Учебники / Auditory Trauma, Protection, and Repair Fay 2008

testicular tumors and a variety of other solid tumors, particularly ovarian, bladder, and head and neck malignancies as well as malignant neoplasms in children (Rozencweig et al. 1977). Cisplatin also has some benefits against brain tumors (Feun et al. 1984).

The major toxicity in a phase I clinical trial was to the kidney, occurring in 25% to 61% of patients depending on the dose (Talley et al. 1972; DeConti et al. 1973). Other toxicities discovered during those trials included nausea and vomiting followed by prolonged periods of anorexia, leukopenia, and thrombocytopenia which manifested itself as late as 4 weeks after treatment, hyperuricemia, and hearing impairment primarily in the high frequencies (Kovach et al. 1973; Lippman et al. 1973).

Today, combination chemotherapy is the cornerstone of cancer treatment regimens. The goal is to achieve a synergistic effect of cisplatin with other antineoplastic agents in order to increase efficiency and lower toxicity. These combinations may also reduce the chance of resistance of tumors by inhibiting repair of platinum–DNA adducts or actually increasing the formation of platinum–DNA adducts. Such drug combinations are useful to treat cancer of the lung, ovary, testis, cervix, head and neck, gastrointestinal tract (stomach, esophagus, colon, and pancreas), and bladder, and metastatic cancers of the prostate or breast, mesothelioma, metastatic melanoma, and malignant gliomas (Boulikas and Vougiouka 2004).

2.2 Aminoglycosides

In contrast to the century-long delay between the synthesis of cisplatin and its clinical application, the therapeutic value of aminoglycoside antibiotics as a potent antibiotic against gram-negative bacteria was recognized within a year following the discovery of streptomycin (Schatz et al. 1944; Hinshaw and Feldman 1945). The first trials also discovered the adverse effects ototoxicity and nephrotoxicity. The characterization of streptomycin was followed by the isolation and semisynthetic production of a wide variety of aminoglycosides including neomycin, tobramycin, gentamicin, kanamycin, dihydrostreptomycin, and netilmicin but the ototoxic and the nephrotoxic potentials were never eliminated. Nevertheless, their broad antibacterial spectrum and efficacy against and hitherto untreatable diseases, such as tuberculosis, made the aminoglycoside antibiotics some of the most successful drugs in the second half of the 20th century.

Aminoglycoside antibiotics are low-molecular-weight compounds with similar structures consisting of several, usually three, rings. These rings are cyclitols (a saturated six-carbon ring) and fiveor six-membered sugars that are linked via glycosidic bonds. The hallmark of aminoglycosides is the presence of amino groups attached to the various rings of the structure (Fig. 8.1b). These amino groups and the additional hydroxyl groups convey the major chemical properties, namely a highly polar basic character and high water solubility. Aminoglycoside antibiotics are produced by different strains of soil actinomycetes, “-mycins” by

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Streptomyces, and “-micins” by Micromonospora. Although all aminoglycosides carry the suffix “-mycin” or “-micin,” this suffix is not a chemical or therapeutic classification and does not exclusively denote an aminoglycoside. The anticancer drug bleomycin, for example, or the macrolide antibiotic erythromycin are unrelated to aminoglycosides in structure, therapeutic indications, and potential side effects.

Although new generations of antibiotics have emerged in the last decades, the aminoglycosides maintain a leading role in the treatment of enterococcal, mycobacterial, and severe gram-negative bacterial infections. Gentamicin is frequently used in newborn infants against potentially life-threatening sepsis and in adults, for urinary tract infections and the prophylactic treatment against pulmonary infections in cystic fibrosis patients are major applications (Pong and Bradley 2005). Further, aminoglycoside antibiotics are recommended by the World Health Organization as part of the combination therapy against multi- drug-resistant tuberculosis (World Health Organization 2005). A critical aspect that drives the worldwide popularity of aminoglycoside antibiotics is their use in developing countries. Aminoglycoside antibiotics are nonallergenic and can be readily used in emergency situations, and, importantly, they can be produced very inexpensively and frequently are the only affordable option in less affluent countries.

3. Mechanisms of Therapeutic Action

3.1 Cisplatin

The dichloro compound cisplatin undergoes hydration inside the cell, which is facilitated by the low intracellular concentration of chloride ions. This hydrated form is more reactive to targets within the cell, the primary target being DNA. Its configuration is reminiscent of the mustard-type bifunctional alkylating agents which can become strong electrophiles and can form covalent linkages by alkylating DNA. The 7-nitrogen atom of guanine is especially susceptible to the formation of a covalent bond with these agents so that these drugs can form interand intrastrand cross-links with DNA (Rozencweig et al. 1977). Platinated DNA adducts inhibit replication, inhibit transcription, arrest the cell cycle, prevent DNA repair, and induce cell death by apoptosis. The distortion of the DNA helix by platinum binding can also cause binding of several classes of proteins to the modified DNA. These include the high-mobility group (HMG) box proteins which may play a role in the antineoplastic activity of cisplatin (Wang and Lippard 2005). Platinated DNA intrastrand crosslinks can be removed by cellular repair mechanisms such as nucleotide excision repair, while interstrand crosslinks are likely eliminated by recombination repair mechanisms (McHugh et al. 2001). The extent of tolerance to DNA lesions caused by cisplatin may decide the fate of a particular cell, survival, or apoptosis (Liedert et al. 2006).

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is due to an accumulation of the drugs in the proximal tubules. As with cisplatin, necrosis of cells occurs in the proximal and distal tubules and the loop of Henle, potentially resulting in renal failure. Nephroand ototoxicity are not necessarily expressed together in experimental animals or in patients.

Ototoxicity generally is bilateral and manifests itself only after days or weeks of systemic treatment, and the severity of hearing loss may still increase after drug administration has ended. An exception to this rule is found in patients carrying mitochondrial mutations that predispose to rapid hearing loss, oftentimes from a single dose of the drugs. Ototoxicity involves either the auditory (cochleotoxicity) or vestibular system (vestibulotoxicity), or both, and the toxic potential and organ preference varies among the different aminoglycosides. Neomycin is regarded as highly toxic; gentamicin, kanamycin, and tobramycin are of intermediate toxicity; and amikacin and netilmicin are somewhat less toxic. As to organ preferences, amikacin or neomycin may target primarily the cochlea while gentamicin is considered more vestibulotoxic in the human and therefore frequently used for vestibular ablation in Ménière’s disease (Blakley 1997). Small changes in structure may greatly influence the pattern of toxicity: streptomycin mostly targets the vestibular system in the human inner ear while dihydrostreptomycin targets the cochlea. Such preferences are not predictable by any structure–activity relationship and are also not related to any site-specific uptake mechanism or drug levels in the tissues (see section on “Pharmacokinetics”).

5. Incidence of Ototoxicity

5.1 Cisplatin

Because the loss of hearing begins at the high-frequency range, it may not be detected with conventional audiometry or initially perceived by the patient. Seventy-one percent of patients with cisplatin-induced hearing loss had hearing deficits at frequencies of 8000 Hz or higher (Fausti et al. 1993). While a highfrequency loss may not result in any communication problems for the patient, such an assessment illustrates the ototoxic potential. In fact, some studies peg the incidence close to 90% or 100% (Benedetti-Panici et al. 1993). A mild and early hearing loss may be partially reversible, but when the hearing loss is profound it tends to be permanent (Vermorken et al. 1983).

5.2 Aminoglycosides

The reported incidence of ototoxicity varies considerably among different clinical studies, due to varying treatment regimens and, mostly, varying assessment and definitions of “hearing loss.” Nevertheless, hearing loss induced by the most commonly used aminoglycosides may occur in about 20% of patients; balance may be affected in about 15% (Fee 1980; Moore et al. 1984; Lerner et al. 1986). In cystic fibrosis patients who receive repeated courses of aminoglycoside

therapy as prophylaxis against pneumonia caused primarily by Pseudomonas aeroginosa, the reported range of cochleotoxicity is quite variable but may approach 17% (Mulheran et al. 2001). An early prospective study of kanamycininduced hearing loss in tuberculosis treatment found hearing loss in 80% of the patients (Brouet et al. 1959) but a lower incidence appears to be associated with current therapeutic regimens (de Jager and van Altena 2002).

Just as with cisplatin, most data must be considered conservative estimates because hearing loss by aminoglycosides also begins at the highest frequencies which are not routinely monitored. Using high-frequency testing procedures, a prospective study in 53 patients determined hearing loss in 47% of the ears studied (Fausti et al. 1992).

The problem of ototoxicity is even more prevalent in developing countries where aminoglycosides are frequently available over the counter and where safety precautions such as monitoring of serum levels or auditory function are rarely available if at all. In the absence of such auditory monitoring, no firm data on the incidence of hearing loss exist but the frequency of ototoxicity must be considerably higher than in industrialized countries, as can be extrapolated from reported cases of complete deafness by aminoglycosides. In an area of southern China, two thirds of all deaf-mutism was due to the administration of aminoglycosides to children (Lu 1987). Even more recently, aminoglycosideinduced deafness was confirmed in 15 of 77 deaf children (Zhang et al. 1997).

6. Risk Factors

6.1 Cisplatin

There is considerable individual variation in susceptibility to cisplatin and aminoglycoside ototoxicity. For cisplatin, the severity of hearing loss appears to be related to the magnitude of the cumulative dose (Bokemeyer et al. 1998) and the rate of intravenous injections (Vermorken et al. 1983). Age appears to be an important risk factor for cisplatin-induced ototoxicity, with both children younger than 5 years of age and elderly patients more susceptible to cisplatin-induced hearing loss than are younger adults (Laurell and Jungnelius 1990; Li et al. 2004). Hearing loss may increase even years after cisplatin therapy has been completed (Bertolini et al. 2004). Nutritional and metabolic status can influence the probability of hearing loss from cisplatin. Patients with hypoalbuminemia and anemia develop more severe hearing losses than do those with normal albumin and hemoglobin levels (Blakley et al. 1994). Additional factors such as renal insufficiency and preexisting hearing loss may increase the probability of cisplatin ototoxicity, as can noise, cranial irradiation, and other concomitant drugs, such as high-dose vincristine (Bokemeyer et al. 1998).

6.1.1 Genetic Predisposition

A genetic predisposition to cisplatin-induced hearing loss may be related to mitochondrial mutations. Five of 20 cancer survivors with cisplatin ototoxicity

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were found to cluster in a rare European J mitochondrial haplogroup that has been associated with Leber’s Hereditary Optic Atrophy (Peters et al. 2003). In contrast, a variety of other mutations including those that predispose to aminoglycoside ototoxicity appear not to contribute to the susceptibility to cisplatin-induced hearing loss (Knoll et al. 2006).

6.2 Aminoglycosides

It is intriguing that studies on diverse patient groups (and hence different drug regimens) do not find a direct correlation between ototoxicity and the level of dosage and the duration of treatment. This includes hearing loss in cystic fibrosis (Mulheran et al. 2001) and tuberculosis patients (de Jager and van Altena 2002) as well as vestibular damage (Black et al. 2004). Thus, individual susceptibility may play a decisive role in addition to very few confirmed risk factors such as concurrent nephrotoxicity (Halmagyi et al. 1994). Another confounding factor, at least in animal studies, is the nutritional and physiological state of the subject. Animals stressed by infections or fed a less than optimal diet display an enhanced susceptibility to aminoglycosides (Garetz et al. 1994; Lautermann et al. 1995). Notably, drug–drug interactions pose a major threat, for example, the concomitant administration of the loop diuretic ethacrynic acid together with an aminoglycoside that can lead to a precipitous severe hearing loss (Mathog and Klein 1969). A detrimental interaction of aminoglycosides with noise exposure has also been discussed, and nontoxic doses of amikacin may indeed impair recovery from noise-induced hearing loss (Tan et al. 2001).

During specific stages in development, the ear appears to be at particular risk from aminoglycoside antibiotics. This “critical period” coincides with the development of the inner ear both in altricial and precocial mammals. For example, the ototoxic effect of kanamycin was weak in the rat before the onset of cochlear potentials (8th postnatal day) but strong thereafter (Marot et al. 1980). Because the drugs also can penetrate the placental barriers, hair cell loss can be induced in utero during and after the functional differentiation of the cochlea in the guinea pig (Raphael et al. 1983).

6.2.1 Genetic Predisposition

Mitochondrial mutations are a well-defined risk factor in aminoglycosideinduced hearing loss, and a single injection of an aminoglycoside may already lead to profound deafness in carriers. The existence of families with multiple individuals with maternally inherited susceptibility to aminoglycoside-induced deafness was noticed several decades ago in China (Hu et al. 1991) and traced to an A1555G mutation in the 12S ribosomal RNA (Prezant et al. 1993). Subsequently, the same mutation was found in families with aminoglycoside ototoxicity from all ethnic backgrounds and geographic origins (reviewed by Fischel-Ghodsian 2005). This mutation may account for deafness in about 20% of patients with aminoglycoside ototoxicity while several other mitochondrial mutations make additional but only minor contributions.

The relationship between mitochondrial mutations and aminoglycoside ototoxicity remains enigmatic, but a consideration of the pattern of pathology is informative. In the absence of the 1555 mutation, an aminoglycoside such as gentamicin can display both cochlear and vestibular toxicity with a preponderance of the latter in the human. In contrast, patients with the 1555 mutation may (in the absence of aminoglycoside treatment) develop nonsyndromic hearing loss but little or no vestibular problems. Profound hearing loss without a concomitant vestibular component is the result of aminoglycoside treatment in patients with mitochondrial mutations. It thus seems that aminoglycoside ototoxicity is not enhanced by the mutation but that aminoglycosides are a stressor that triggers the phenotypic expression of the 1555 mutation. Requiring a trigger for its pathology would not be surprising, as the 1555 mutation has variable expression and onset in the carrier population, sometimes never manifesting itself.

7. Animal Models

7.1 Cisplatin

Most of the in vivo studies of cisplatin ototoxicity have been performed in rodent models including the rat, guinea pig, gerbil, hamster, mouse, and chinchilla. Systemically applied cisplatin causes high-frequency hearing loss and loss of outer hair cells (OHCs) primarily in the basal turn of the cochlea (Fig. 8.2). Deterioration in the distortion product otoacoustic emissions and a reduction in the endocochlear potential suggest dysfunction of both the OHCs and stria vascularis (Alam et al. 2000). Unfortunately, a major difficulty with most models

Figure 8.2. Cochlear damage by cisplatin. Left panel: Scanning electron micrograph of the hook region of the cochlea of a rat harvested 72 h after treatment with intraperitoneal saline. The single row of inner hair cells and the three rows of outer hair cells are well preserved. Right panel: Scanning electron micrograph of the hook region of the cochlea of a rat processed 72 h after treatment with 16 mg/kg of cisplatin by intraperitoneal infusion. Note the extensive loss of outer hair cells with preservation of the inner hair cells. Scale bar: 10 μm. Initial damage by aminoglycoside antibiotics shows a similar pattern.

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is the high mortality rate of 33% to 50% (Ravi et al. 1995; Campbell et al. 1996; Reser et al. 1999; Li et al. 2002; Iraz et al. 2005).

Since clinical regimens for cisplatin consist of several courses of injections with rest intervals in between treatment days, a two-cycle model for cisplatininduced ototoxicity in rats was developed. Each cycle consists of 4 days of cisplatin intraperitoneal injections (1 mg/kg twice a day) on days 1–4 (first cycle) and days 15–18 (second cycle). In addition, animals were injected with 10 ml of saline subcutaneously daily for hydration. These animals develop 40– 50 dB elevation of auditory brainstem response (ABR) threshold at 16 and 20 kHz after the second cycle with no mortality. This model eliminates potentially confounding factors that may select the survival of a particular cohort of animals and may represent the best current model for cisplatin ototoxicity (Minami et al. 2004).

Several in vitro models to demonstrate cisplatin ototoxicity have also been used as tools to investigate mechanisms of toxicity. These include organotypic cultures of the organ of Corti of the neonatal rat (Kopke et al. 1997; Zhang et al. 2003), acutely isolated hair cells from the guinea pig cochlea (Dehne et al. 2001), hair cell lines derived from the immortomouse cochlea (Devarajan et al. 2002), mouse spiral ganglion cell cultures, and cultured type I spiral ligament fibrocytes (Liang et al. 2005). Such models can serve as useful screening techniques but because of genetic, pharmacokinetic, and metabolic differences, they may not necessarily be relevant to the processes that occur with cisplatin in the whole animal or in the human. Results obtained using these tissues would need to be confirmed in the appropriate animal model. A similar concern arises for the study of mechanisms of aminoglycoside ototoxicity in such in vitro systems.

7.2 Aminoglycosides

Inner ear pathology induced by aminoglycosides in experimental animals and humans is remarkably similar. Hair cells in the inner ears of all vertebrate classes as well as those of the lateral line organs of fish and larval amphibia are susceptible to aminoglycosides. Traditionally, the guinea pig had been the major animal model for aminoglycoside-induced hearing loss, and the cochlear pathology and pathophysiology have been well characterized. Thanks to recent developments in molecular biology, the mouse has become the favorite biomedical research subject. However, early studies in adult mice produced little if any auditory or vestibular deficits from doses of aminoglycosides that would induce severe trauma in other animal models (Henry et al. 1981).

An adult mouse model that avoids confounding aminoglycoside ototoxicity with developmental issues has only recently been developed (Wu et al. 2001). Interestingly, mice would not survive gentamicin treatment but tolerated kanamycin very well. Much higher doses of kanamycin were necessary in these mice than in previous model animals but the familiar pattern of base to apex loss of hair cells and high-frequency threshold shifts were identical to other animal models and the human as well. Although the required dosing with kanamycin

was high in comparison, serum levels of the drug were comparable to levels achieved with other antibiotics in models such as the guinea pig, suggesting a higher rate of elimination rather than an intrinsic resistance to aminoglycosides was the reason behind earlier failures in establishing an adult mouse model of aminoglycoside ototoxicity.

8. Pathophysiology

8.1 Cisplatin

Although cisplatin ototoxicity is mostly documented as increased thresholds in ABR recordings, with greatest effects in the higher frequencies (Rybak et al. 2000), its actions may also include a reduction of the endocochlear potential (Ravi et al. 1995; Tsukasaki et al. 2000; Klis et al. 2000) and elevation of the thresholds for both the compound action potential and cochlear microphonic (van Ruijven et al. 2005a). Likewise, distortion product otoacoustic emissions are diminished, indicative of damage to OHCs (Alam et al. 2000). Vestibular disturbances are rare.

8.2 Aminoglycosides

Akin to the damage found with cisplatin, hearing loss is initially confined to the high frequencies and then progresses to lower frequencies (Aran and Darrouzet 1975) including frequencies in the human speech range. Historically, vestibular disturbances were noted and investigated before the cochlear effects (Caussé et al. 1949) because streptomycin, the first aminoglycoside to be discovered, is primarily vestibulotoxic. Following systemic drug administration, the compound action potential threshold is elevated, and the depression of the cochlear microphonic potential and distortion product otoacoustic emissions suggest the involvement of OHCs.

In contrast to cisplatin, the endocochlear potential (EP) may be maintained close to normal during the early course of aminoglycoside treatment (Davis 1958; Komune et al. 1987). A significant decline in EP occurs only after the stria is substantially atrophied, usually weeks after the manifestations of hair cell damage.

9. Cochlear Pathology

9.1 Cisplatin

Only a few human temporal bone studies have been published for cisplatin pathology (Wright and Schaefer 1982; Strauss et al. 1983; Schuknecht et al. 1993; Hinojosa et al. 1995; Hoistad et al. 1998). Despite differences in dose and duration of cisplatin treatment, they clearly delineate OHC loss as the hallmark

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Table 8.1. Patterns of cisplatin-induced damage to the cochlea in humans and experimental animals.

of pathology (Table 8.1). The loss begins in the basal turn and progresses to the apex of the cochlea with the innermost row of hair cells being more susceptible. Spiral ganglion cells also degenerate, again mostly in the basal turn. Inner hair cells (IHCs) are less consistently affected, and damage to stria vascularis is variable. The extent of damage appears dose-dependent and may, in extreme cases, include supporting cells of the cochlea. In contrast, the neuroepithelium of the saccule, utricle and the semicircular canals as well as the vestibular ganglion seem to be spared.

Studies in experimental animals largely reflect the pathology seen in humans with at least three major targets in the cochlea: the organ of Corti, the spiral ganglion cells and the tissues of the lateral wall, stria vascularis and spiral ligament. The loss of OHCs occurs initially in the most basal region of the cochlea (Fig. 8.2). As the ototoxic damage increases, the loss of OHCs progresses more apically (Schuknecht et al. 1993). OHC loss is most pronounced in the first row and least in the third row in the basal turn. With increasing doses or prolonged administration, the hair cell loss progresses apically and eventually involves the IHCs and supporting cells. IHCs show damage and degeneration only after all three rows of OHCs in the same region have degenerated (MarcoAlgarra et al. 1985; Barron and Daigneault 1987). When hair cells are lost, protrusion of the supporting cells into the space of Nuel and into the tunnel of Corti occurs. This can eventually result in complete replacement of the sensory cells by a layer of epithelial cells (Estrem et al. 1981; Laurell and BaggerSjöbäck 1991).

Cisplatin ototoxicity is frequently accompanied by deleterious effects on the stria vascularis of the basal turn, such as strial edema, bulging, and depletion of organelles from the cytoplasm. Such damage and eventual atrophy occurs primarily in the marginal cells, with some mild changes in the intermediate cells (Meech et al. 1998; Campbell et al. 1999). On the other hand, some studies have failed to detect any morphological changes in the stria vascularis after cisplatin treatment (Fleischman et al. 1975; Boheim and Bichler 1985; DeGroot et al. 1997). These discrepancies could be owed to dose-intensity and timing.

The type I spiral ganglion cells can undergo detachment of their myelin sheaths. The time sequence of damage to spiral ganglion cells and the OHCs