Patent Publication Number: US-9889156-B2

Title: Method for treating noise-induced hearing loss (NIHL)

Description:
RELATED APPLICATIONS 
     This patent application is a continuation-in-part of U.S. patent application Ser. No. 14/847,178 filed on Sep. 8, 2015. U.S. patent application Ser. No. 14/847,178 is a continuation-in-part of U.S. patent application Ser. No. 13/839,760 filed on Mar. 15, 2013, which is now U.S. Pat. No. 9,144,565. U.S. patent application Ser. No. 13/839,760 is a continuation-in-part of U.S. patent application Ser. No. 13/679,224 filed on Nov. 16, 2012, which is now U.S. Pat. No. 8,927,528. U.S. patent application Ser. No. 13/679,224 is a continuation-in-part of U.S. patent application Ser. No. 12/761,121 filed on Apr. 15, 2010, which is now U.S. Pat. No. 8,338,397. U.S. patent application Ser. No. 12/761,121 is a continuation-in-part of U.S. patent application Ser. No. 11/623,888 filed on Jan. 17, 2007, which is now U.S. Pat. No. 7,951,845. U.S. patent application Ser. No. 11/623,888 claims priority to and all advantages of U.S. Provisional Patent Application No. 60/760,055, filed on Jan. 19, 2006. U.S. patent application Ser. No. 13/679,224 is also a continuation-in-part of U.S. patent application Ser. No. 13/091,931 filed on Apr. 21, 2011, which is now U.S. Pat. No. 8,338,398. U.S. patent application Ser. No. 13/091,931 is a continuation of U.S. patent application Ser. No. 11/623,888 filed on Jan. 17, 2007, which is now U.S. Pat. No. 7,951,845. U.S. patent application Ser. No. 11/623,888 claims priority to and all advantages of U.S. Provisional patent App. Ser. No. 60/760,055, filed on Jan. 19, 2006. 
    
    
     GOVERNMENT LICENSE RIGHTS 
     This invention was made with Government support under DC004058 awarded by the National Institutes of Health. The Government has certain rights in this invention. 
    
    
     BACKGROUND OF THE DISCLOSURE 
     Field of the Disclosure 
     The present disclosure generally relates to method for treating noise-induced hearing loss (NIHL) and, more particularly, to a method for treating NIHL from repeated exposure(s) to high noise levels. The method includes administering a composition to a mammal throughout a period of the repeated exposure to noise or sound levels sufficient to cause hearing loss. The composition consists essentially of a biologically effective amount of vitamin A, vitamin E, vitamin C, a vasodilator comprising magnesium, and, optionally, a withanolide, and/or resveratrol. 
     Description of the Related Art 
     Extensive studies have been performed on compositions for treating various types of hearing loss or damage. NIHL is a leading cause of acquired hearing impairment in the industrialized world. Activities that result in persons having NIHL include, but are not limited to, occupational- and military-related activities. For example, more than twenty million United States workers are typically exposed to potentially hazardously high noise levels while working. In addition, about one-third of returning military personnel have suffered disabling hearing impairments. The number of people actually at risk for NIHL worldwide is much higher, which may be due to an increasing use of digital music players and increasing attendance at night clubs, discos, and music concerts. 
     Noise levels high enough to cause hearing loss (i.e., intense noise) can cause hearing impairment by two mechanisms: 1) direct mechanical damage to delicate structures of the inner ear and/or 2) intense metabolic damage. Noise-induced mechanical damage to the inner ear is caused from energy of sufficient intensity to break apart membranes of the inner ear tissue(s), such as exposure to a blast injury or explosion. Noise-induced mechanical damage to the inner ear is typically prevented by decreasing the energy that reaches the inner ear. Noise-induced metabolic damage is typically caused from oxidative stress in the cells of the inner ear, the formation of free radicals, and a resulting decrease in blood flow to the inner ear. 
     In some instances, NIHL can occur rapidly from a single exposure to a very intense noise, such as an explosion. More typically, NIHL occurs over time, such as with repeated exposures to high levels of noise, e.g. at the work place and/or with regular and repeated use of a digital music player or by attending concerts. Certain risk factors identified as contributing to NIHL include malnutrition, cardiovascular disease, diabetes, smoking, exposure to heavy metals, and exposure to noise. The impact of noise and NIHL are amplified in the population by its significant influence on age-related hearing loss (ARHL), and NIHL contributes to about 25 to 50% of the burden of ARHL. Said differently, noise exposure enhances ARHL, where about 25 to 50% of people with ARHL have a history of exposure to high levels of noise. Urban environments are increasingly noisy, and there is a rapidly increasing percentage of the world&#39;s population living in urban environments. For example, in the year 1800, about three percent of the world&#39;s population lived in urban environments. By the year 1900, almost fourteen percent of the world&#39;s population lived in urban environments. By the year 1950, about thirty percent of the world&#39;s population lived in urban environments. In 2008, the world&#39;s population was about evenly split between urban and rural areas, with more than four hundred cities with over one million people and nineteen cities with over ten million people. In addition, in 2008, about seventy-four percent of the population lived in urban environments in more developed countries, while forty-four percent of the population lived in urban environments in less developed countries. However, urbanization is occurring rapidly in many less developed countries, and about seventy percent of the world&#39;s population should be living in urban environments by the year 2050 with most of this urban growth occurring in the less developed countries. 
     In addition to increasing environmental and industrial noises, high levels of noise exposure from rapid adoption of digital music technologies and personal listening devices places about 1.1 billion people worldwide at risk for early onset of noise-induced hearing loss. 
     Intense noise is an environmental stress factor for the ear that may cause damage to the ear, which may lead to cell death. For instance, intense noise can cause damage to micromechanical properties of sensory transducers in the ear, changes in blood flow in the ear, modification in intracellular ion transport properties, depletion of sensory cell transmitter substances (e.g. glutamate), changes in post synaptic membrane transmitter receptors (e.g. gluR) on afferent nerve fibers, modification of dispersion and uptake properties of transmitters in extracellular, synaptic spaces, and/or changes in postsynaptic membrane biophysical properties that may affect space- and time-constant properties modifying depolarization. Intense noise can also cause changes of an excitotoxic nature in postsynaptic membranes, causing destruction of afferent neural tissues. Any one or more of these changes may result in modification of spontaneous activity in individual or small populations of afferent nerve fibers, which can result in a change in the sensitivity of hearing and/or the perception of tinnitus. 
     Additionally, intense noise (i.e., high noise levels sufficient to cause hearing loss) demands greater activity of the respiratory chain to create adenosine triphosphate (ATP), resulting in excess free radical formation. Typically, in the normal ear under normal stress, the endogenous antioxidant systems are sufficient to maintain normal homeostatic function of the inner ear. However, under high levels of stress, increased free radical formation may contribute to excitotoxicity and can damage the DNA. Through the process of lipid peroxidation, the increased free radical formation can destroy inner and extracellular membranes that can lead to temporary loss in hearing sensitivity, distortions of sounds, muffling of sounds, and permanent hearing loss associated with cell death. While a number of factors may influence these changes, intercellular redox properties of cells and blood flow to the inner ear may be of particular importance in causing these changes. Stress induced hearing loss becomes evermore prevalent in the ageing inner ear with reduced blood flow and reduced efficiency of antioxidant systems. 
     Dietary supplements including  ginkgo biloba , melatonin, zinc, lipoflavenoids, and vitamin supplements are available for treating hearing loss; however, no evidence is available that any of these supplements are actually beneficial for NIHL or tinnitus. In addition, no chemical treatments have been attempted to prevent repeated exposure to high noise levels leading to NIHL. Accordingly, there remains an opportunity to develop an effective composition and method for treating noise-induced hearing loss. 
     SUMMARY OF THE DISCLOSURE 
     The subject disclosure provides a method for treating noise-induced hearing loss (NIHL) that includes the step administering a composition to the mammal, wherein the composition consists essentially of a biologically effective amount of vitamin A, vitamin E, vitamin C, a vasodilator comprising magnesium, and, optionally, a withanolide, and/or resveratrol. 
     The composition of the present disclosure may be used for treating noise-induced hearing loss, and includes components that function through different biological mechanisms to provide an additive effect that is equal to or greater than a sum of the effect of the individual components. In essence, the composition includes a biologically effective amount of at least one scavenger of singlet oxygen, a donor antioxidant, a third antioxidant, and a vasodilator. The at least one scavenger of singlet oxygen may be present for reducing free radicals that contribute to hearing loss. The donor antioxidant may be present for reducing peroxyl radicals and inhibiting propagation of lipid peroxidation that also contributes to hearing loss. The vasodilator may be present for preventing decreases in both cochlear blood flow and oxygenation that also contribute to hearing loss. 
     The composition, when presented daily during repeated noise exposures, is significantly effective in reducing damage to the inner ear and NIHL from repeated exposure to high noise levels. As used herein, high noise levels include those at which hearing loss is induced, such as repeated exposure to noise at 85 decibels (dB) and above for about 8 hours per day. Typically, as the noise level increases above 85 dB, the time for exposure decreases. In an example, for every 6 dB increase in noise level, the exposure time decreases by about half. For instance, a person exposed to a noise level of about 115 dB (such as at a high level rock concert), hearing loss may occur at an exposure of about 15 minutes. It is to be appreciated that, in certain instances, hearing loss may be induced at noise levels less than 85 dB, such as at 80 dB or even as low as 75 dB. The noise level that induces hearing loss may depend, at least in part, on genetics and/or a person&#39;s sensitivity toward particular noises that result in hearing loss. 
     The method for treating noise-induced hearing loss from repeated exposure to high noise levels, described in detail below, may be used to reduce or even prevent NIHL of in mammals, particularly humans, that are repeatedly exposed to noise at levels that typically result in hearing impairment. Examples of noise levels that typically result in hearing impairment include, but are not limited to, occupational noise exposures, military noise exposures, and repeated noisy leisure time activities. As a result, the composition and method for treating noise-induced hearing loss provide great promise to minimizing hearing loss resulting from trauma to the inner ear(s) of a mammal. Given the high incidence of noise-induced hearing loss in the general population worldwide, there is a great need for the composition and method of the present disclosure to minimize socioeconomic effects that persist due, at least in part, to noise-induced hearing loss. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Advantages of the present disclosure will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawing. 
         FIGS. 1A-1D  are graphs representing auditory brainstem response (ABR) thresholds in mammals before and 1-10 days following four days of repeated exposures to high noise level (e.g., 118 dB SPL for 4 hrs/day for 4 days) in two groups of rats fed on non-supplemented rat diet (ND) or a supplemented rat diet (ED) diet prior to and throughout the noise exposure and ten day recovery period.  FIGS. 1A and 1B  illustrate threshold sensitivity in dB SPL across frequency.  FIGS. 1C and 1D  illustrate noise-induced threshold shifts observed. 
         FIG. 2  is a graph representing ABR thresholds (measured in decibels of sound pressure level (dB SPL)) for mammals of 6-8 months of age at noise frequencies ranging from 0 to 32 kHz. 
     
    
    
     DETAILED DESCRIPTION 
     A composition for treating noise-induced hearing loss (NIHL) includes components that may function through different biological mechanisms to provide an additive effect that is equal to or greater than a sum of the effect of the individual components. The composition used by the method is typically used for treating NIHL that may result from repeated exposure to high levels of noise or sound stress to an inner ear of a mammal. The stress may be further defined as mechanically-induced metabolic trauma, mechanical/metabolic trauma, stress trauma, stress-induced damage, and/or environmental stress. The stress may also reflect a downregulation of inner ear blood flow and endogenous antioxidant systems that occur gradually with malnutrition, cardiovascular disease, certain genetic disorders, smoking, and aging. This may increase the sensitivity of the inner ear to pathology that would be well below a threshold for damage to a healthier and/or younger ear. It is possible that the composition may be used to treat or prevent all types of NIHL, including repeated exposure to high levels of noise from any source. 
     A method in accordance with the instant disclosure includes the step of administering a composition to a mammal that includes components that function through different biological mechanisms. In the method, the composition is typically used for preventing and treating NIHL, in short, preserving the hearing capacity of a mammal&#39;s ear. 
     In various embodiments, the trauma may be further defined as stress to the inner ear over a period of time, where the stress is defined as environmental or endogenous actions that demand relatively high level of energy production by the cells of the inner ear. Intense noise (i.e., high noise levels) can cause damage to micromechanical properties of sensory transducers in the ear, changes in blood flow in the ear, modification in intracellular ion transport properties, depletion of sensory cell transmitter substances (e.g. glutamate), changes in post synaptic membrane transmitter receptors (e.g. gluR) on afferent nerve fibers, modification of dispersion and uptake properties of transmitters in extracellular, synaptic spaces, and/or changes in postsynaptic membrane biophysical properties that may affect space- and time-constant properties modifying depolarization. In addition, intense noise can cause changes of an excitotoxic nature in postsynaptic membranes causing destruction of afferent neural tissues. Any one of these changes can result in modification of spontaneous and evoked activity in small or large populations of afferent nerve fibers, which can result in temporary loss of hearing sensitivity, distortion of sound, or permanent loss of hearing. While a number of factors may influence these changes, intercellular redox properties of cells, blood flow to the inner ear and calcium uptake by postsynaptic afferent nerve fiber membranes may be of particular importance in causing these changes. 
     The baseline physiological function of systems that underlie normal homeostasis sets the conditions upon which stress agents may effect the inner ear. In a young inner ear, blood flow is typically resilient and endogenous antioxidant systems are robust with the exception of certain hereditary hearing losses. With age, these systems are typically compromised, with age itself being a stress factor of the inner ear. One result of noise, age, and/or drug stressors on the inner ear is the formation of excess free radicals in association with the induced metabolic trauma. The free radicals typically damage sensitive structures, such as hair cells within the ear and can initiate processes that can lead to hearing loss. Vasoconstriction may also occur as a result of the noise, which can lead to decreased blood flow to the inner ear and cause cell death that results in NIHL and associated tinnitus. It has been found that the underlying cause of vasoconstriction is noise-induced free radical formation. Specifically, one of the molecules formed in the inner ear as a result of the presence of free radicals is 8-isoprostane-2F alpha, which is a bioactive agent. The bioactive agent induces a constriction of blood vessels in the inner ear, which causes a reduction in blood flow. In order to counteract the free radical formation and the vasoconstriction, the composition of the subject disclosure typically includes at least one scavenger of singlet oxygen, a donor antioxidant, a third antioxidant, and a vasodilator. Unexpectedly, it was found that the composition including the at least one scavenger of singlet oxygen, the donor antioxidant, the third antioxidant, and the vasodilator produce an additive effect that is not only greater than the effect of any one of those components alone, but at least equal to or greater than a sum of the effects of each of the components. 
     Many genetic hearing disorders are dependent upon a defect in a single gene leading to hearing impairment or deafness, and many reflect defects in more that one gene, and may be associated with other, non hearing, clinical disorders. Greater sensitivity to NIHL may be associated with either class of genetic hearing disorders and thus associated with one (non-syndromic) mutation or with mutations in more than one gene (e.g. syndromic) and may be associated with other inherited clinical disorders. 
     Greater sensitivity to repeated exposure of high noise levels, NIHL may reflect a defect in a gene resulting in flawed gene copies which mis-instructs production of a protein important for homeostasis and transduction processes in the inner ear. This could lead to disruption of micromechanical properties of hair cells, depolarization defects, inadequate production and or assembly of transmitter substances, and/or compromised synaptic activity. This may also result in disruption of ion metabolism, e.g. potassium, their distribution and movement in the inner ear and particularly in the lateral wall and sensory cells, and a disruption of cellular homeostasis in these cells, frequently leading to cell death. 
     Since potassium homeostasis in the inner ear is important for normal function of the sensory cells of the inner ear (hair cells), any disruption tends to result in hearing impairment and may induce hearing loss or tinnitus. Knockout mouse models of genetic hearing impairment may show early widespread degeneration of both inner and outer hair cells, presumably secondary to defects that upregulate apoptotic cell death pathways. In humans, hearing impairment may begin sometime following birth and progress until profound deafness occurs. However, gene defects may be expressed as mild to moderate hearing impairment. 
     More specifically, one or more genetic defects may lead to disruption of potassium homeostasis in an inner ear of a mammal. Genotypic characteristics of potassium channels have been associated with sensitivity to NIHL. Potassium homeostasis may regulate apoptosis such that stress-driven disruption of inner ear potassium homeostasis may lead to increase production of free radicals by mitochondria directly leading to upregulation of apoptotic cell death pathways as well as support direct potassium induced cytochrome c release and apoptosis. Potassium channels Kv1.3, mitochondrial Ca 2+  regulated potassium channel, mitoBKCa, and mitochondrial ATP-regulated potassium channel—mitoKATP have been demonstrated in mitochondrial membranes. Mitochondrial potassium channels effect energy production by the mitochondrion. In addition, there may be a direct dependence of free radical formation on potassium channel function during the respiratory chain in mitochondrial function. Moreover, increased mitochondrial K+ influx may result in release of cytochrome c and caspase-3 followed by apoptosis. These events could be blocked by bcl-2, which upregulated the mitochondrial K/H-exchanger, leading to increased removal of K + . In addition, Bcl-2 and tBid proteins may counter-regulate mitochondrial potassium transport. By removing/eliminating excess free radicals antioxidants may restore mitochondrial function. Antioxidants act through a variety of mechanisms. The at least one scavenger of singlet oxygen and the donor antioxidant are two different classes of antioxidants that act through different mechanisms. The third antioxidant, while typically a scavenger of singlet oxygen, may be a different antioxidant that acts through a different mechanism. Scavengers of singlet oxygen reduce free radicals that contribute to inner ear pathology and thus to hearing loss and/or tinnitus. These free radicals may also cause side effects of antibiotic treatment such as kidney damage and loss of balance. More specifically, by reducing free radicals, the scavengers of singlet oxygen prevent, among other damaging effects, the singlet oxygen from reacting with lipids to form lipid hydroperoxides. Lipid hydroperoxides may play a role in causing NIHL. 
     Even within the class of scavengers of singlet oxygen, it is believed that various antioxidants react at different sites within the body, and in particular, within cells to attenuate free radical formation. For example, one of the scavengers of singlet oxygen is typically vitamin A. In various non-limiting embodiments described herein, the terminology Vitamin A and beta-carotene may be used interchangeably. However, these embodiments in no way limit this disclosure. Vitamin A is a generic term that captures a number of molecules with a biological activity of retinol or carotenoids. Primary dietary forms of vitamin A/retinol include retinol esters and beta-carotene. The beta-carotene is made up of a polyene chain of 11 conjugated double bonds with methyl branches spaced along the polyene chain, capped at both ends by cyclohexenyl rings with 1,1,5-trimethyl substitution. Other forms of vitamin A include xanxthophylls, astaxanthin, canthxanxin, lutein, and zeaxanthin, which include a backbone of beta-carotene with hydroxyl and/or carbonyl substitution on one or more of the cyclohexenyl rings. For purposes of the subject disclosure, the vitamin A is typically present as beta-carotene. Beta-carotene is a powerful scavenger of singlet oxygen, as well as nitric oxide and peroxynitrite, and may also scavenge lipid peroxyl radicals within a lipophilic compartment of a mitochondrial membrane. Beta-carotene is an excellent scavenger of free radicals under normal physiological conditions present in most tissues. 
     In addition to vitamin A, other scavengers of singlet oxygen may also be present in the composition of the subject disclosure. For example, another scavenger of singlet oxygen that may be present is resveratrol. Resveratrol is more efficient at scavenging hydroxyl radicals than vitamin C, and the addition of resveratrol to the vitamins A may have additive effects. 
     The at least one scavenger of singlet oxygen may be present in the composition in a biologically effective amount. For purposes of the subject disclosure, the biologically effective amount may be further defined as an amount that is sufficient to produce an additive effect in a reduction in stress induced threshold shift or tinnitus when used in combination with other antioxidants and the magnesium. Additive effect, as used herein, refers to an effect that is equal to or greater than a sum of the effects of the individual components. In order to produce additive effect and the reduction in threshold shift or tinnitus, the at least one scavenger of singlet oxygen is typically present in the composition in a total amount of at least 830 international units (IU), more typically from 830 to 120,000 IU, most typically from about 2,100 to 70,000 IU for an adult dosage. 
     The amount of the vitamin A present in the composition is dependent upon the form of vitamin A that is used. For example, in one embodiment, vitamin A is present as retinol in an amount of at least 830 IU, more typically from 830 to 10,000 IU, more typically from 2,100 to 10,000 IU, most typically from 2,100 to 8,000 IU. As known in the art, a conversion of IU to weight for vitamin A (as retinol) is 3.33 IU/μg. Thus, at least 830 international units (IU) of vitamin A (as retinol) is equivalent to at least 0.25 mg of vitamin A, from 830 to 10,000 IU of vitamin A (as retinol) is equivalent to from 0.25 to 3 mg of vitamin A, and from 2,100 to 8,000 IU of vitamin A (as retinol) is equivalent to from 0.63 to 2.4 mg vitamin A. 
     Alternatively, the vitamin A may be present in the composition as beta-carotene, as opposed to retinol. The retinol activity equivalents (RAE) for retinol conversion to beta-carotene, which is a pro-vitamin A carotenoid, is 1 mg to 12 mg. In terms of conversion of the amounts set forth above for the vitamin A present in the composition as retinol to the vitamin A present in the composition as beta-carotene, in one example, a total amount of at least 3.0 mg or at least 830 international units (IU) of vitamin A as beta-carotene, more typically from 3.0 to 180 mg or 830 to 50,000 IU vitamin A as beta-carotene, most typically from about 7.2 to 108 mg or 2000 to 30,000 IU of vitamin A as beta-carotene is typically present for an adult dosage. In another example, a total amount of at least 3.0 mg or at least 10,000 international units (IU) of vitamin A as beta-carotene, more typically from 3.0 to 36 mg or 10,000 to 120,000 IU vitamin A as beta-carotene, most typically from about 7.5 to 21 mg or 25,000 to 70,000 IU of vitamin A as beta-carotene is typically present for an adult dosage. 
     Specific amounts of the vitamin A present in the composition may be dependent on the body weight of the mammal. In one specific example, the amount of vitamin A present as retinol in the composition is about 0.0178 mg/kg body weight. Thus, for an average human weighing about 70 kg, the amount of vitamin A present as retinol in the composition may be about 1.25 mg. If the vitamin A is in the form of beta-carotene, in one example, the beta carotene in the composition is about 0.257 mg/kg body weight may be present in an amount of about 18 mg. In another example, the beta-carotene in the composition may be about in an amount of about 15 mg. 
     It is to be appreciated that, when additional scavengers of singlet oxygen such as resveratrol are present in the composition in addition to vitamin A, the total amount of scavengers of singlet oxygen may be greater than the ranges set forth above for the at least one scavenger of singlet oxygen, so long as at least one scavenger of singlet oxygen is present in the amounts set forth above. In addition, other scavengers of singlet oxygen may be used in place of vitamin A, so long as the amount of the at least one scavenger of singlet oxygen is present within the amounts set forth above. When present, the resveratrol is typically included in the composition in an amount of at least 1 mg, more typically in an amount of from 10 mg to 1500 mg, most typically in an amount of from 15 mg to 1000 mg. 
     Whereas the at least one scavenger of singlet oxygen tends to prevent the initial formation of lipid peroxides, the donor antioxidant tends to reduce peroxyl radicals and inhibits propagation of lipid peroxidation that contributes to inner ear pathology and NIHL. More specifically, the donor antioxidant reacts with and reduces peroxyl radicals and thus serves a chain-breaking function to inhibit propagation of lipid peroxidation. As is evident from the chain-breaking function of the donor antioxidant in lipid peroxidation, the donor antioxidant functions within cell membranes. A specific donor antioxidant that is contemplated for use in the composition of the subject disclosure is vitamin E. Vitamin E is a generic term for all tocols and tocotrienol derivatives with a biological activity of alpha-tocopherol. Primary dietary forms of vitamin E include vitamin E itself and alpha-tocopherol. Trolox®, a water-soluble analogue of alpha-tocopheral commercially available from Hoffman-Laroche, Ltd. of Basel, Switzerland, is a research agent that is typically used as a source of vitamin E. 
     The donor antioxidant is typically present in the composition, for example, in an amount of at least 75 IU, more typically from 75 IU to 2,000 IU, more typically from 150 to 1,500 IU, most typically from 150 IU to 800 IU. In another example, the donor antioxidant is present in the composition in an amount of at least 75 IU, more typically from 75 IU to 1,500 IU, most typically from 150 IU to 800 IU. As known in the art, a conversion of IU to weight for synthetic vitamin E is 0.66 mg/IU and for natural vitamin E is 0.45 mg/IU. Thus, when the donor antioxidant is synthetic vitamin E, in on example, at least 75 IU of vitamin E is equivalent to at least 50 mg of vitamin E, from 75 to 2,000 IU of synthetic vitamin E is equivalent to from 50 to 1,320 mg of vitamin E, from 150 to 1,500 IU of synthetic vitamin E is equivalent to from 100 to 1,000 mg of vitamin E, and from 150 to 800 IU of synthetic vitamin E is equivalent to from 100 to 536 mg of vitamin E. In another example, when the donor antioxidant is vitamin E, at least 75 IU of vitamin E is equivalent to at least 50 mg of vitamin E, from 75 to 1500 IU of vitamin E is equivalent to from 50 to 1000 mg of vitamin E, and from 150 to 800 IU of vitamin E is equivalent to from 150 to 600 mg of vitamin E. As with the amount and type of vitamin A, specific amounts of the vitamin E present in the composition may be dependent on the body weight of the mammal. In one specific example, the amount of synthetic vitamin E present in the composition is about 3.8 mg/kg body weight. Thus, for an average human weighing about 70 kg, the amount of vitamin E present in the composition may be about 266 mg. In another specific example, the amount of synthetic or natural vitamin E present in the composition is about 2.6 mg/kg body weight. Thus, for an average human weighing about 70 kg, the amount of vitamin E present in the composition may be about 182 mg. 
     In addition to the at least one scavenger of singlet oxygen and the donor antioxidant, the composition further includes the third antioxidant. While the third antioxidant may be a scavenger of singlet oxygen, the third antioxidant may also be an antioxidant that functions through a different mechanism. When the third antioxidant is a scavenger of singlet oxygen, the at least one scavenger of singlet oxygen is still present in the composition as a separate component from the third antioxidant, and is still present in the composition in the amounts set forth above for the at least one scavenger of singlet oxygen. As a result of the third antioxidant being another scavenger of singlet oxygen, the resulting composition would have at least two scavengers of singlet oxygen. 
     The third antioxidant is typically vitamin C, which is a scavenger of singlet oxygen and reactive nitrogen species. It is to be appreciated that, although the third antioxidant is typically vitamin C, other antioxidants may be used in place of the vitamin C, and the other antioxidants may function through different mechanisms than vitamin C. The term vitamin C applies to substances that possess antiscorbutic activity and includes two compounds and their salts: L-ascorbic acid (commonly called ascorbic acid) and L-dehydroascorbic acid. In addition to being known as ascorbic acid and L-ascorbic acid, vitamin C is also known as 2, 3-didehydro-L-threo-hexano-1, 4-lactone, 3-oxo-L-gulofuranolactone, L-threo-hex-2-enonic acid gamma-lactone, L-3-keto-threo-hexuronic acid lactone, L-xylo-ascorbic acid and antiscorbutic vitamin. Vitamin C is known to scavenge both reactive oxygen species and reactive nitrogen species. It can be oxidized by most reactive oxygen and nitrogen species, including superoxide, hydroxyl, peroxyl and nitroxide radicals, as well as such non-radical reactive species as singlet oxygen, peroxynitrite and hypochlorite. Vitamin C thus inhibits lipid peroxidation, oxidative DNA damage, and oxidative protein damage. 
     In contrast to vitamin A, which functions best under conditions present in most tissues, water-soluble vitamin C is an excellent free radical scavenger in an aqueous phase to thus reduce free radicals at a site different from that of vitamin A. More specifically, ascorbic acid functions to reduce free radicals in fluid, such as in cytoplasmic fluid and/or blood, before the free radicals reach cell membranes. 
     The third antioxidant is typically present, for example, in an amount of at least 4,000 IU, more typically from 4,000 to 60,000, more typically from 8,000 to 40,000 IU, most typically from 8,000 to 20,000 IU. In another example, the third antioxidant is typically present in an amount of at least 4,000 IU, more typically from 6,000 to 40,000 IU, and most typically from 8,000 to 20,000 IU. Using vitamin C as an example for converting IU to weight units for the third antioxidant, as known in the art, a conversion of IU to weight for vitamin C is 0.05 mg/IU. Thus, at least 4,000 IU of vitamin C is equivalent to at least 200 mg of vitamin C, from 6,000 to 60,000 IU of vitamin C is equivalent to from 300 to 3,000 mg vitamin C, from 6,000 to 40,000 IU of vitamin C is equivalent to from 300 to 2,000 mg, from 8,000 to 40,000 IU of vitamin C is equivalent to from 400 to 2,000 mg vitamin C, and from 8,000 to 20,000 IU vitamin C is equivalent to from 400 to 1,000 mg vitamin C. As with vitamins A and E, specific amounts of the vitamin C or other third antioxidant present in the composition may be dependent on the body weight of the mammal. In one specific example, the amount of vitamin C present in the composition is about 7.14 mg/kg body weight. Thus, for an average human weighing about 70 kg, the amount of vitamin C present in the composition may be about 500 mg. 
     As set forth above, the composition further includes a vasodilator. Typically, the vasodilator includes magnesium; however, the vasodilator, for purposes of the subject disclosure, may include other vasodilators in place of or in addition to magnesium, in place of or in addition to those including magnesium, or may include only magnesium or only magnesium-containing compounds. Vasodilators can be used for treating NIHL. Vasodilators including magnesium prevent decreases in cochlear blood flow and oxygenation via biochemical mechanisms involving changes in calcium concentration and prostaglandins. Deficient cochlear blood flow and lack of oxygenation can contribute to NIHL by causing metabolic changes in lateral wall tissues important for maintaining normal homeostasis of the inner ear, e.g. endocochlear potential, and normal transduction; and may cause cell death in sensitive hair cells within a cochlea of the ear. Vasodilators including magnesium have also been found to improve the efficacy of immunosuppressant therapy or carbogen inhalation therapy in recovery from sudden hearing loss and/or tinnitus. Furthermore, it has been found that magnesium deficiency leads to increased calcium channel permeability and greater influx of calcium into cochlear hair cells and afferent nerve endings, increased glutamate release, and auditory nerve excitotoxicity, each of which play a role in health of the inner ear. 
     When the ear is under oxidative stress, the vasodilators, particularly vasodilator magnesium, exhibit an unexpected additive effect when combined with the biologically effective amounts of the at least one scavenger of singlet oxygen, the donor antioxidant, and the third antioxidant, especially when the at least one scavenger of singlet oxygen is vitamin A, the donor antioxidant is vitamin E, and the third antioxidant is vitamin C. The additive effect referred to above is greater than not only the most efficacious of the components for treating inner ear pathology that causes NIHL, ARHL and tinnitus, but also typically greater than the sum of the effects of each of the components. While vasodilators other than those including magnesium are envisioned for purposes of the present disclosure, additive effects are not observed with all vasodilators. For example, betahistine, which is another known vasodilator, does not exhibit an additive effect. 
     The vasodilator including magnesium typically includes a magnesium salt or magnesium salt complex and, more specifically, magnesium sulfate or magnesium citrate. Other vasodilators including magnesium that may be suitable for purposes of the subject disclosure include; magnesium acetate, magnesium aspartate, magnesium carbonate, magnesium chloride, magnesium fumarate, magnesium gluconate, magnesium glycinate, magnesium hydroxide, magnesium lactate, magnesium oxide, magnesium salicylate, magnesium stearate, and magnesium sulfate. Other representative salts include but are not limited to; hydrobromide, hydrochloride, bisulfate, nitrate, arginate, ascorbate, oxalate, valerate, oleate, palmitate, laurate, borate, benzoate, phosphate, tosylate, maleate, fumarate, succinate, taurate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate and laurylsulphonate salts. 
     Typically, the vasodilator is present in the composition in an amount of at least 50 mg. For example, when the vasodilator is magnesium, the magnesium is typically present in an amount of from 50 to 450 mg, most typically from 100 to 350 mg. As with vitamins A, C, and E, specific amounts of the vasodilator present in the composition may be dependent on the body weight of the mammal. In one specific example, the amount of the vasodilator including magnesium present in the composition is about 4.46 mg/kg body weight. Thus, for an average human weighing about 70 kg, the amount of the vasodilator including magnesium present in the composition may be about 312 mg. In another example, the amount of the vasodilator including magnesium present in the composition is about 2.14 mg/kg body weight. Thus, for an average human weighing about 70 kg, the amount of the vasodilator including magnesium present in the composition may be about 150 mg. 
     Non-limiting examples of amounts of the typical components included in the composition, along with more and most typical amounts, are summarized in Table 1 below. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                   
                   
                 Typical 
               
               
                   
                   
                 More 
                 Most 
                 Dosage, 
               
               
                   
                   
                 Typical 
                 Typical 
                 mg/kg body 
               
               
                 Component 
                 Amount 
                 Amount 
                 Amount 
                 weight 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Vitamin A 
                   
                 ≧830 
                 IU 
                 830-10,000 
                 IU 
                 2100-8,000 
                 IU 
                 0.0178 mg/kg  
               
               
                   
                 Vitamin A 
                 ≧830 
                 IU 
                 830-50,000 
                 IU 
                 2,000-30,000 
                 IU 
                 0.257 mg/kg  
               
               
                   
                 As beta- 
               
               
                   
                 carotene 
               
               
                 Vitamin C 
                   
                 ≧4,000 
                 IU 
                 4,000-60,000 
                 IU 
                 8,000-20,000 
                 IU 
                 7.14 mg/kg 
               
               
                 Vitamin E 
                   
                 ≧75 
                 IU 
                 75-2000 
                 IU 
                 150-800 
                 IU 
                  3.8 mg/kg 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 (synthetic) 
               
               
                 Magnesium 
                   
                 ≧50 
                 mg 
                 50-450 
                 mg 
                 100-350 
                 mg 
                 4.46 mg/kg 
               
               
                   
               
            
           
         
       
     
     With respect to Table 1, the amounts specified for the antioxidants and the vasodilator correlate, in terms of biological effectiveness, to amounts used for humans. Furthermore, it is to be appreciated that the biologically effective amounts of the antioxidants and vasodilator may be lower within the above ranges for children than for the average human, based on lower US recommended daily allowances and maximum intake levels for children. This is evident based on the typical dosages in Table 1 based on mg/kg. 
     Other non-limiting examples of amounts of the typical components included in the composition, along with more and most typical amounts, are summarized in Table 2 below. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                   
                   
                 Typical 
               
               
                   
                   
                 More 
                 Most 
                 Dosage, 
               
               
                   
                   
                 Typical 
                 Typical 
                 mg/kg body 
               
               
                 Component 
                 Amount 
                 Amount 
                 Amount 
                 weight 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Vitamin A 
                   
                 ≧830 
                 IU 
                 830-120,000 
                 IU 
                 2,100-70,000 
                 IU 
                 — 
               
               
                   
                 Vitamin A 
                 ≧830 
                 IU 
                 830-50,000 
                 IU 
                 2,100-5,900 
                 IU 
                 0.0178 mg/kg  
               
               
                   
                 As Retinol 
               
               
                   
                 Vitamin A 
                 ≧10,000 
                 IU 
                 10,000-120,000 
                 IU 
                 25,000-70,000 
                 IU 
                 0.214 mg/kg  
               
               
                   
                 As beta- 
               
               
                   
                 carotene 
               
               
                 Vitamin C 
                   
                 ≧4,000 
                 IU 
                 6,000-40,000 
                 IU 
                 8,000-20,000 
                 IU 
                 7.14 mg/kg 
               
               
                 Vitamin E 
                   
                 ≧75 
                 IU 
                 75-1,500 
                 IU 
                 150-800 
                 IU 
                  2.6 mg/kg 
               
               
                 Magnesium 
                   
                 ≧50 
                 mg 
                 50-450 
                 mg 
                 100-350 
                 mg 
                 2.14 mg/kg 
               
               
                   
               
            
           
         
       
     
     With respect to Table 2, the amounts specified for the antioxidants and the vasodilator correlate, in terms of biological effectiveness, to amounts used in animal studies on guinea pigs. Furthermore, it is to be appreciated that the biologically effective amounts of the antioxidants and vasodilator may be lower within the above ranges for children than for the average human, based on lower U.S. recommended daily allowances and maximum intake levels for children. This is evident based on the typical dosages in Table 2 based on mg/kg. 
     In addition to the antioxidants and vasodilator, other components may also be present in the composition for treating NIHL. These components may be used for treating the side effects of the antibiotic treatment also. For example, in one embodiment, the composition further includes a withanolide. With anolides have been suggested for use in anti-inflammatory, anti-tumor, cytotoxic, and immunological applications. One example of a specific withanolide that may be included in the composition of the subject disclosure is the withanolide extracted from day lily plants. The extract is a powerful natural antioxidant which may be effective in preventing cell death in the inner ear by interrupting the cell-death pathway initiated by deafferentation of the auditory nerve. When included in the composition, the withanolide may be present in an amount of at least 10 ppm, more typically from 10 to 1000 ppm. Additional components, besides with anolides, can also be included. Typically, the composition is free of components that interfere with the biological mechanisms through which the at least one scavenger of singlet oxygen, the donor antioxidant, the third antioxidant, and the vasodilator function. The composition is also typically free of additional components that could degrade or neutralize the at least one scavenger of singlet oxygen, the donor antioxidant, the third antioxidant, and the vasodilator function when mixed therewith prior to internally administering the composition to the mammal. Those of skill in the art can readily identify such components in view of the mechanisms by which the individual components in the composition function as set forth above (e.g., components that cause vasoconstriction, various oxidizing agents, etc.). 
     It is also to be appreciated that, even if additional components are present in the composition that could interfere with the mechanisms by which the at least one scavenger of singlet oxygen, the donor antioxidant, the third antioxidant, and the vasodilator function, the composition described above is may still be effective for purposes of treating side effects of the antibiotic treatment. As one example, and as described in further detail below, the composition including the at least one scavenger of singlet oxygen, the donor antioxidant, the third antioxidant, and the vasodilator may be effective for treating hearing loss and/or tinnitus and other side effects of antibiotic treatment when administered in conjunction with aminoglycoside antibiotics. This is true even though aminoglycoside antibiotics, themselves, are responsible for causing hearing loss and may add to the sensitivity to NIHL and other side effects such as kidney damage and loss of balance through free radical formation. 
     Since components as detrimental as aminoglycosides can be administered in conjunction with the composition described above, it is clear that additional components that are less detrimental to the specific mechanisms by which the at least one scavenger of singlet oxygen, the donor antioxidant, the third antioxidant, and the vasodilator function can also be present in the composition with an expectation that the composition maintains effectiveness for purposes of treating side effects of other stresses that may contribute to repeated exposure NIHL. Examples of some additional components that may be included in the composition include, but are not limited to, excipients, flavoring agents, fillers, binders, and additional vitamins or minerals not specifically mentioned herein. 
     As alluded to above, with the method for treating NIHL from repeated exposure to high noise levels and the method for treating side effects of other stressors to the inner ear, the instant disclosure includes the step of internally administering the composition of the subject disclosure to a mammal. The composition may be orally administered to the mammal, such as in the form of a tablet, liquid, gel, etc. Alternatively, the composition may be intravenously administered to the mammal through an IV or an injection of the composition and may also be locally administered via the round window membrane of the cochlea. As a specific example, the vitamins A, C, and E, the vasodilator including magnesium, and the optional components may be first combined to form the composition, with the composition then administered to the mammal. Alternatively, the vitamins A, C, and E, the vasodilator including magnesium, and the other optional components may be separately administered, in which case the composition forms within the mammal. In all scenarios, the treatment is typically provided daily throughout the period of repeated exposure to high levels noise or other stressors to the inner ear. 
     For purposes of the subject disclosure, NIHL is typically associated with stress (such as, e.g. noise, but may be compounded by other stressors including antibiotics and genetic factors)-induced hearing loss as objectively measured in terms of differences in threshold shift, or through measurement of a percentage of hair cell loss. In small mammal studies, NIHL is objectively measured as a change in hearing threshold measured behaviorally, or as more commonly known as a change in the threshold of the Auditory Brainstem Response (ABR). This is clearly demonstrated in a consistent and reliable manner after application of stress agents such as noise and drugs and observed NIHL. 
     For purposes of the subject disclosure, NIHL may be objectively measured in terms of threshold shift from a baseline (prior to noise exposures) to the threshold measured following noise exposures, or through measurement of a percentage of hair cell loss. In studies utilizing rats, NIHL and the efficacy of the composition for treating NIHL may be measured as the threshold shift from baseline threshold sensitivity at 0.5, 1, 2, 4, 8, 16, and 32 kHz, measured following repeated noise exposures in a group of rats provided a daily diet supplemented with the vitamins A, C, and E, and the vasodilator including magnesium. This is compared to a similar group of rats exposed daily to the same noise exposure, but fed a controlled, non-supplemented normal rat diet. The normal or control rat diet includes carotene, vitamins A, D, and E, ascorbic acid, and magnesium in amounts which are considered appropriate to maintain health. It is noted that the amounts of the individual components of the normal rat diet is significantly less than for the supplemented diet. Larger differences in threshold shift correlate to less NIHL and greater efficacy of the composition for treating the hearing loss from repeated exposures to high intensity of noise. 
     It is has been shown that hair cell loss correlates to threshold shift. For example, in experiments utilizing guinea pigs having ears that recover from temporary threshold shift, morphological damage may be limited to tips of stereocilia in a third row of outer hair cells (OHCs). However, ears from animals with permanent threshold shift typically have damage to all three rows of OHCs and, in some cases, the inner hair cells (IHCs), with damage throughout the length of the stereocilia as well as the to the body of the hair cell. 
     In one example, and as shown in  FIGS. 1A-1D , the composition of the present disclosure is administered to three month old mammals beginning ten days preceding exposure to noise, and continuing throughout the exposure to a high level of white noise at 118 dB SPL, about equivalent to noise produced at a boiler plant or a loud rock concert, for 4 hr/day for four consecutive days. For comparison, a similar group of animals on a normal control (non-supplemented) diet were exposed to a similar noise. The results demonstrate that in both groups of animals, there was a significant NIHL at day 1 and day 10 following the noise exposures at all frequencies tested. However, the threshold shift observed in the group of rats treated with the supplemented diet was significantly lower that that observed in the animals on a normal rat, non-supplemented, diet. More particularly, at baseline (control) and prior to noise exposure, the mean values of the auditory thresholds for both the normal diet (ND) and the supplemented diet (ED) are shown in  FIGS. 1A and 1B , respectively. The mean values following acute noise over-exposure is significantly higher for rats on ND and rats on ED compared to the baseline (control), the mean thresholds found in rats on ED are significantly lower than that observed for rats on ND at the same survival time. In comparison to the baseline, the threshold shift in the rats on ND ranged from 31.11 (+/−1.11) to 43.98 (+/−2.61) dB at day 1 and from 27.14 (+/−1.84) to 40.71 (+/−2.02) dB at day 10 post-stimulation (as shown in  FIG. 1C ). In contrast, the threshold shift is significantly lower for rats on ED, which ranged from 21.42 (+/−3.03) to 35.00 (+/−2.86) dB at day 1 and from 20.00 (+/−2.31) to 30.63 (+/−3.95) dB at day 10 post-stimulation (as shown in  FIG. 1D ). Accordingly, the results show that the supplemented diet is effective in reducing NIHL throughout the frequency range of hearing. 
     In a second example, two groups of three month old rats, with one group on a supplemented diet and the other group on a controlled, non-supplemented diet, were exposed to a moderately high white noise at 118 dB SPL for about 1 hr/day for 5 days/week for about 3-5 months. As shown in  FIG. 2 , the animals on the supplemented rat diet (ED) demonstrated a significant reduction in the NIHL, compared to animals on the normal, non-supplemented rat diet (ND), particularly within the 0.5 to 4 kHz range which is particularly important for speech discrimination with auditory brainstem response (ABR) thresholds shown for mammals at 6-8 months of age. Clear elevations in threshold sensitivity to all frequencies greater than 16 kHz are also observable over time. For example, and as shown in  FIG. 2 , the noise-induced threshold shift for rats on ND ranged from 16.25 (+/−2.27) to 29.38 (+/−3.05) dB, while the threshold shift for rats on ED ranged from 7.50 (+/−3.13) to 38.88 (+/−2.82) dB. From these results, there is a significant difference between the threshold shifts observed in rats on ND compared to rats on ED at 0.5, 1, and 2 kHz frequencies, which are particularly relevant to speech discrimination in humans. For example, the noise-induced elevations observed in the rats on ED are clearly less (e.g. in the 0.5-4 kHz range, which are in the speech-related frequency range) than that observed in ND. Accordingly, the supplemented diet (ED) is effective for reducing NIHL. 
     In this example, the component diet was fed to the mammals throughout the study paradigm. The noise level given was significant, but was not greater than that experienced in many occupations and leisure time activities. The mammals were exposed for about 1 hr/day for about 3-5 months. Given the differences in life span, this may equate to less than a decade of the human lifespan and is well below the working life of the average person in a noisy occupation or the period that a person exposes himself/herself to loud levels of music amplified in live performance (such as at a concert), though personal listening devices, or other noise stresses associated with urban recreational, industrial, and/or manufacturing environments. 
     After initial administration, the composition is typically administered to the mammal each day throughout the study, or for humans, the composition is typically administered to the human throughout a period of repeated exposure to loud sound. Although excellent results have been achieved through such treatment, it is to be appreciated that other treatment regimens may also prove efficacious for purposes of the present disclosure. 
     For the method for treating side effects of antibiotic treatment, which may contribute to NIHL, the composition may be internally administered to the mammal in conjunction with administration of the antibiotic. In this regard, the method also includes the step of internally administering the antibiotic, which antibiotic is capable of inducing hearing loss in the mammal. It is to be appreciated that, even though the antibiotic with which the composition is administered is capable of inducing hearing loss, the method of the instant disclosure is not strictly limited to treatment of NIHL that is enhanced by the antibiotics. More specifically, the method of the instant disclosure proscribes the step of administering the subject composition in conjunction with administration of the antibiotic for any purpose including for treating any side effect of the antibiotics including not only antibiotic-induced NIHL, but also kidney damage, loss of balance, among other side effects. 
     To maximize effectiveness of the treatment described herein, it is desirable to establish stable serum levels of the at least one scavenger of singlet oxygen, the donor antioxidant, the third antioxidant, and the vasodilator at the time that the stress induces increase free radical formation and damage to the inner ear. Typically, the composition is administered to the mammal immediately, which is sufficient to achieve the stable serum levels of the at least one scavenger of singlet oxygen, the donor antioxidant, the third antioxidant, and the vasodilator before the antibiotics begin to materially increase the sensitivity of the ear to NIHL. In one embodiment, the composition is internally administered prior to administration of the stress. 
     Once administration of the composition has begun, the composition is typically administered each day during stress administered to maintain adequate serum levels of the at least one scavenger of singlet oxygen, the donor antioxidant, the third antioxidant, and the vasodilator. Additionally, the composition is typically administered daily for the duration of stress. This is typically performed for purposes of ensuring that adequate serum levels of the at least one scavenger of singlet oxygen, the donor antioxidant, the third antioxidant, and the vasodilator are maintained until the serum levels of the antibiotic decrease, and typically until all of the additional free radicals formed secondary to the stress are eliminated. 
     In varying embodiments, an average difference in threshold shift in mammals from baseline threshold sensitivity at greater than 4 kHz, as compared to an untreated control, is improved by at least three fold (10 decibels). To obtain those results, the composition is orally administered ten days prior to the beginning of the exposure to the noise and administered again each day until the mammals were assessed at 6-8 months of age. The threshold shift is measured after exposure to the noise using auditory brainstem response (ABR) testing. Similar results would be anticipated using other alternative measures of auditory or sensory cell function, such as psychophysical tests or otoacoustic emission measures. 
     One or more of the values described above may vary by ±5%, ±10%, ±15%, ±20%, ±25%, etc. so long as the variance remains within the scope of the disclosure. Unexpected results may be obtained from each member of a Markush group independent from all other members. Each member may be relied upon individually and or in combination and provides adequate support for specific embodiments within the scope of the appended claims. The subject matter of all combinations of independent and dependent claims is herein expressly contemplated. The disclosure is illustrative including words of description rather than of limitation. Many modifications and variations of the present disclosure are possible in light of the above teachings, and the disclosure may be practiced otherwise than as specifically described herein. In additional non-limiting embodiments, all values and ranges of values within any aforementioned range of numbers are hereby expressly contemplated.