentrations, but with higher proportions and concentrations of UDCA and conjugates, possibly due to reduced intestinal biotransformation of TUDCA, suggesting enhanced bioavailability (Crosignani et al. 1996; Setchell et al. 1996).

Though oral dosing with UDCA or TUDCA greatly increases serum levels of UDCA conjugates, bile acid compositions and levels in blood vary greatly across subjects. Oral treatment of PBC patients with either TUDCA or UDCA (750 mg/ day for 2 months) results in higher serum levels of UDCA conjugates, but of great concentration range (Invernizzi et al. 1999). UDCA serum levels were 24.1 ± 15.1 mmol/L (mean ± SD) following UDCA treatment and 26.1 ± 19.9 following TUDCA treatment. (Pretreatment levels were 0.2 ± 0.3 and 0.1 ± 0.2 mmol/L, respectively.) (Invernizzi et al. 1999).

Similar variability was observed in other studies. Feeding UDCA (12–15 mg/kg body weight per day) to PBC patients for 6 months results in UDCA and its conjugates in becoming the most prevalent bile acids both in serum and urine with a corresponding decrease in the endogenous bile acid concentrations, with absolute levels of serum UDCA increasing from 1.7 mmol/L prior to treatment to 24.5 mmol/L. However, serum UDCA concentrations across patients ranged from 2.3 to 51.3 mmol/L, a remarkable variation in response to the same dosing regimen (Batta et al. 1989).

Variability in serum levels following oral UDCA administration may result in differences at neuronal tissue targets. In subjects who are free of known hepatobiliary disease, oral dosing with 15-, 30-, and 50-mg/kg body weight for 29 days led to significantly increased serum UDCA concentrations that correlated with dose concentration and with concentration of UDCA in CSF (Parry et al. 2010). However, for each dose, CSF UDCA concentrations varied greatly (fourfold, 3.5-fold, and 2.7-fold, respectively), suggesting that even in subjects without hepato-biliary compromise, the amount of UDCA “spilled” into the circulation varies greatly from subject to subject.

Where might this variability originate and could it have consequences for the utility of TUDCA or UDCA use as neuroprotectants in the ophthalmic clinic? Several transporters and metabolic enzymes mediate the regulation of endogenous bile acid concentrations in circulation. One of these, organic anion transporting polypeptide 1B1 (OATP1B1), is an influx transporter that mediates hepatic uptake of endogenous compounds such as bile acids and bilirubin and also uptake of several drugs from the portal blood (reviewed in Xiang et al. 2009). Polymorphisms in SLCO1B1, the gene that codes for OATP1B1, are linked to differences in the pharmacokinetics and effects of several drugs. Recently, SLCO1B1 polymorphisms were similarly linked to differences in plasma levels of bilirubin and bile acids, including UDCA and TUDCA (Xiang et al. 2009). In particular, reduced plasma concentrations of UDCA and TUDCA were associated with the SLCO1B1*1B/*1B genotype, leading the study’s authors to suggest that this is likely due to enhanced hepatic uptake mediated by OATP1B1 during enterohepatic circulation (Xiang et al.

2009). It is of course not clear that processes that regulate fasting levels of bile acids will similarly mediate circulating levels of UDCA and its conjugates during therapeutic intervention. At the dosages given, the regulatory capacity of such mediators may be overwhelmed.

In agreement with these human subject trials, experiments with mouse strains lacking all mouse Oatp1a/1b transporters show markedly increased plasma levels of unconjugated bile acids. These mice also have decreased hepatic uptake and thus increased systemic levels following i.v. or oral administration of the OATP substrate drugs methotrexate and fexofenadine (van de Steeg et al. 2010). It is thus possible that substrates of OATP such as UDCA or TUDCA, when given systemically as drugs, indeed may have their pharmacokinetics mediated by these transporters.

We have observed strain differences in fasting serum levels of TUDCA of mice. Serum of Balb/C mice had very low TUDCA concentrations (0.0293 ± 15 mmol/L, N = 17; mean ± SEM), whereas C57BL/6J mice had easily measured levels, but over a large range (27.4 ± 12 mmol/L, range of 0.007–170 mmol/L; N = 16). However, we have not been able to identify strain-specific polymorphisms in the mouse homolog of SLCO1B1, SLCO1b2, that correspond to those of SLCO1B1 associated with altered circulating bile acid levels in humans (Foster et al. 2009). Obviously numerous other mediators of circulating bile acid levels could be at play here. We continue to explore the source of this strain difference.

22.4  Preliminary Studies of Ocular Delivery of Bile Acids

Individual or subpopulation differences in the regulation of circulating levels of TUDCA and UDCA following systemic administration could confound assessment of their efficacy as neuroprotectants in treatment of posterior ocular disease. Thus, it may be useful to test local delivery. We have initiated such studies and find that a single intravitreal injection of TUDCA provides protection in the LIRD mouse model comparable to that provided by multiple systemic injections reviewed above and elsewhere (Boatright et al. 2006a, b, 2009a).

In these experiments, Balb/C mice were intravitreally injected with 1 mL of varying doses of free acid TUDCA 0.5, 5, 15, 30, 50 mg/mL in phosphate-buffered saline (PBS) in one eye, and with sterile PBS in the other. ERGs were taken weekly. Mice sacrificed at various times after injection to assess morphology and TUNEL signal in retina sections. Doses higher than 5 mg/mL showed reduced a-wave and b-wave amplitudes in the ERG waveforms, and increased apoptotic signal that corresponded to reduced ONL thickness (Kendall et al. 2008). Doses of 5 mg/mL and below, however, showed similar ERG amplitudes to that of the PBS treated eyes, along with similar retinal morphology. Based on this, we tested the effects of a single intravitreal injection on LIRD as described above and elsewhere (Boatright et al. 2006a, b, 2009a). Balb/C mice were intravitreally injected with 1 mL of 5 mg/mL of TUDCA or PBS in each eye, dark-adapted overnight, and exposed to bright light (10,000 lux) or dim light (50 lux) for 7 h on the following day. ERGs were taken

weekly for 3 weeks after light damage. Dim-adapted ERG a-wave and b-wave amplitudes were greater in eyes that had been injected with TUDCA-treated eyes compared to amplitudes generated by the PBS-injected contralateral eyes. Similarly, TUDCA-treated eyes showed reduced TUNEL signal compared to their PBS counterparts (Kendall et al. 2008). Thus, a single intravitreal injection of 5 mg/mL of TUDCA protected against LIRD.

In addition to intravitreal injection, preliminary data suggests that TUDCA should be able to be delivered transsclerally as it has predictable scleral diffusion parameters. We assessed whether TUDCA in balanced salt solution (BSS) or balanced salt solution plus (BSS+) can diffuse across human sclera. Donor sclera was mounted in a Lucite block perfusion chamber. The outer surface of the sclera was exposed to 200 mL of TUDCA (50 mg/mL) in either BSS or BSS+ for 24 h. Perfusate fractions were collected every 2 h over a 24 h period. Ultra performance liquid chromatography (UPLC)/tandem mass spectrometry was used to quantitate TUDCA and unconjugated UDCA in perfusates. We found that TUDCA readily diffused across sclera. The transscleral permeability constant (Kconst) for TUDCA was 1.89 × 10−6 cm/s in BSS, 1.97 × 10−6 cm/s in BSS+, and 4.63 × 10−7 cm/s in fibrin sealant. These perfusion rates are in agreement with other compounds of similar molecular weight (e.g., penicillin G, Doxil, rhodamine, dexamethasone-fluorescein, etc.) (Boatright et al. 2009b).

22.5  Conclusion

The hydrophilic bile acids UDCA and TUDCA are anti-apoptotic and protective in many neurodegeneration models. Protective effects in ocular disease models are reported by several independent laboratories using models of ADRP, AMD, and other diseases and injuries (Arora et al. 2009; Boatright et al. 2009a). In nearly all of the studies testing in vivo models of neurodegeneration and retinal degeneration, systemic treatment provides marked protection. Such efficacy coupled with the lack of notable side effects in animals or humans suggests that systemic delivery is an adequate delivery modality for these therapeutic compounds. As such, it is worthwhile to consider that the few studies that have directly examined circulating levels of UDCA and its conjugates, either in the resting state or following bile acid therapy, indicate that humans and mice can have vastly differing levels of these bile acids. This variability may be due to individual or subpopulation differences in bile acid physiology and could have ramifications for clinical trial design and eventual neuroprotective therapeutic use, particularly if subpopulations are refractory to attempts to increase circulating levels via systemic administration of these bile acids in order to provide therapeutically sufficient concentrations at target tissues. Local delivery might be required. Our initial experiments testing in vivo intraocular injections and in vitro transscleral permeability indicate that this will be no more challenging than for other ophthalmic therapeutic compounds currently being tested or already in the clinic.