Potential Therapeutic Agents

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Potential Therapeutic Agents

Kathleen C.M. Campbell, Ph.D.,¹and Colleen G. Le Prell, Ph.D.²

ABSTRACT

Acquired hearing loss, which can develop after noise insult or ototoxic drug treatment, is a significant clinical, social, and economic issue. A major advance in our understanding came with the discovery that intense metabolic activity in the inner ear drives the formation of free radicals (short-lived, unstable, highly reactive clusters of atoms) in multiple types of cells in the inner ear after both noise and drug insult.

Animal studies have now shown that free radicals formed during and after metabolic stress importantly contribute to acquired hearing loss.

These new mechanistic insights provided for the first time a rationale for directly treating the inner ear to prevent hearing impairment.

Consequently, use of free radical scavengers, or antioxidants, to prevent acquired hearing loss became a clinically relevant research goal. Many laboratories have now demonstrated that a variety of free radical scavengers reduce the potential for acquired hearing loss in animal subjects. Scientific data, supporting the use of these agents to prevent environmentally acquired hearing loss, is reviewed. Translational in- vestigations are now essential to confirm the potential utility of these agents in the human inner ear. This article reviews the pharmacological otoprotective agents in or approaching clinical trials to prevent noise-, aminoglycoside-, and cisplatin-induced hearing loss.

KEYWORDS:Noise-induced hearing loss, ototoxicity, otoprotection, D-methionine, N-acetylcysteine, ebselen, amifostine, sodium thiosulfate, aspirin, b-carotene, vitamin C, vitamin E, magnesium

Learning Outcomes: As a result of this activity, the participant will be able to (1) list similarities in the mechanisms underlying drug and noise-induced hearing loss and (2) describe putative pharmacological otoprotective agents in or approaching clinical trials to prevent drug- and noise-induced hearing loss.

1Division of Otolaryngology–Head and Neck Surgery, Department of Surgery, Audiology Research, Southern Illinois University School of Medicine, Springfield, Illi- nois; ²Department of Speech, Language and Hearing Sciences, University of Florida, Gainesville, Florida.

Address for correspondence and reprint requests:

Kathleen C.M. Campbell, Ph.D., Division of Otolaryng- ology–Head and Neck Surgery, Department of Surgery, Audiology Research, Southern Illinois University School

of Medicine, P.O. Box 19629, Springfield, IL 62536 (e-mail: [email protected]).

Current Perspectives on Ototoxicity; Guest Editor, Steven P. Smith, Au.D.

Semin Hear 2011;32:281–296. Copyright # 2011 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.

DOI: http://dx.doi.org/10.1055/s-0031-1286622.

ISSN 0734-0451.

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Both ototoxic drugs and noise have the potential to damage the cells in the inner ear.

Some drugs are cochleotoxic, meaning they damage sensory cells in the cochlea and induce hearing loss; other drugs are vestibulotoxic, meaning they damage cells in the semicircular canals and induce balance deficits. Most aminoglycoside antibiotics are both cochleotoxic and vestibulotoxic, as is cisplatin, a common anti- neoplastic agent. Noise also has the potential to damage both auditory and sensory cells. Devel- opment of therapies that reduce noise-induced hearing loss (NIHL) as well as drug-induced hearing loss during aminoglycoside antibiotic treatment or cisplatin administration would not only protect hearing, but might also ameliorate vestibulotoxicity. A better understanding of the mechanisms of cellular and molecular changes subsequent to noise or ototoxic drug insult has advanced the potential to identify and develop such agents. Identification of oxidative stress has been particularly significant for the development of novel therapeutic agents to reduce noise and ototoxic drug insults. For the first time, there is a potential opportunity not just to monitor developing toxicity, but to actually intervene and prevent the onset and/or progres- sion of acquired hearing loss.

OXIDATIVE STRESS AND ENDOGENOUS DEFENSE

Biological energy is built in cell mitochondria from nutrients (such as glucose) and oxygen, which are combined to form adenosine triphos- phate (ATP). ATP is the main form of energy used by cells. When oxygen is used to produce ATP, highly reactive oxygen-based molecules are produced as a by-product of the metabolic process. These molecules are appropriately termed ‘‘reactive oxygen species’’ (ROS). Other highly reactive molecules are nitrogen-based, and these species are termed ‘‘reactive nitrogen species’’ (RNS). A generic term for the ROS and RNS molecules produced during metabolic processes is ‘‘free radicals.’’ Free radicals, simply defined, are atoms, molecules, or ions with unpaired electrons; these unpaired electrons make the radicals highly reactive. Free radical production inside hair cells and other cells in the inner ear has been reviewed in detail else-

where.¹ In brief, under normal physiological conditions, 1 to 5% of the oxygen consumed during cell metabolism is converted to free radicals, including superoxide (O₂^ÿ), hydrogen peroxide (H₂O₂), or other free radical species (for additional evidence see Chow et al²).

These free radicals must be neutralized, and endogenous defense mechanisms for the neu- tralization of toxic free radicals do exist. En- dogenous defense is mediated by superoxide dismutases (SODs), catalase, glutathione (GSH), and glutathione peroxidase (GPx), an enzyme that speeds the oxidation of endogenous GSH.

ENDOGENOUS DEFENSE Superoxide Dismutase

Specific proteins can increase the rate of (or, catalyze) certain chemical reactions. These proteins are called enzymes. SODs are enzymes that speed the breakdown of superoxide into oxygen (O2) radicals and hydrogen peroxide radicals, which are less toxic than superoxide.

Specific SOD enzymes are distinguished based on the metal cofactors they bind (copper and zinc, manganese, or nickel) and where they are found (extracellularly, in cell cytoplasm, or in cellular mitochondria). In humans and other mammals, SOD1 (Cu-Zn-SOD) is found in cytoplasm, SOD2 (Mn-SOD) is found in mitochondria, and SOD3 (Cu-Zn-SOD) is found in extracellular areas. SOD1 is found at high levels in cochlear tissues including organ of Corti and stria vascularis.³There is a variety of evidence for a direct role for SOD1 (and to a lesser extent SOD2) in vulnerability of the inner ear to noise insult⁴ (see Le Prell and Bao and McFadden et al for reviews^1,5).

SOD1 (and to a lesser extent SOD2) also have been implicated in mediating damage to cochlear and vestibular tissues after ototoxic drug administration^6–15 (see ‘‘Oxidative Stress and Ototoxic Drugs’’ for additional detail).

Catalase

Catalase is a second key enzyme in the endogenous defense network, and like SOD, it is found in the cochlea.³ After SOD converts

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superoxide to hydrogen peroxide, catalase speeds the decomposition of hydrogen peroxide into water and oxygen. Like SOD enzymes, catalase deficiency has been linked to vulnerability to NIHL^16,17and to drug-induced oto- toxicity6,12,15,18,19 (see ‘‘Oxidative Stress and Ototoxic Drugs’’ for additional detail).

Glutathione

GSH is the third endogenous antioxidant defense system, and it is present in the inner ear.²⁰ GSH is produced from three amino acid pre- cursors: cysteine, glutamic acid, and glycine.

The antioxidant activity of GSH is specifically attributed to hydrogen atom donation from the cysteine sulfhydryl (thiol) group. In other words, GSH protects cells against electron leaks by donating a hydrogen atom to stabilize free radicals. When electron donation leaves GSH in an oxidized (reactive) state, two reactive GSH molecules then bind to each other to form glutathione disulfide (GSSG). The enzyme glutathione reductase then reduces GSSG back to GSH. An increase in the ratio of GSSG relative to GSH indicates increased oxidative stress; under normal conditions, GSH is the predominant form with low GSSG detected. GPx is an enzyme that cata- lyzes the reduction of free radicals by GSH.

There are at least eight major GPx isoforms, and glutathione peroxidase 1 (GPx1) is the major isoform within the cochlea, where it is highly expressed in organ of Corti, spiral gan- glia, stria vascularis, and spiral ligament.²¹ Knockout of the GPx1 enzyme in mice leaves them more vulnerable to noise insult than mice that produce GPx1.²²

OXIDATIVE STRESS AND OTOTOXIC DRUGS

The evidence that cell death after noise insult can be mediated by free radical production and that manipulation of antioxidant status can reliably influence postnoise outcomes has been reviewed in significant detail.¹ In this section, specific evidence that cell death after ototoxic drug treatment is mediated at least in part by free radical production and that endogenous antioxidant status can be used to influ-

ence cell survival and hearing outcomes during and after ototoxic drug treatment is described.

These preliminary studies made it possible to define antioxidant interventions that attenuate ototoxicity in animals. Effective strategies are reviewed in the next section.

Aminoglycoside Antibiotics

Many of the aminoglycoside antibiotics, such as neomycin, tobramycin, kanamycin, amikacin, and gentamicin, can affect both hearing and balance. However, some of these ototoxic drugs are almost exclusively vestibulotoxic (such as streptomycin) or cochleotoxic (such as dihydrostreptomycin). In general, for those drugs that affect the cochlea, the outer hair cells (OHCs) are affected most, with OHC losses progressing from base to apex (i.e., from high to low frequencies). The frequency-specific hearing losses associated with aminoglycoside antibiotic drugs develop slowly and may arise even weeks after cessation of treatment (see, for example Stebbins et al²³). The differential vulnerability from base to apex was not well understood until more recently, when the pro- gressive deficits were finally attributed to intrinsic differences in GSH production along the cochlea.¹⁰Recent data also have revealed a difference in the rate of accumulation of ROS within OHCs, with basal cells accumulating ROS faster than more apical cells, and cells in the first row of OHCs accumulating ROS more quickly than cells in the second or third row of OHCs.²⁴

Free radicals form when cochlear cells are treated with aminoglycoside antibiotics. Both hydrogen peroxide and hydroxyl radicals have been measured after cochlear cells were treated with gentamicin or kanamycin.²⁵In a second in vivo study from the same group, both superoxide radicals and hydrogen peroxide radicals were shown to decrease auditory function; superoxide-induced functional deficits were alleviated by coadministration of SOD with superoxide, and hydrogen peroxide-induced deficits were alleviated by coadministration of catalase with hydrogen peroxidase.²⁶These in vivo data confirmed earlier in vitro observations in which SOD and catalase were applied to cultured OHCs to reduce the effects of superoxide

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anions, hydrogen peroxide, and hydroxyl radicals.²⁷

Other groups have shown ROS formation in the inner ear after aminoglycoside antibiotic administration. For example, ROS immunostaining, shown using immunostaining for 4- hydroxynonenal, was robustly increased in hair cells in tissues harvested from kanamycin- treated mice (although RNS labeling, using nitrotyrosine, was not; see Jiang et al²⁸). Robust ROS was seen after gentamicin was applied to immortalized organ of Corti cells in vitro; in those cells, there was a decrease in SOD levels, and apoptotic cell death was confirmed using caspase-3 antibodies.²⁹Taken together, a variety of evidence suggests oxidative stress is a key early event preceding cell death in the inner ear after aminoglycoside antibiotic therapy.

These early stress events presumably over- whelm endogenous defenses. There is a robust decrease in endogenous antioxidant enzymes after aminoglycoside treatment. For example, in cochleas harvested from guinea pigs treated with amikacin, all antioxidant enzymes measured, including SOD, catalase, GSH, GPx, and oxidized glutathione (GSSR), were significantly lower in the amikacin group than the control group.¹² Consistent with the notion that endogenous antioxidant systems provide some defense against aminoglycoside insult, transgenic mice that overexpress SOD1 are more resistant to ototoxicity associated with kanamycin than their wild-type counterparts.¹¹ In vitro treatment with a SOD2 mimetic also has been effective in reducing gentamicin-induced cell death.

Although the above data all suggest a key role for classic oxidative stress-driven apoptotic cell death pathways after aminoglycosides just as after noise (for review, see Le Prell et al³⁰), it is important to note that many of the classic markers for apoptotic cell death after oxidative stress were ‘‘missing’’ in several studies. For example, markers for cytochrome c, caspase-9, caspase-3, as well as Jun-terminal protein kin- ase (JNK), and terminal deoxynucleotidyl transferase dUTP nick end labeling, failed to show any changes in kanamycin-treated mice.³¹ Other molecular events that were detected included the translocation of endonu- clease G from the mitochondria to the

nucleus³¹and also a disruption of phosphoino- sitide signaling, which the authors suggested provides one potential explanation for changes in the Rac/Rho signaling pathways and drug-induced changes observed in the actin cytoskeleton.³² Taken together, oxidative stress is a critical event, but may not be the only significant pathway to cell death activated by aminoglycoside drug treatment.

Drug effects may vary with the specific drug tested, the species it is evaluated in (for additional evidence, see Poirrier et al³³), as well as the severity of the insult delivered. Systematic manipulation of drugs and doses within and across species are needed to better resolve questions regarding mechanisms of cell death after drug treatment.

Chemotherapeutics

Cisplatin and carboplatin are common antineo- plastic drugs used during lifesaving chemotherapeutic treatments. Like the aminoglycosides, they are highly ototoxic, killing hair cells in a pattern that progresses from base to apex. Also like aminoglycoside treatments, these drugs have the potential to result in hearing loss that continues to progress long after the drug treatment has ended. We now know that another shared feature of these two drug classes is a shared early event: oxidative stress. The role of ROS in cisplatin-induced hearing loss has been reviewed³⁴ and parallels that of aminoglycosides in many ways. We therefore only briefly note here that cisplatin (1) specifically increases the production of superoxide anions inside the OHCs³⁵; (2) effects cochlear SOD and catalase, with increased activity^6,36 and decreased levels^8,37 reported; and (3) as observed after aminoglycoside therapy, cisplatin significantly decreases the measured levels for GSH, GPx, and GSSR.^6,8,37

There are some critical differences between aminoglycoside-induced ototoxicity and cisplatin-induced ototoxicity. First, vulnerability to cisplatin appears to vary as a function of polymorphisms in GSH-related genes,³⁸ whereas those polymorphisms were not able to predict vulnerability to aminoglycoside ototoxicity in a more recent study.³⁹ Second, unlike aminoglycoside treatment, cisplatin

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treatment effects were not effectively reduced by the SOD2 mimetic that reduced aminoglycoside-induced toxicity.¹³ A third key difference between aminoglycoside insult and cisplatin insult is observed when drugs are evaluated in the avian model. It is known that the sensory cells on the avian basilar papilla regenerate after noise and after aminoglycoside insult.^40–43Unlike after noise and aminoglycosides, the avian basilar papilla does not regenerate after cisplatin insult.⁴⁴Thus, despite the shared evidence for a key role of oxidative stress in toxicity after different drug insults, there is the potential for additional mechanisms of insult that differ across drug agents.

With the discovery that cell death after noise or ototoxic drug treatment is mediated at least in part by free radical production and that endogenous antioxidant status can be used to influence cell survival and hearing outcomes during and after noise or ototoxic drug treatment, it has become possible to define antioxidant interventions that attenuate NIHL ototoxicity in animals, and many of these strategies have proven effective. Although these pharmacological strategies require multiple years of safety testing and dose development to ensure that hearing protection is maximized while antibiotic/chemotherapeutic or other lifesaving benefits remain uncompromised, there are new drugs under development. New drugs under development for protection of the inner ear are discussed next.

PHARMACOLOGICAL OTOPROTECTIVE AGENTS

Several different pharmacological otoprotective agents have shown promise for reducing or even eliminating hearing loss secondary to excessive noise exposure, platinum-based chemotherapeutic drug exposure, or aminoglycoside-induced drug exposure. Some agents work for all three insults. Others work specifically for one insult, but not another. Developing otoprotective agents for drug-induced hearing loss can be particularly challenging because the protective agent must protect normal cells but not tumor cells as in the case of chemotherapeutics or bacteria as in the case of aminoglycoside antibiotics. Other types of toxic

insults can cause hearing loss but because excessive noise exposure, platinum-based chemotherapeutics, and aminoglycoside antibiotics are three of the most common toxic insults to the ear seen clinically, most research on otoprotective agents has focused on those three areas. NIHL otoprotection may move forward most rapidly because no potential exists for reducing a drug’s therapeutic action as with drug-induced ototoxins such as platinum-based chemotherapeutics and aminoglycosides. Ad- ditionally, subjects needing otoprotectants for NIHL are generally healthy, but patients re- ceiving chemotherapy or aminoglycosides are more fragile because of their illnesses.

Otoprotective agents may be prophylactic agents, rescue agents, or both. Prophylactic agents are started prior to or in conjunction with the noise or drug exposure. Rescue agents are started within hours or days after the noise or drug exposure to prevent permanent damage, whether or not temporary damage has already occurred. Protective and rescue agents do not provide hair cell regeneration for long- standing hearing loss. Hair cell regeneration is an exciting but separate research area. Not all protective and/or rescue agents that are effective in animal studies are appropriate for clinical development. Some agents may have side effects that render them unsafe for human use.

Others may require difficult or expensive formulation or may have storage or delivery requirements that reduce the likelihood of clinical acceptance. For example, drugs that can only be administered directly to the round window would be more expensive, less convenient, and more risky to use. For NIHL, daily round window treatment, for individuals with daily noise exposure, is unlikely to gain acceptance. Still other potential new otoprotective agents may interact with other drugs the patient may be taking or reduce the efficacy of the target drug such as cisplatin.

Patent issues also can play a role in drug development. If an otoprotective agent cannot be patented, it is unlikely that anyone will invest the hundreds of millions or more to take that drug through the U.S. Food and Drug Administration (FDA) approval process because they will not be able to protect their market. Nutraceutical agents can have lower

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costs relative to the traditional new drug development process; however, clinical data are often less available as the data were not required to have medical claims approved as part of the new drug approval process. Nutraceut- icals are regulated by the FDA through the Dietary Supplement Health and Education Act of 1994, and these products are required to be labeled with the disclaimer that the FDA has not evaluated any health-related claims for the product. The disclaimer also must state that the dietary supplement product is not intended to

‘‘diagnose, treat, cure, or prevent any disease,’’

because only a drug can legally make such a claim. Some of the agents being developed as otoprotectants are being produced as traditional pharmaceutical products, and others as nutraceuticals. However, the line differentiat- ing between nutraceuticals and drugs can sometimes be difficult to ascertain.

Antioxidants may be the most promising class of otoprotective agents for several reasons.

First, many antioxidants are well characterized in humans because they have been studied in the nutritional literature and thus are not foreign to the human system. Second, many antioxidants can be delivered orally, which is more convenient, less costly, and less risky than injection or round window administration.

And last, many antioxidants have few or min- imal side effects.

Of course, all drugs have multiple actions, and it is unlikely that any one agent will be optimal for all patients in all settings. Although antioxidants are discussed as a single class of agents, antioxidants can vary in their specific mechanisms of action, they can act on different pathways or different cellular areas, their distribution through the body can vary, their safety profiles can vary, and their therapeutic indices can vary.

Therefore, it is important that we develop and obtain FDA approval for a variety of otoprotective agents to prevent both drug-induced hearing loss and NIHL, even within the class of antioxidants. Just as we need more than one analgesic, diuretic, antibiotic, and so on, we need patients and physicians to have a wide array of approved otoprotective and rescue agents available to them. Currently, no agent has been FDA approved to prevent or treat

NIHL or tinnitus. Some agents that are being investigated as otoprotective agents have been approved by the FDA for other purposes;

however, that does not mean that the agent is safe or effective as an otoprotective agent.

Moreover, the dosing requirements may be markedly different for the inner ear and other biological systems.

Otoprotection for NIHL

Several otoprotective and rescue agents are being tested, and it is our hope that eventually more than one agent will be FDA approved for use in humans. This discussion is limited to oral agents that are in or approaching clinical trials. Other agents in early development or that cannot be safely administered systemically are not included. Agents that can only be administered by transtympanic injection or direct round window application may have limited applicability for prevention of NIHL, although agents that can be administered either orally or via the round window are reviewed.

All of these agents are antioxidants but they vary in their antioxidant mechanisms.

D-METHIONINE

D-methionine (D-met) can be administered orally as a suspension and by injection or by direct application to the round window.^45–47 An orange-flavored oral formulation of D-met with flavor-matched placebo, prepared according to FDA Good Manufacturing Practices (GMP) for clinical trials, has been used in previous small-scale clinical trials to prevent cisplatin-induced hearing loss and radiation- induced oral mucositis. Clinical trials to prevent NIHL have been designed and submitted for funding. For adults, volume per dose is approximately one teaspoonful depending on subject weight. This formulation is stable for at least 18 months at up to 408C. For clinical trials, it is packaged in individual doses for easy and contamination-free dose distribution. An- imal studies in multiple laboratories have demonstrated D-met efficacy against NIHL,^45,47–49 cisplatin-induced hearing loss,46,47,50–52 aminoglycoside-induced hearing loss,^9,47and radiation-induced oral mucositis.⁵³Additionally, a phase I study in humans has been published.⁵⁴

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In studies to date, D-met provides virtually complete protection from permanent threshold shift (PTS) and cochlear hair cell loss in chinchillas^45,47,55and mice⁴⁸for the noise exposures tested thus far. For temporary threshold shift (TTS), results have been more variable, showing no significant protection from TTS at 24 hours in chinchillas⁴⁵ but showing virtually complete protection from TTS immediately postnoise and 1 day after noise in guinea pigs.⁴⁹ Dosing protocols have varied across studies but D-met has provided virtually complete protection from PTS whether administered every 12 hours starting 2 days prior to noise exposure and continuing 2 days after noise exposure,⁴⁵ starting 1 hour after noise exposure and continuing every 12 hours for an additional 2 days,⁴⁷ or if simply given 1 hour before and 1 hour after the noise exposure.⁴⁸ To date, all studies have shown D-met protection from NIHL with no studies showing a lack of protection or exacerbation of permanent NIHL. Additionally, almost complete protection from permanent NIHL and OHC loss has been reported in chinchillas even when D-met is first administered up to 7 hours after noise cessation.^47,55To date, D-met has been effective for noise exposures ranging from a 105-dB sound pressure level (dB SPL) octave band of noise centered at 4 kHz for 6 hours^45,47,55to a 110-dB SPL octave band of noise centered at 4 kHz for 4 hours⁴⁸and for a 105-dB SPL broad band of noise for 10 minutes.⁴⁹Noise protection studies for other noise exposures are still needed. Because studies for different otoprotective agents have often used different noise exposure paradigms, studies comparing different otoprotective agents need to be conducted using the same noise exposure conditions in the same species.

ACE Mg

A combination of b carotene, which can be metabolized to form vitamin A, plus vitamins C and E and magnesium (ACE Mg) is another otoprotective agent for NIHL that can be delivered orally and that has demonstrated consistent protection from NIHL in studies by Le Prell and colleagues.^56–58 A capsule- based formulation and matched placebo have been developed for use in a series of clinical

trials, and this formulation meets the GMP standards for dietary supplements. This agent combination is currently funded for clinical trials in Sweden, Spain, and Florida, through a grant from the National Institutes of Health awarded to Dr. Miller of the University of Michigan. To date, the animal work with ACE Mg seems promising.^30,56–58 Although Mg alone or the ACE combination alone did not confer significant hearing protection with treatments starting 1 hour prenoise, the combination of ACE Mg provided partial but significant protection from permanent NIHL in the guinea pig³⁰and the mouse.⁵⁸ACE Mg also partially reduced TTS in the guinea pig after shorter, less intense exposure.⁵⁷

In animals, ACE Mg has shown efficacy against NIHL in guinea pigs.^56,57 In mice, dose-dependent protection from PTS was obtained when the nutrients were delivered via custom dietary formulations for noise exposures of 113- to 115-dB SPL, 8- to 16-kHz octave band of noise for 2 hours.⁵⁸ TTS in guinea pigs secondary to a 110-dB SPL noise band centered at 4 kHz for 4 hours was reduced at 8 to 32 kHz to 55 dB from 65 dB and PTS was reduced from 20 dB to 5 to 10 dB.⁵⁷

One limitation of this combination is that there is some evidence that b carotene may increase the risk of lung cancer,^59,60and thus smokers should be advised to not take high- level b-carotene supplements. In addition, individuals with certain gastric disorders such as inflammatory bowel disease should not be advised to consume Mg supplements given that high doses of Mg have a laxative effect. As previously discussed, there is a need to develop a variety of treatment and prophylaxis regimens to provide patients and physicians with options.

It is unlikely that any one otoprotective agent will be optimal for all patient populations and only clinical trials for all otoprotective agents will clearly identify benefits and risks and define the appropriate patient populations for use.

EBSELEN

Reportedly, ebselen is approaching phase II clinical trials. Ebselen can be administered orally, and the clinical trial preparation is a dry blend capsule, formulated according to

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FDA GMP standards. Ebselen, a selenium- containing compound, has consistently shown either partial or complete protection from PTS in rats and guinea pigs,^61–64 with no studies reporting a lack of protection or exacerbation of NIHL. Only one study has been published addressing TTS, and that study showed significant reduction of TTS in the guinea pig.⁶³ Significant protection from PTS has been noted in the guinea pig after a 5-hour, 125- dB SPL, 4-kHz octave band noise exposure⁶² and TTS reduction after a 3-hour, 115-dB SPL, 4-kHz octave band exposure.⁶³ In rats, partial but significant protection from PTS has been reported for a 4-hour, 4- to 16-kHz noise band exposure at 110-, 113-, or 115-dB SPL.^62,64

N-ACETYLCYSTEINE

N-acetylcysteine (NAC) has been the most widely studied agent for protection from NIHL but results have been variable. Most studies show at least partial protection from permanent NIHL in animals when administered either before or within 24 hours of noise exposure,^65–69 but other studies have shown that NAC provides no protection or even exacerbation of NIHL.^70,71 NAC appears to work best in combination with other agents.

Kopke et al^72,73 attributed the protection against NIHL to NAC but only found significant protection from NIHL obtained with combinations of agents with NAC. For example, they only found significant protection from NIHL when NAC was combined with high- dose salicylate. Salicylate itself has otoprotective properties,⁷⁴ and thus may have contrib- uted to the results observed. High-dose salicylate (aspirin) is probably not advisable for a variety of patient populations because of its side effects. In children, aspirin cannot be used because of the risk of Reye’s syndrome.

Because of its gastrointestinal toxicities, it is inadvisable for individuals with any gastric disorder such as ulcers. For military personnel, use would be inadvisable because it can increase the risk of bleeding. Some studies, however, do show partial protection from permanent NIHL using NAC in isolation.^75–77

Three human clinical trials with NAC have been conducted, but none showed signifi-

cant threshold protection from TTS or PTS.^78,79 Toppila et al⁷⁸ used 400 mg NAC per day and Kramer et al⁷⁹used 900 mg NAC per day in a double-blind placebo clinical trial of 31 normal-hearing subjects before and after 2 hours of night club noise. Neither study showed threshold protection for the TTS models used. Another study by Kopke using 900 mg NAC administered three times per day in 566 U.S. Marine recruits at Camp Pendleton exposed to 300 rounds of M-16 weapon fire reported no significant protection from PTS.

The results have not been published.

For the latter clinical trial, the NAC was oral in a ‘‘fizzy tab’’ dissolved in a glass of water.

Whether or not increased dosing or a different noise exposure paradigm will show protection is unknown currently.

Despite the lack of positive clinical data from human subjects, NAC is being sold over the Internet, perhaps highlighting the difference in required proof of efficacy between agents sold as supplements as opposed to FDA-approved drugs.

COMPARATIVE STUDIES OF OTOPROTECTIVE AGENTS FOR NIHL

To date, studies simultaneously comparing all the various otoprotective agents approaching clinical trials have not yet been performed.

Comparative studies will be useful in helping to identify the most promising agents to test in clinical trials. However, it is unlikely that only one otoprotective agent will be needed to address the needs of all individuals exposed to high noise levels.

D-met and NAC are among the most widely studied protective agents for NIHL. In comparative studies thus far, D-met seems to provide better otoprotection than NAC. Clif- ford et al⁸⁰reported that preadministration of a combination of low-dose D-met and NAC markedly reduced permanent noise-induced threshold shift in chinchillas, but that the protection afforded by the D-met component alone was similar to the combined administration of D-met and NAC. When the NAC component was delivered alone, no protection was provided.⁸⁰ Kopke et al⁴⁵ reported that both D-met and NAC prenoise administration⁷² could protect against NIHL in the

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chinchilla. However, D-met provided superior cochlear OHC protection. Over 90% of OHCs were preserved with D-met protection as compared with only 50 to 60% with NAC/salicylate preadministration. The reason they administered NAC with salicylate is reportedly to improve the stability of NAC but as reviewed above, the salicylate may have served as the otoprotective agent.

Protection from Aminoglycoside- Induced Ototoxicity

SALICYLATE

Thus far, the only agent that has undergone clinical trials for otoprotection against aminoglycoside-induced ototoxicity is high-dose aspirin. Salicylate has multiple mechanisms, which include actions as an antioxidant and as an iron chelator in tissues undergoing oxidative stress.⁸¹ In animal studies, salicylate-reduced gentamicin-induced OHC loss and auditory brain stem response threshold shift without interfering with antimicrobial efficacy.⁸¹ Two clinical trials have been reported, one in China and one in Saudi Arabia.^82,83In China, 14/106 patients in the placebo group experienced threshold shifts of 15 dB or greater at 6 and 8 kHz, but only 3/89 in the aspirin-treated group (3 g per day) did. Of the 14 subjects in the control group who obtained hearing loss, 11/14 experienced only 15- to 25-dB threshold shifts.⁸²It is unknown whether salicylate would protect from greater degrees of aminoglycoside-induced hearing loss. Similarly, Behnoud et al⁸³ showed mild protection from aminoglycoside-induced hearing loss using 1.5 g of aspirin per day in 30/60 patients. Salicylate has some clinical limitations in that it has potential gastrointestinal, hematologic, and ototoxic side effects particularly at the high dosage levels apparently required for otoprotection. Also as stated previously, it cannot be used in children because of the possibility of Reye’s syndrome.

Another limiting factor for the development of salicylate as an otoprotective agent is the ab- sence of patents, licensure, and company funding to carry it forward through FDA-approved clinical trials. However, if a salicylate can be clinically developed and approved for this ap-

plication, it could be a useful option for some patient populations.

D-MET PROTECTION FROM AMINOGLYCOSIDE- INDUCED OTOTOXICITY

To our knowledge, the only otoprotective agent other than salicylate approaching clinical trials for protection against aminoglycoside-induced ototoxicity within the next 5 years is D-met.

Sha and Schacht⁹administered twice-daily in- jections of 200 mg/kg D-met 7 hours apart in guinea pigs administered 120 mg/kg/d gentamicin. Gentamicin-induced threshold shifts were reduced from 60- to 20-dB SPL at 18 kHz and from 50- to 30-dB SPL at 9 kHz. In that same study, histidine, another antioxidant, provided substantially less otoprotection.

Campbell et al⁴⁷ administered 300 mg/kg D- met once per day prior to 200 mg/kg amikacin for 28 days in guinea pigs. They obtained less, but still statistically significant, otoprotection as compared with the twice-daily dosing regimen of Sha and Schacht.⁹Further studies of dosing regimens are being conducted to optimize otoprotection and to determine if safe and effective otoprotection can be obtained against tobramycin-, kanamycin- and neomycin-induced ototoxicity without interference with antimicrobial activity. In studies to date, D-met does not appear to interfere with the antimicrobial efficacy of aminoglycoside antibiotics,⁸⁴nor does it reduce aminoglycoside serum levels.⁹

OTHER OTOPROTECTIVE AGENTS FOR AMINOGLYCOSIDE OTOTOXICITY

Other otoprotective agents have been success- fully tested for preventing aminoglycoside-induced ototoxicity in animal models, but none appear to be approaching clinical trials. Sys- temic lipoic acid, another antioxidant, reduced amikacin-induced ototoxicity, in guinea pigs⁸⁵; but Sinswat et al⁸⁶ found that lipoic acid administration enhanced weight loss and animal mortality. Round window administration of lipoic acid slowed neomycin-induced ototoxicity, but significant cochlear damage still occurred.⁸⁷ Round window administration of an otoprotective agent for daily aminoglycoside administration could be clinically problematic.

Whether lipoic acid reduces the therapeutic efficacy of aminoglycosides has not been tested.

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Iron chelators, including deferoxamine and 2,3-dihydroxybenzoate, have been reported to reduce aminoglycoside-induced ototoxicity and free radical formation,^88–90 but these iron chelators can also be ototoxic.^91,92 Iron chelators may not be advisable for chronic administration in many patients (e.g., women of childbearing age), and it is unknown if iron chelators interfere with the antimicrobial activity of aminoglycoside antibiotics.

CEP-1347 and D-JNKI-1, which block JNK pathway activation and thus may block caspase activation, protected against neomycin- induced ototoxicity in in vitro and in vivo experiments.^93,94 Caspase inhibitors such as z-VAD-fmk⁹⁵and neurotrophins have shown variable protection from aminoglycoside-induced ototoxicity in animals.^96–98 However, it has not been determined whether JNK pathway activation blockers or caspase inhibitors interfere with the antimicrobial action of aminoglycoside antibiotics or if they are safe for long- term systemic administration in humans.

Although the studies provide valuable infor- mation in establishing the mechanisms of aminoglycoside-induced ototoxicity, they may not directly lead to clinical therapies.

Gene therapies in transgenic mice¹¹ or delivered directly into the cochlea¹⁴ have shown some promise in reducing aminoglycoside ototoxicity by increasing expression of catalase or SOD. Similarly, transgenic glial cell-derived neurotrophic factor (GDNF) expression can reduce aminoglycoside-induced⁹⁹ or combined aminoglycoside- and loop diuretic-induced¹⁰⁰ototoxicity. These studies provide important support for the oxidative stress theory for aminoglycoside-induced ototoxicity.

They also may eventually lead to new therapies.

However, they will probably not undergo clinical trials in the near future.

Otoprotective Agents for Platinum- Based Chemotherapy (e.g., Cisplatin and Carboplatin

SODIUM THIOSULFATE

Protective agents for cisplatin ototoxicity have been studied for decades but at this point, none have FDA approval. However, several

agents are in or approaching clinical trials for otoprotection from cisplatin and/or carboplatin therapy. This discussion is limited to those agents. One of the earliest otoprotective agents studied for protection from cisplatin- induced ototoxicity is sodium thiosulfate (STS).¹⁰¹ Although systemic administration of STS can reduce cisplatin-induced hearing loss, it also can reduce the antitumor efficacy of the platinum-based chemotherapy.^102,103 Therefore, alternative approaches to STS delivery have been attempted in clinical trials to retain the protective action while retaining cisplatin’s tumoricidal activity. These approaches include delaying STS delivery several hours after delivery of the platinum-based chemotherapeutic agents,^104–106 cochlear application of STS,^107–109 and administering cisplatin intra-arterially and STS intrave- nously to avoid interference.¹¹⁰Clinical trials of simultaneous intravenous STS with cisplatin yielded conflicting findings on otoprotection.^111–114In a phase III clinical trial, Zuur et al¹¹⁰ reported that intravenous STS with intra-arterial cisplatin reduced cisplatin-induced hearing loss by 10%. Thus far, blood-brain barrier disruption for carboplatin delivery with delayed STS administration looks promising in humans^115,116 but will need to be further tested in clinical trials.

D-METHIONINE

D-met was first reported to effectively prevent cisplatin-induced ototoxicity in animals in 1996.⁵⁰Since that time, that finding has been confirmed and expanded in several studies for both carboplatin and cisplatin^47,51,52 with no studies showing a lack of protection. D-met can be effectively delivered by systemic injec- tion47,50,51,117–120 or delivery to the round window.^46,108 One advantage of D-met is that it also can be effectively delivered orally with equal efficacy.⁴⁷Animal studies have not shown significant antitumor interference.¹²¹A small-scale phase II clinical trial demonstrated significant otoprotection when oral D-met was delivered prior to cisplatin in cancer patients without antitumor interference.¹²² These results will need to be confirmed in larger-scale clinical trials but thus far the agent seems promising.

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EBSELEN

Another agent approaching but not yet in clinical trials for cisplatin-induced ototoxicity is ebselen, a selenium-containing compound.

Ebselen, in conjunction with or without allo- purinol, can reduce cisplatin-induced cochlear hair cell loss in vitro¹²³and in vivo^124,125with- out antitumor interference. Like D-met, ebselen also has the advantage that it can be delivered orally.^21,125

AMIFOSTINE

Amifostine is mentioned here because it is a nephroprotective agent sometimes used in clinical practice. However, the preponderance of evidence suggests that it is not otoprotective and it is not recommended by the American Society of Clinical Oncology 2008 Clinical Practice Guideline Update:

Use of Chemotherapy and Radiation Ther- apy Protectants¹²⁶for either otoprotection or neuroprotection.

SUMMARY

In summary, there are many promising otoprotective agents in or approaching clinical trials to prevent NIHL and aminoglycoside-, cisplatin-, and carboplatin-induced hearing loss. Every audiologist should have some familiarity with the mechanisms of ototoxicity and otoprotection and the agents in development. Moreover, every audiologist should be familiar with the http://clinicaltrials.gov Web site, which lists current registered clinical trials. Hopefully in the not too distant future, noise- and drug- induced hearing loss will be markedly reduced.

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