Previously, our laboratory used the zebrafish lateral line to screen a small molecule library for protective agents (Owens et al. 2008
). That study identified two chemicals (PROTO1 and 2) with protective effects against hair cell death. While PROTO1 and 2 are candidates for drug development, FDA approval of new drugs requires an average of 12 years and the odds of a new drug becoming approved are approximately one out of 5,000 (Wierenga and Eaton 1993
). Applying the same screen to the NINDS Custom Collection is potentially more efficient. Compounds in this library have proven drug-like properties such as solubility and cell permeability in contrast to the less well-characterized small molecule libraries. Use of this library bypasses certain aspects of drug development and moves more rapidly towards identifying an “ideal” candidate protective drug as defined above. By evaluating the seven candidate protective drugs identified in the screen with regards to these six characteristics, tacrine was identified as a protective drug worthy of future study.
Safety of administration
In searching for a protective compound for the inner ear, ease of administration is an important factor. Some compounds are not practical for systemic administration or may only be effective when delivered directly to the inner ear (e.g. JNK inhibitors and caspase inhibitors; Wang et al. 2003
; Zine and van de Water 2004
). Systemic administration avoids the need for invasive intracochlear or transtympanic drug applications. All seven of the candidate drugs have been used systemically in humans, although only carvedilol, tacrine, and phenoxybenzamine are currently FDA-approved. The others have been used in experimental protocols or countries outside of the USA.
The clinical uses for these drugs provide insight into their potential as protective drugs. Amsacrine is a topoisomerase II poison and is generally cytotoxic (Rowe et al. 1986
) and thus impractical for systemic use as an inner ear protectant. Hexamethyleneamiloride is a Na/H-exchange blocker used as a diuretic (Davies and Solioz 1992
). Phenoxybenzamine (alpha-adrenergic blocker) and carvedilol (beta-adrenergic blocker) are both used to treat hypertension (Osnes et al. 2000
). Cepharanthine has membrane-stabilizing activity and is used for treatment of nasal allergy and snake venom hemolysis (Furusawa and Wu 2007
; Kohno et al. 1987
). Drofenine and tacrine are acetylcholinesterase inhibitors used for muscle relaxation and Alzheimer’s dementia, respectively (Bodur et al. 2001
; Drukarch et al. 1987
Blood–brain barrier penetration
All seven drugs cross the blood–brain barrier (Table ). The kinetics of inner ear penetration for these drugs is unknown. It is also unknown whether crossing the blood–brain barrier is critical for a potential inner ear protectant, however this would seem to improve the odds of diffusion into the perilymph and possibly endolymph.
Protection against a wide-range of aminoglycoside doses
The ideal protective drug would protect hair cells over a wide range of aminoglycoside dosages. The efficiency of testing in the zebrafish permits thorough investigation of hair cell protection. We tested a range of concentrations of each protectant and tested each protectant against a range of neomycin concentrations. Testing a range of protectant concentrations is important because some protectants have been found to be toxic at higher concentrations. In addition, protective compounds effective against a narrow range of aminoglycoside concentrations may have limited clinical utility (Sugahara et al. 2006
). In this study, all candidate drugs were protective against a wide range of neomycin doses in the zebrafish lateral line, preserving a range of 60% to 100% of the hair cells.
We hypothesize that the variability in the magnitude of protective effects is due to differential effects on the multiple death pathways activated by aminoglycoside exposure. The mechanisms underlying aminoglycoside-induced hair cell death remain controversial, with both caspase-dependent and caspase-independent pathways implicated (Cunningham et al. 2002
; Matsui et al. 2003
; Jiang et al. 2006
). Inhibition of one death pathway likely still allows (or possibly even facilitates) death through alternate pathways (Lin et al. 1999
; Yu et al. 2004
). Although some of the drugs identified in this study achieved complete protection in the lateral line, ultimately, a protective “cocktail” composed of multiple drugs may be a more effective regimen for prevention of hair cell death.
Effects on aminoglycoside uptake
Three candidate drugs, drofenine, tacrine, and cepharanthine, did not affect TR-Gent uptake. Ideally, a protective drug identified in this neomycin-driven screen would inhibit intracellular death pathways triggered by an aminoglycoside rather than inhibiting aminoglycoside uptake. This drug would have more potential applications against a wider variety of causes of hair cell death.
While our tendency is to focus on drugs that affect cell death pathways, it is important to recognize that drugs that blocked aminoglycoside uptake may also have clinical relevance. These drugs could be administered systemically or locally to block ototoxicity of the aminoglycoside without affecting its bactericidal capacity or perturbing intracellular pathways. Hexamethyleneamiloride, amsacrine, carvedilol, and phenoxybenzamine blocked the uptake of TR-Gent. Hexamethyleneamiloride, as an amiloride derivative, is likely to block the mechanotransduction channel. The mechanisms of blockade by carvedilol, amsacrine, and phenoxybenzamine are unknown. Of these drugs, only phenoxybenzamine also affected FM1-43 uptake. This differential blockade between FM1-43 and TR-Gent suggests either different mechanisms of uptake, differing sensitivity to channel blockade, or different sensitivities of our detection methods.
Effects on bactericidal activity of aminoglycoside
None of the seven drugs affected the bactericidal activity of neomycin. This has obvious importance for potential use as an aminoglycoside-specific protective drug.
Protection in mammalian tissue
Hair cell protection in the zebrafish lateral line does not assure protection in mammalian hair cells, and thus testing was performed in the mouse utricle.
Due to the more time-consuming nature of mammalian testing, only two of the seven drugs were tested in the utricle, cepharanthine and tacrine. These two drugs were chosen because they were effective protective drugs that did not inhibit aminoglycoside uptake. Tacrine demonstrated significant protection against neomycin-induced hair cell death in mouse utricle explants. In contrast, cepharanthine was found to cause hair cell death at moderate concentrations, and thus further experiments examining its protective effects were not conducted. Tacrine is particularly interesting because its derivative, bis(7)-tetrahydroacridine, stabilizes the mitochondrial membrane potential and has been used experimentally as a neuroprotectant (Fu et al. 2006
; Fu et al. 2007
The lack of hair cell protection by cepharanthine in the mouse utricle demonstrates the importance of confirming findings from zebrafish in mammalian systems. However, there are critical differences between the exposure conditions of lateral line hair cells and free-floating utricle hair cells. Free-floating utricles have been removed from the organism, and thus any neural input has been removed. Secondly, lateral line hair cells are exposed to drugs predominantly at their apices, while free-floating utricles are bathed circumferentially. Lastly, one can hypothesize that hair cells in vitro may be more fragile and susceptible to cell death than hair cells in vivo.
This study represents the first screen of a library of compounds with known bioactivity for drugs that protect against aminoglycoside-induced hair cell death. Further evaluation of the screen identified tacrine as a particularly promising drug. Tacrine demonstrates protection against a wide range of neomycin doses, can be administered systemically, crosses the blood–brain barrier, does not inhibit aminoglycoside uptake, does not interfere with the bactericidal activity of neomycin, and is effective in mammalian utricle explants. Tacrine also has known targets as an acetylcholinesterase inhibitor and stabilizer of mitochondrial membrane potential. Tacrine is thus an excellent candidate for further investigation through in vivo testing, evaluation of its mechanism of action (cholinergic versus off-target effects), and evaluation of its potential as a protective drug against other challenges such as cisplatin, noise, and even aging.
The results of this screen are not meant to suggest that none of the other compounds in the library have protective effects. In particular, many antioxidants are known to protect against hair cell death and were not detected in the screen. Varying the screening conditions would likely identify additional protective drugs and is worthy of future study. For example, protection against hair cell death in the zebrafish lateral line has been shown to require a 24-h exposure to antioxidants such as D-methionine and glutathione (Ton and Parng 2005
Some protection may be due to global effects on the organism (e.g. increasing renal clearance of neomycin) rather than specific effects on the hair cell. We think this is unlikely since aminoglycosides appear to enter lateral line hair cells directly from the medium rather than depending on circulation or tissue accumulation.
It is important to note that tacrine has known hepatotoxicity that has hindered its use for the treatment of Alzheimer’s dementia. In a multicenter clinical trial of tacrine, 49% of patients demonstrated an elevation of liver transaminases at a mean of 50 days after initiation of therapy (Watkins et al. 1994
). Most of these patients were asymptomatic and had a return to normal liver function levels after withdrawal of the drug. Of the patients that had tacrine therapy resumed, 88% were able to resume long-term use of the drug. A number of possible mechanisms for this hepatotoxicity have been implicated, including poisoning of topoisomerases (Mansouri et al. 2003
). It is likely that this toxicity occurs via a separate mechanism from its protective effect seen in hair cells. It may be possible to decrease the tacrine dose to minimize toxicity while maintaining its protective effects in hair cells. In addition, it is likely that for hair cell protection protocols, tacrine would be administered for days to weeks, rather than the months to years required for treatment of Alzheimer’s dementia. Tacrine hybrid compounds are currently being developed (Fang et al. 2008
) with potent activity but reduced hepatotoxicity. It remains to be seen whether these modifications would also reduce the protective effects seen in hair cells.
Limited mammalian data are presented due to the time-consuming nature of mammalian experiments. Additional studies are imperative for further progress. Lateral line hair cells have important differences from inner ear hair cells. There is no separation of fluid spaces in the lateral line so the apical surfaces of hair cells extend into the surrounding water. In addition, since there is no stria vascularis, drugs that act through strial mechanisms will not be identified. We view the screen as an efficient method for rapidly identifying candidate drugs that must then be confirmed in the mammalian inner ear.