SCIENCE

The Science of ACEMg

For nearly a century scientists believed most hearing loss was caused by biomechanical damage. Our research from 1985 to 2005 demonstrated excess inner ear free radicals and reduced inner ear blood flow are responsible. Those findings changed the way medical science understands the problem. Today, it is accepted science that the common fundamental cause of many types of hearing loss is biochemical, not biomechanical. The problem is oxidative stress.

Further, our research discovered the way to block the first cause of oxidative stress – excess free radicals – eliminating all biochemical paths to cell damage and death. Hearing Health Science developed the first and the only patented antioxidant micronutrient for hearing preservation. ACEMg is a safe, pure, high potency antioxidant micronutrient formula of antioxidant vitamins and a mineral – vitamins A, C, E and magnesium.

Free radical formation associated with environmental stress from intense noise or sound energy, drugs, aging and trauma play a key role in hearing loss and cell death in the inner ear. This white paper explains the scientific concept; research leading to the ACEMg formula; and the ACEMg mechanism or mode of action.

Growing knowledge of the role of free radicals in other diseases and pathology lend credence to their important role in hearing loss and the promise of ACEMg to treat hearing impairment. Free radicals play a key role in pathology from a variety of environmental stress factors. While free radicals are essential for normal cellular biochemistry, in excess free radicals lead to pathology and are now well recognized as important factors in sensorimotor disorders, as well as other neurologic diseases associated with the aging process. 

 

Free radicals function as triggers to upregulate necrotic and apoptotic pathways to cell death. Thus, an over guiding driving hypothesis and scientific rationale underlying ACEMg is that free radical formation defines a ‘final common cell death pathway’ inducing pathology from a large variety of etiological factors. Moreover, for the ear and hearing, we hypothesize that these factors are potentiated by reduced organ circulation, which is a dramatic feature of noise-induced stress and a significant feature of the aging process.

Given this knowledge, an important feature of the strategy underlying the efficacy of ACEMg is that it removes the initial cause of cellular stress and pathology.  Thus, given the knowledge of the biochemical pathways to cell death, there are a number of sites along any of these pathways that could block cell death.  We could block the upregulation of one of the caspases or the insertion of BCL-2 genes into the mitochondrial membrane, or perhaps block release of cytochrome c.  However these are each parallel pathways to cell death and if one is blocked others may take its place. By blocking the first-cause, free radicals, we eliminate all biochemical paths to cell damage and death.

Discussion
Free radical formation associated with environmental stress from intense noise, drugs, aging and trauma play a key role in hearing loss and cell death in the inner ear. Our studies in the auditory system have demonstrated that antioxidants plus a vasodilator reduce by 75% both noise and drug (aminoglycoside)-induced inner ear pathology and hearing loss (Le Prell et al., 2007; Le Prell et al 2012). This formulation is β-carotene (converted in the body to vitamin A), ascorbic acid (vitamin C), trolox (vitamin E) and the vasodilator magnesium. Together known as ACEMg or ACEMg++ these agents synergistically attenuate stress induced hearing loss. ACEMg has been under NIH- and European Commission-supported preclinical and clinical trials to demonstrate its efficacy to prevent NIHL in humans. These trials involve teams of scientists from Spain, Germany, Sweden, Canada and the USA. A European Commission supported clinical trial is ongoing to assess the efficacy of ACEMg to prevent the loss of residual hearing in patients undergoing cochlear implant surgery and to enhance performance benefits of cochlear implantation.

These studies have demonstrated efficacy of ACEMg to prevent age-related hearing loss in animal models of presbycusis; to define the relationship between level of stress (noise) and ACEMg dosing; and provided further knowledge on the basic mechanisms of stress-related hearing loss and its treatment by ACEMg.  The results of these trials hold promise for the prevention of age-related hearing loss (ARHL) and other stress-related hearing loss, where free radical formation is a known component. Thus data indicate that free radicals play a key role in sensory and neural cell death and loss of residual hearing, with implantation (Abi-Hachem et al, 2010, Dinh and Van De Water, 2009). Hence, ACEMg may both protect residual hearing and reduce nerve loss with the stress of implantation, which will impact implant benefit outcomes in young and old patients.

Growing knowledge of the role of free radicals in other diseases and pathology lend credence to their important role in hearing loss and the promise of ACEMg to treat hearing impairment. Free radicals play a key role in pathology from a variety of environmental stress factors. While free radicals are essential for normal cellular biochemistry, in excess free radicals lead to pathology and are now well recognized as important factors in sensorimotor disorders, as well as other neurologic diseases associated with the aging process. Free radicals function as triggers to upregulate necrotic and apoptotic pathways to cell death.  They may be either generated as part of the metabolic processes (Ames et al, 1993) of the cell or frequently as side effects of environmental stress factors, as visible light (Agarwal et al., 1993;Oleinick and Evans, 1998) solar and ionizing radiation (Godar and Lucas, 1995; Godar, 1999; Zhao, et al;, 2007), cigarette smoke (Aoshiba et al 2001; Carnevali et al., 2003) , hyper- and hypoxia  (Budinger et al., 2002; Wang et al., 2003; Wang et al., 2004), drugs (Forge and Schacht, 2000; Rybak and Ramkumar, 2007), intense noise exposure (Ohinata et al., 2003; Le Prell et al., 2007).  Models detailing the pathways to cell death initiated by free radicals have been developed, across a broad range of pathologies in the peripheral and central nervous systems; and importantly the enhanced production of free radicals secondary to cell metabolism and environmental stress agents with aging has been well documented (for review see: Ryter et al, 2007).

Thus, an over guiding driving hypothesis and scientific rationale underlying ACEMg is that free radical formation defines a ‘final common cell death pathway’ inducing pathology from a large variety of etiological factors.  Moreover, for the ear and hearing, we hypothesize that these factors are potentiated by reduced organ circulation (Miller, et al, 2003; Le Prell et al, 2007b), which is a dramatic feature of noise-induced stress and a significant feature of the aging process.  Evidence on the key role of free radicals in cell death in these many fields provides strong theoretical and empirical support for the likelihood of their importance in hearing impairment.  Evidence for efficacy of antioxidant treatment to prevent free radical-induced pathology in the eye (ARED study) and the ear (Sha and Schacht, 2006) supports our expectation that this antioxidant intervention will prevent loss of hearing from noise exposure, drugs that induce hearing loss, will prevent loss of residual hearing following implantation and prevent or delay the onset of presbycusis. 

A model of the biochemistry underlying cell death from stress is illustrated in Figures 1, 2 and 3 below. The source of cellular energy for normal homeostasis and function are the mitochondria.  And in the process of their normal function they produce both molecular oxygen and partially reduced forms of oxygen (See Figure 1, Mechanism of action for ROS-induced cell death.  The electron transport chain of mitochondria ends with cytochrome c and an oxidase-dependent tetravalent reduction of oxygen to form water.  In this process redox carriers leak additional electrons to oxygen generating free radicals. As mentioned a certain number of free radicals are essential for normal cellular processes: and if there are too many, there are built-in antioxidant systems that reduce or scavenge the excess, detoxifying the cell.

However, with cellular stress, such as intense noise or direct surgical trauma, there is an increase demand for energy to maintain a greater level of metabolic activity required of the cell under stress. The ROS-initiated genetic cascade can lead to necrosis or apoptosis; and induces mitochondrial dysfunction, disrupting the cell‘s respiratory chain, creating more ROS leading to apoptotic cell death.

In response to that demand mitochondria produce more energy (ATP) and with that generate excess free radicals. With intense noise exposure we have found a remarkable 40-fold increase in free radical formation in the tissues of the inner ear (Ohinata el al., 2000). Such vast amounts of free radicals overwhelm the endogenous antioxidant system and initiate processes that damage the cell.  While mitochondrial activity likely accounts for the major increase in free radical formation with noise, additional sources include excitotoxic events in the hyperexcited auditory nerve, and ischemia/reperfusion, which as mentioned occurs with intense noise exposure in the inner ear.

Excess free radicals may cause cell death by initiating lipid peroxidation of nuclear and cell membranes, destroying the integrity of the cell and leading to necrotic cell death.  This is likely the path to death in the presence of extreme concentrations of free radicals.  In the presence of excess, but less extreme, free radical cell death is likely by apoptotic mechanisms.  Thus free radicals may up regulate genetic pathways that lead to programmed, apoptotic, cell death via a number of biochemical pathways. See Figures 2 and 3.  

We now know that oxidative stress first initiates an influx of calcium leading to calcium-dependent-calcineurin/calpain activation, initiating dephosphorylation of NFAT and activation of the BCL-2 family gene Bad. Bad causes release of cytochrome c, activation of caspases 9 and 3, and cell death.  Second, a caspase-2 dependent pathway to cell death can be triggered by free radical-induced DNA damage. Third, caspase-independent pathways to cell death include release of AIF and EndoG from the mitochondria. Translocation of EndoG to the cell nucleus results in chromatin condensation and high-molecular mass-chromatin fragmentation and cell death. Fourth, receptor-mediated cell death is initiated with ligation of death receptors on the surface of the cell, forming a death inducible signaling complex, which activates pro-caspase-8. Caspase-8 activates caspase-3, leading directly to cell death, and/or cleaves the gene Bid, resulting in translocation and insertion of the Bax-Bak complex into the mitochondrial membrane and release of cytochrome c, in turn activating caspases 9 and 3, and cell death. The caspase-2 dependent pathway differs from the caspase-8 and caspase-9 dependent pathways in that pro-caspase-2 is activated by DNA damage. Upregulation of a number of these pathways have been demonstrated in our laboratories in the noise-stressed inner ear, and the efficacy of interventions that block them have been demonstrated (Minami et al, 2004, Yamashita et al, 2004, Le Prell et al, 2007, Minami et al, 2007, and Yamashita et al, 2008).  This model of the biochemistry of free radical initiated cell death is entirely consistent with similar models of free radical initiated pathology in the cardiovascular system, brain and stress induced cell death from the many etiologies mentioned above.  This internal consistency adds to the face validity of the model for the inner ear and to the generality and potential implications for ACEMg-strategy for other systems, disorders and diseases.

Given this knowledge, an important feature of the strategy underlying the efficacy of ACEMg is that it removes the initial cause of cellular stress and pathology.  Thus, given the knowledge of the biochemical pathways to cell death, there are a number of sites along any of these pathways that could block cell death.  We could block the upregulation of one of the caspases or the insertion of BCL-2 genes into the mitochondrial membrane, or perhaps block release of cytochrome c. However these are each parallel pathways to cell death and if one is blocked others may take its place.  By blocking the first-cause, free radicals, we eliminate all biochemical paths to cell damage and death.

 

ACEMg Mechanism or Mode of Action
Vitamin C detoxifies by reducing free radicals (for review, see Evans, Halliwell 1999).  Scavenging of oxygen radicals by vitamin C occurs in the aqueous phase (Niki 1987a; Niki 1987b).  Vitamin E, present in lipids in cells (see Burton et al. 1983), is a donor antioxidant that reacts with and reduces peroxyl radicals and inhibits the propagation of lipid peroxidation (for review, see Schafer et al. 2002).  The primary antioxidant action of β-carotene (metabolized to vitamin A in vivo) is to scavenge singlet oxygen; because singlet oxygen reacts with lipids to form lipid hydroperoxides, the removal of singlet oxygen prevents lipid peroxidation (for review, see Schafer et al. 2002).  Thus vitamin C removes free radicals from the water compartments of the cell, while vitamin E removes them from the lipid compartments, and vitamin c blocks the lipid peroxidation that may be initiated by free radicals not removed by vitamins C and E.

Why add magnesium?
In most tissues, increased metabolism increases blood flow, which provides additional oxygen.  However, high levels of noise reduce blood vessel diameter and red blood cell velocity and thus decrease cochlear blood flow (Miller et al. 2002; for review, see Le Prell et al. 2006). Reduced cochlear blood flow has significant implications for metabolic homeostasis, as cellular metabolism clearly depends on adequate supply of oxygen and nutrients as well as efficient elimination of waste products (e.g., Miller et al. 1996). In addition the reduction in blood flow with noise is followed by an overshoot with off-set of the noise, causing reperfusion-induced formation of additional free radicals, which synergistically add to those formed during noise. Providing agents that reduce noise-induced vasoconstriction, such as magnesium, betahistine, or hydroxyethyl starch attenuates NIHL (for review, see Le Prell et al. 2006).

 

  1. There are a number of other mechanisms for the production of free radicals; and while we are only discussing oxygen free radicals or reactive oxygen species (ROS), there are parallel pathways for the production of reactive nitrogen species (RNS).  Both ROS and RNS, regardless of source, act similarly to trigger cell death pathways to necrosis or apoptosis.

 

  1. In addition to well-known effects of magnesium on blood flow, other biochemical mechanisms may further contribute to the protective effects of magnesium.  Magnesium modulates calcium channel permeability, influx of calcium into cochlear hair cells, and glutamate release (Gunther et al. 1989; Cevette et al. 2003), each of which may reduce NIHL. Mg is also a NMDA-receptor antagonist. That the NMDA-receptor antagonist MK-801 reduces the effects of noise, ischemia, or ototoxic drugs (Janssen 1992; Basile et al. 1996; Duan et al. 2000; Konig et al. 2003; Ohinata et al. 2003), suggests another potential protective mechanism for Mg. Regardless of the specific mechanism of action, Mg supplements clearly attenuate NIHL

.

FIGURE 1 - Mechanism of action for ROS-induced cell death

FIGURE 2 - The ROS-initiated genetic cascade can lead to necrosis or apoptosis.

FIGURE 3 - Cyt C in the cytosol induces mitochondrial dysfunction, disrupting the cell‘s respiratory chain, creating more ROS leading to apoptotic cell death.

Peer reviewed published papers providing the scientific rationale for this work include:

Yamasoba et al 1998a; Yamasoba et al 1998b; Yamasoba et al, 1999; Shoji et al, 2000a; Ohinata et al, 2000; Shoji et al 2000b; Ohinata et al, 2000; Yamasoba et al; 2001; Altschuler et al; 2002; Ohinata et al, 2003; Zou et al; 2003; Le Prell et al, 2003; Yamashita et al, 2004a; Minami et al, 2004; Yamashita et al. 2004b; Yamashita et al, 2005; Miller et al, 2006; Le Prell et al, 2007a; Le Prell 2007b; Minami et al, 2007; Yamashita et al, 2008

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Effectiveness

In the theoretical, basic and empirical science, we and others have found that intense noise causes a great increase in metabolic demand for cellular energy. Cells of the inner ear undergo metabolically induced oxidative stress.  With that there is a very large increase in formation of free radicals (40 fold over normative baseline).

Free radicals can directly attack the proteins of the cell causing damage to cell membranes and cellular DNA. The increase in free radicals also causes an upregulation of selective gene activity to remove (scavenge) the free radicals and (apoptotic) cell-death genes that lead to “programmed cell death” and (anti-apoptotic) genes to prevent this programed cell death.  Each of these genes initiate a specific biochemical cascade of events that either lead to cell death or to recovery from the free radical overload and return to normal function.

Importantly other stresses to the inner ear, e.g. aminoglycoside toxicity or age-related changes, also lead to increases in free radical formation and the same sequence of events leading to cell pathology; this oxidative stress in the inner ear is a common sequence of events leading to pathology in many organs of the body. Fundamentally, oxidative stress-induced free radical-initiated cascades define a common pathway to cell death. And while there is always more to learn, the bottom line is we well understand specifically what happens and why and how intense noise leads to pathology and hearing impairment.

Adding to this, we discovered that the free radicals that form lead to a unique byproduct – the isoprostane 8-iso prostaglandin F2a – that causes vasoconstriction and reduces blood flow to the inner ear.  Thus just as the cells of the inner ear are under metabolic demand there is a decrease in oxygen to the cell, causing it to work less efficiently and causing increased formation of free radicals, which then leads to additional formation of the vasoconstrictive byproduct and less blood flow, a positive feedback loop of significant negative consequences. Moreover when the noise terminates there is a large overshoot in blood flow, which, like a stroke or heart attack, leads to yet a further increase in free radicals.

Based on this basic science research and understanding we defined a micronutrient formulation of three antioxidants – vitamins A, C and E (ACE) – that function in different compartments of the cell and by different mechanisms to remove or block the free radicals before they can directly damage the cell or upregulate gene pathways that can lead to cell death.  To these we added a mineral – magnesium (Mg) – that increases blood flow to block that noise-induced vasoconstriction and the subsequent rebound that further increases free radical formation. We demonstrated that this formulation – ACEMg – could block the noise-induced reduction in blood flow and overshoot and could significantly reduce the noise-induced formation of free radicals.

Thus the theoretical basis and empirical science of the fundamental events that underlie so-called noise-induced hearing loss (NIHL) from intense sound and noise exposure have been well elaborated and provide a strong scientific rationale for expecting this micronutrient formulation to effectively prevent NIHL.

General Efficacy

In preclinical animal studies by many laboratories, antioxidants have been demonstrated to be effective in preventing NIHL, and for that matter aminoglycoside-induced hearing loss as well. In our work we compared a variety of antioxidants and other agents that increase cellular energy production efficiency and found that many can reduce NIHL. By far, most effective in NIHL reduction was the micronutrient formulation of three antioxidants – vitamins A, C and E (ACE) and the mineral magnesium (Mg) that increases blood flow to the inner ear.

 

The demonstrated key to the greater efficacy of the ACEMg formulation was the synergistic interaction between the antioxidants and vasodilator in protecting the cells from oxidative stress and preserving normal function.

 

ACEMg has now been studied in a variety of small mammal animal models, including mice, rats and guinea pigs, under a variety of noise conditions and in all have been show efficacious in the prevention of NIHL. More recently we have also shown ACEMg prevents age-related hearing impairment and can reduce the symptoms of tinnitus that frequently accompany exposure to intense sound.

Thus empirical data provides a robust scientific rationale for the effectiveness of ACEMg to preserve normal function of the tissues of the inner ear under stress and prevent NIHL.

Clinical
Research
Human Efficacy

Clinical research is drug research; randomized, controlled trials (RCT) conducted using double-blind, placebo-controlled methods. In RCT trials, neither the person giving the medicine nor the person getting it knows if they are getting the active or placebo.

ACEMg is manufactured as an active and placebo clinical trial medicine (CTM) and approved by drug regulatory agencies for use in clinical trials in the EU and USA.

 

Moreover, the addition of additional actives that effect energy production or other metabolic factors did not add to the efficacy of ACEMg, they distracted. These empirical demonstrations are the underlying justification for five issued and three pending patents on the ACEMg formulation.

The ACEMg clinical trial program includes testing ACEMg to preserve hearing in cochlear implant patients; preserving hearing in children with the Connexin 26 gene defect; blocking hearing loss caused by the ototoxic side effect of gentamicin antibiotics; blocking the ototoxic side effect of cytomegalovirus. The clinical trials program continues to expand with new preclinical discoveries.

Studies to demonstrate the efficacy of ACEMg to preserve hearing under environmental stress are underway in the ACEMg real world studies. We are optimistic they will return positive results. Our optimism is based upon:

The Age Related Eye Disease Study (AREDS) by the US National Institutes of Health (NIH) demonstrated that a formulation of vitamins A, C and E, plus iron chelator minerals prevent formation of free radicals and thus prevent age-related macular degeneration. Importantly, the mechanism of cellular damage by age-related macular degeneration and NIHL share the fundamental oxidative stress-induced free-radical-initiated cell death cascade and pathway to cell death.

Studies in humans have shown that magnesium along significantly prevents NIHL observed in military recruits undergoing basic training in the Israeli army. (Attitus et al.)

A variety of studies in humans have shown antioxidants in diets and as supplements are associated with healthy hearing. in particular, a study showing that specifically dietary supplements of vitamins A and E were associated with a reduction in hearing loss in the elderly. (Choi et al).

Finally, we have demonstrated that administration of ACEMg in a dose that is safe and within the guidelines of the Institute of Medicine (IOM) at the National Academy of Sciences (NAS) for the recommended daily limits elevates the plasma level of these micronutrient agents by an amount equivalent to that seen in the guinea pig that prevented NIHL.

Epidemiological research is real world research, science conducted for the public good by scientists partnering with citizens.

It is best to use epidemiological or real world research to study age-related hearing loss (ARHL) and noise-induced hearing loss (NIHL) because they are linked. Both typically develop over many years or decades, and are influenced by environmental, genetic, nutritional and lifestyle factors.

The ACEMg real world studies are designed to assess efficacy among a broad range of populations globally.

Real world (epidemiological) studies

Pilot tests are the first step. Our modest goal at the moment is to recruit participants to a four-week pilot test with professional musicians and sound engineers regularly and routinely exposed to loud music.

These tests help evaluate and refine the study design and tools before starting recruitment to placebo-controlled randomized study described above, and subsequent studies.

What We Know

ACEMg has been shown in lab studies to delay the onset of ARHL. ACEMg reduces ARHL in subjects exposed to a moderate level of noise, demonstrate that ACEMg reduces NIHL in situations of continuous exposure of noise, extending the original work showing efficacy with a single noise exposure.

Age-related hearing loss (ARHL) and noise-induced hearing loss (NIHL) are linked. Both typically develop over many years or decades, and are influenced by environmental, genetic, nutritional and lifestyle factors. Our research shows ACEMg can be used to treat both, and researchers are able to study the intervention in scientifically rigorous ways for the first time.

Second, evidence from previous epidemiological research on the key role of free radicals in cell death in many fields provides strong theoretical and empirical support for the likelihood of their importance in hearing impairment and cognitive dysfunction, and treatment with antioxidants. This includes evidence for efficacy of antioxidant treatment to prevent free radical-induced pathology in the eye (NIH AREDs study), using a very similar formulation of antioxidants to that used in ACEMg, and in the ear (Sha and Schacht, 2006), using a different antioxidant formulation.

Third, previous epidemiological studies have demonstrated a relationship of the antioxidants vitamins A & E and magnesium and reduction in hearing impairment in the elderly (Choi et al, 2013); while nutrition studies have shown a similar relationship of vitamins A,C and E and hearing impairment (Spankovich et al 2011; Seidman, 2000; Shargorodsky, et al 2010).

What we want to measure next.

The first phases of the studies will explore the use of ACEMg to attenuate hearing loss in participants with regular exposure to loud music. Importantly, the studies happen on an app.

The primary objective is to measure ACEMg effectiveness reducing temporary threshold shifts and tinnitus in music professionals who are regularly exposed to loud music.

The proposed primary outcome is reduction of temporary threshold shifts (TTS), which occur during the course of each time period of sustained exposure to loud music and persist after cessation of exposure for a period of time up to roughly equivalent to the length of time of exposure.

Prevention of TTS is known to reduce the risk of permanent threshold shifts (PTS, or, simply, permanent hearing loss). Although individuals chronically exposed to noise are known to develop permanent hearing loss (PTS) with resulting declines in measurable TTS, evidence exists (Kähärit et al., 2003; Axelsson et al., 1995) indicating that a substantial proportion of the music professional community targeted in this phase of the studies may have preserved enough hearing to be susceptible to TTS, especially those early in their careers.

 

TTS can now be measured with relatively good accuracy and precision using a rapid, easy-to-use, self-administered app-based audiometry test (the app) delivered by iPhones and other Apple iOS devices. This technical development makes it possible to collect repeated measures at very little cost, and at the convenience of the study participant. The app can also be used for self-administered questionnaires that gather survey data from potential recruits, feedback and notifications from participants during the study period, for assessments of qualitative data on tinnitus, and more. Over time, the app can be expanded to monitor ambient sound in the participant’s environment, and to deliver dosimeter data.

We aim to build a digital platform for hearing.

We’re building a platform for collecting and expanding the largest data set on hearing that will be shared with customers and accredited research partners who wish to confirm and compare expectations, analyze effectiveness and map sound around the world. Think Google maps for sound.

You will be able to test your hearing accurately. Compare your hearing to the world. Compare your environment to the world and monitor your exposure. How long is it safe to listen? To stay? Know the difference ACEMg is making for you and the world.