December 2007 : Pharmacological OtoProtection strategies against Cisplatin-induced Ototoxicity

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Category: Guest editorial
Written by Sathiyaseelan Theneshkumar MD, Ph.D.
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Dr. Theneshkumar

Introduction

Cisplatin induced ototoxicity is one of the main dose-limiting side-effect of anti-neoplastic treatment. Even though the histopathology of cisplatin ototoxicity has been well understood, the molecular mechanisms underlying the hearing loss are not fully understood. It is believed that reactive oxygen species (ROS) play a role in cisplatin ototoxicity. Cisplatin chemotherapy induces a decrease in plasma antioxidant levels and suppresses the formation of endogenous anti-oxidants, which may reflect a failure of the antioxidant defense mechanism against the ROS mediated oxidative damage [46]. The deleterious consequences of excessive oxidation and the patho-physiological role of ROS have been studied intensively [12, 24, 34]. The hearing impairment in cisplatin ototoxicity has been demonstrated by the effect of cisplatin on the outer hair cells (OHC) located in the organ of Corti in the inner ear [3,40,44,45]. This phenomenon is more severe at the first row of OHCs in the basal turn of the cochlea and then progresses to the other two rows of OHCs [3]. The apoptotic damage has been partly explained by a decrease of the endogenous anti oxidant- glutathione level in these cells. OHCs have lower antioxidant capacity compared with other cell types in the organ of Corti [43]. The level of glutathione in the OHCs is lower than of the level in other cell types in the organ of Corti, and there is a gradient of glutathione levels in the OHCs positioned from the base to the apex of the cochlea. Apical OHCs have much higher levels of glutathione than basal OHCs [39]. A secondary target for cisplatin is the spiral ganglion and the stria vascularis [44, 45].
           In this review we present information on a number of pharmacological otoprotectors which had shown some promising results and we will present  other molecules which show good otoprotection potential and they might be incorporated in future otoprotection strategies.

 

1. Cisplatin

Cisplatin is a platinum-based chemotherapeutic agent, known to be one of the most active platinum compounds in cancer treatment that was introduced into clinical chemotherapy in the early 1970s. It is highly effective in the treatment of head and neck cancer, soft-tissue neoplasms, lung cancer, squamous cell cancer, testicular, ovarian, cervical, and bladder cancer. Although it is not completely understood how cisplatin acts intra-cellularly, there are evidence that cisplatin is intra-cellularly converted into its monohydrated complex, which is the most important cytotoxic agent mediating the reaction with DNA [22]. Also it is believed that this monohydrated complex is responsible for cisplatin’s common side effects, which are nephrotoxicity, neurotoxicity and ototoxicity [11, 19, 36, 46]. In a study by Li et al [14] have explained another mechanism where the high-mobility group protein (HMG1) and inducible nitric oxide-synthase (iNOS) play a big role in cisplatin toxicity. They found that elevated levels of expression of HMG1 and iNOS in response to cisplatin chemotherapy occurred in the spiral ganglion cells of the basal cochlear turn in comparison to HMG1 and iNOS levels in the spiral ganglion cells of the basal cochlear turn of untreated control animals. In contrast, HMG1 expression was not detected by immuno-histochemistry in the myocardial tissue of either control or cisplatin-treated animals [ 14].
Clinically cisplatin administration can cause tinnitus and high frequency sensorineural hearing loss, which can be permanent or progressive, involving also lower frequencies. There is evidence in the literature that with an increase of the total administration dose of cisplatin, almost every patient has the risk of developing at least some degree of hearing loss [2, 6].

 

2. Reactive oxygen species

Reactive oxygen species (ROS) are defined as oxygen molecules having an unpaired electron in the outer orbit [15]. ROS are generally unstable and very reactive. They can be converted to other non-radical reactive species, such as hydrogen peroxide, hypochlorous acid (HOCl), hypobromous acid (HOBr), and peroxynitrite (ONOO_) by antioxidants. ROS are produced in animals and humans under physiologic and pathologic conditions [9].

 

3. Possible pathway of cisplatin induced ototoxicity:

 Cisplatin results in depletion of glutathione and antioxidant enzymes (Superoxide dismutase, catalase, glutathione-peroxidase and glutathione-reductase) in cochlear tissues, with a corresponding increase of the malondialdehyde levels [35]. These effects are mainly caused by the generated ROS. Cisplatin chemotherapy also induces a decrease in the plasma antioxidant levels, which may reflect a failure of the intra and extra cellular antioxidant defense mechanism against oxidative damage induced by cisplatin. The latter might be caused from the consumption of antioxidants due to the induced oxidative stress as well from the renal loss of the water-soluble, low molecular-weight antioxidants [46]. These conditions eventually lead to the oxidation of biomolecules by ROS causing cellular damage. Oxidative damage can manifest in different ways; It might affect the DNA, leading to nucleotide oxidation and dimerization, and ultimately to mutations during the replication process. It might alter protein function by changing the structure and the function of enzymes.             Intracellular toxic products such as nitrotyrosine are produced when peroxinitrites react with cellular proteins. Peroxinitrites are formed when ROS interact with nitric oxide in the cells. Lastly, Membrane lipids are preferred targets of oxygen free radicals and their oxidation may lead to membrane dysfunction and cell lysis. Damage to the mitochondrial membrane will result in the release of cytochrome C which for its part will activate caspase 9 and then 3 that will end in apoptosis of the outer hair cell [3.

The following flow-chart summarizes the various steps leading to cellular apoptosis caused by cisplatin administration:

 

4. Classification of Antioxidants

 

4.1 Compounds that directly scavenge the formed free-radicals

Antioxidants chemically bind to the ROS and this chemical binding does not depend on endogenous enzymes. But to some extent, there is also an interaction with intracellular enzymes since some of these compounds should be recycled [1]. These antioxidants are also known as direct antioxidants or chain-breaking antioxidants.

  • Monophenolic (has a single phenolic group in its chemical structure) E.g.: Vitamin E, Estrogen, and Serotonin
  • Polyphenolic (has more than one phenolic group in its structure) E.g.: Flavonoids, Hydroquinones , Stilbenes.

 

 4.2 Compounds that reduce the formation of free radicals.

       These compounds chemically bind with the molecules that participate in the ROS formation. They are also known as indirect antioxidants. Examples include calcium antagonists, glutamate receptor antagonists and iron chelators.

 

4.3 Compounds that support the endogenous antioxidant production.

      These compounds take part in the endogenous antioxidant production or take part in antioxidant recycling. Examples include: N-Acetyl-cysteine, D-Methionine and Lipoic acid.

 

5. Otoprotectors which have shown protection effects 

 

5.1 N- Acetylcysteine (NAC):

 

          N-Acetylcysteine (NAC) is an amino acid form best utilized by the body and widely used in clinical practice as a mucolytic agent. NAC is a cysteine analog with strong antioxidant activity. Induces synthesis of glutathione which contributes to long-term protection against ROS and its sulf-hydryl group is thought to play a main role in the observed otoprotection. As a glutathione precursor and an antioxidant, it has many important functions including the preservation of hearing [12, 33, and 28]. In a study by Dickey et al. rats those were treated with 400 mg/kg NAC I.V. for 15 min before cisplatin therapy (6 mg/kg) showed a very good auditory brainstem response (ABR), while the control group showed a poor ABR response reflecting a clear ototoxicity, mainly at higher frequencies [8].

 

5.2 Methionine: MET

 

Methionine is an amino acid with antioxidant properties, MET has a protective effect both on auditory hair cells and auditory neurons from various types of ototoxic hearing loss, especially from cisplatin, ionic platinum compounds, and aminoglycosides. These effects are explained by MET’s antioxidant capacity as a precursor for glutathione. In addition upregulation of HMG1 and iNOS in response to cisplatin chemotherapy could be prevented by systemic delivery of MET [4, 14]. Various animals models (Wistar and Sprague-Dawley rats) have been tested in the evaluation of the otoprotection capacity of D- MET against cisplatin induced ototoxicity [5, 16]. Data from these studies have showed that animals which received 16 mg/kg cisplatin, and various dosages (75, 150 and 300 mg/kg) of D- MET presented various degrees of otoprotection. The best results were observed in the animals receiving the higher dosage of D-MET.

 

5.3 Vitamin E

 

Vitamin E is the main lipid-soluble, chain-breaking antioxidant found in membranes and in plasma. Being a free radical scavenger it also shows potential otoprotection and it can be used in cisplatin therapy [41]. Rats receiving a single dose of vitamin E (4 g/kg) for 30 min before a cisplatin injection (16 mg/kg) showed a remarkable preservation of ABR thresholds at 8, 16, and 32 kHz, when compared to the control group. These results were confirmed by electron microscopy of the cochlea where a significant preservation of OHCs was observed in the group injected with vitamin E [23].

 

5.4 Ebselen

 

Ebselen is an anti-inflammatory, antioxidant compound, which acts as a glutathione peroxidase mimic. Ebselen shows a neuroprotection and inhibits free radical induced apoptosis. Increases in both reduced glutathione (GSH) and oxidized glutathione (GSSG) have been demonstrated with ebselen treatment [18, 21, 25, 31, 32, 38]. A study from Ryback et al. already confirmed the cytoprotective effect from ebselen after cisplatin administration in rats [35]. A recent study tested individual (16 mg/kg) and combined formulations (8 mg/kg for both) of allopurinol and ebselen in an attempt to reduce the formation of ROS during cisplatin exposure (16 mg/kg) in rats. The results showed that a combined administration of these two agents gave an otoprotective effect with an improved response at lower doses than could be achieved by either agent alone [29].  These results were supported by of the OHCs preservation and ABR thresholds.

 

5.5  Interaction-effects between Cisplatin and Otoprotectors

 

The otoprotector compounds referenced so far have shown some good otoprotection against cisplatin, but additional studies have shown that these protectors reduce the antineoplastic effect of cisplatin and they are toxic at high dosages.
        In the case of N- Acetylcysteine and D-Methionine, their complexes formation wih cisplatin may reduce the anti-tumor effect of cisplatin when administrated systemically. According to Schweitzer [37], sulfur containing compounds may prevent cisplatin from interacting with intracellular target molecules. This is because the nucleophilic oxygen or sulfur atoms interacting with the electrophilic site of the cisplatin [42]. It is known that cisplatin reacts with methionine's sulfhydryl group [26]. In the studies conducted by Campbell et al., 1999 [4] D-Methionine has shown protective effect against cisplatin-related side-effects in animal studies. But in the study by Ekborn et al [11] it was found that i.v. administration of D-methionine lowered the systemic exposure to cisplatin. Even the pre-administration of D-methionine does not reduce the ototoxic or nephrotoxic effects of cisplatin in the guinea pig after dose adjustment compared with similar cisplatin exposure in treated and control animals [10,11]. Also L-methionine in vitro [20]  and in vivo [7]  may reduce the anti-tumor effect of cisplatin when administrated systemically [7, 20]. The dose of vitamin E (4g/kg) given to the animal models is extremely high. The oral median lethal dose found in several species is 2 g/kg. At high dose vitamin E could cause increase in mortality due to subarachnoid hemorrhage in human [27]. High dose of vitamin E can depress leukocyte oxidative bactericidal activity and mitogen-induced lymphocyte transformation. This is not preferable for clinical cases where the subjects are undergoing cisplatin treatment. In theory the selenium group in the ebselen structure is even more nucleophilic due to its high degree of polarization than the sulfur, therefore ebselen is also suspected for complex formation hence ebselen may reduce the anti-tumor effect of cisplatin by forming a complex.

 

6. Future Strategies

 

        Chemo-protectants are limited in clinical use due to concerns about the potential for negative interaction with chemotherapy of the tumor, resulting in reduced chemotherapeutic efficacy. A possible way to avoid interaction effects is to administer the protector drugs in different routes. For example, when cisplatin is given intravenously an otoprotector should be given intramuscularly, subcutaneously or intraperitonealy, the latter one being more convenient in animal models such as mice, rats and guinea pigs. On the other hand, the most advantageous administration modality remains the local administration where a higher concentration of compound can be directly administrated to the inner ear without affecting other organs, and avoidance of side-effects in the rest of the body [30].

        The major disadvantage in local administration is the presence of scar tissue in the middle ear of a patient which blocks the access and the technique is not very patient friendly which for the moment presents many practical and unresolved issues [30]. Another concern is related to the dosage of the protector given in order to obtain a good protection and the time necessary to reach the inner ear. In this context, it might be very advantageous to formulate a cocktail-combination of protectors. Data from ebselen studies  [29]  have shown that it is possible to reduce the dosage of ebselen (minimizing possible side-effects) with the simultaneous administration of allopurinol and the anti-tumor effect of cisplatin in vivo tumor models at the mean time by combining ebselen and allopurinol the protection was obtain at lower dose than could be obtained by either agent alone [29]. Vitamin E is a lipid-soluble chain-breaking antioxidant in membranes and in plasma [17]. It is quickly oxidized in a high ROS filled environments and vitamin C is the main reductant of oxidized vitamin E [46]. Therefore by combining vitamin E with vitamin C may reduce the vitamin E dosage and increase the effectiveness of vitamin E. The possible advantages in introducing a cocktail are that ccocktails may give much better results as it can act on different cell targets, correctly combined drugs in the cocktail will interact with each others to increase the efficiency of each other and cocktails are easy to administrate.
                 Finally it is important not only to work on mono protector therapy but also to work on combined protector therapy since this type of study may show good outcome which could be used in future clinical practices as clinical application is our final destination.

 

Effects of protective agents against cisplatin ototoxicity:

 

Protector                  Administration      Animal model                      Protection                        Refs
N- Acetylcystine                    I.V.                Rat                                         +++                             [8]
D-Methionine                        I.P.                Rat                                         +++                             [5]
Tocopherol                           I.P.               Rat                                            ++                             [23]
Tocopherol                           I.P.               Guinea pig                                  ++                             [41]
Tocopherol + tiopronin          I.P.               Guinea pig                                  ++                             [13]
Ebselen                                I.P.               Rat                                          +++                            [35]
Ebselen & Allopurinol              P.O.             Rat                                            ++                             [29]

Abbreviations:      I.P.: intraperitoneal;          ++: Moderate protection;
                         I.V.: intravenous;           +++: Good protection;
                         P.O.:Orally

 

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