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A novel micronutrient-based strategy to prevent hearing impairments: test and road to market for age-related hearing loss and preservation of residual hearing

Final Report Summary - PROHEARING (A novel micronutrient-based strategy to prevent hearing impairments: test and road to market for age-related hearing loss and preservation of residual hearing)

Executive Summary:
In the course of the ProHearing project we have tested a proprietary combination of vitamins A, C, E and magnesium (Mg) (ACEMg) which synergistically combines antioxidation (A,C,E) and vasodilator (Mg) properties. Preclinical results in experimental animals strongly support that oral ACEMg protects against noise-induced hearing loss with improvements in auditory thresholds (brainstem auditory evoked potentials) of around 15%. There is also very significant protection against a combination of age-related hearing loss and noise. In experimental animals, the safety margin seems wide, although effective dosages are relatively narrow. An interesting collateral finding is that irradiation of the compound (supplied as animal chow) results in lack of otoprotection activity, which raises cautions about its use in the pharmaceutical and nutraceutical industries.
These preclinical results have generated scientific publications and intellectual property which will be used for exploitation and commercialization purposes.
The ultimate goal of tests in humans, was addressed through an extensive clinical trial aimed at protecting residual hearing in cochlear implant users. 51 patients have been recruited. At this point, only interim results are available. Although possibilities of alternative funding were explored none was viable. Due to lack of alternative funding, the trial had to be stopped and available data analyzed and reported later.

Project Context and Objectives:
Ten per cent of the world population, around 600-700 M people, is hearing impaired, which makes hearing loss the most prevalent impairment worldwide. For 360 M, hearing impairment is disabling or highly disabling. In the EU alone, 55M are hearing impaired. This condition greatly decreases quality of life, particularly in the elderly, and constitutes an unmet health and medical need, as well as a highly significant burden for the economy. Current WHO estimates of this economic burden are approximately 1.3T€/year. Just for reference, the estimated cost of global warming predicted in the year 2070 amounts to 1.15T€/year.
Timely regeneration of damaged sensory auditory cells, an ideal ultimate cure for hearing loss, is not currently available as therapy for humans. In concordance with the aims of the 7th Framework Program, we proposed a novel strategy based on the development of a micronutrient combination implemented as an oral formulation, to prevent damage, and perhaps promote recovery of auditory hair cells. This program (PROHEARING) is based on the rationale that free radical formation and blood flow reduction are central in the pathophysiology of cell death in sensory systems in response to environmental stress and aging. In the auditory system, stress associated with inner ear damaging agents and conditions such as intense noise, drugs, interventions for implantation of cochlear prosthesis and aging, all lead to excess free radical formation, blood flow reduction, inner ear sensory cell loss and irreversible hearing impairment. Our goal is to gather evidence that a proprietary combination of antioxidants (vitamins A,C and E) and a vasodilator (Mg++) (ACEMg) effectively prevents/treats: 1) hearing loss in the elderly and 2) loss of residual hearing in the elderly and patients receiving cochlear implants. A combination of human trials and animal studies should create a pathway to effectively translate and take to the market this new technology to human applications.
Therefore, the global objective of PROHEARING is to provide clinical and laboratory data to support the otoprotection efficacy of ACEMg as an immediate translational basis to market. This global objective is better specified in the following specific objectives:
1) Gain foreground knowledge to demonstrate that ACEMg preserves residual hearing in cochlear implant patients.
2) Gain foreground knowledge to demonstrate that ACEMg prevents age-related hearing loss in animal models.
3) Define the relation between ACEMg dosage and preservation of inner ear function following acoustic stress.
4) Gain foreground knowledge to demonstrate that free radical formation and activation of cell death pathways follows acoustic stress in adult and aged animals.
5) Gain foreground knowledge to demonstrate that the former is prevented/reversed by ACEMg.
6) Provide proof of safety, biocompatibility and interoperability of the formulation.
7) Fulfil all regulatory aspects to justify commercialization and provide a clear and compelling pathway to market.

Project Results:
The main S&T results and foreground relate to Work packages 2, 3, 4 and 5. Project coordination aspects belong to Work package 1, which will not be reported here.
Work package 2.-
Partner 2 (HHSE), Partner 3 (MHH) and Partner 4 (HCTC) participated in work package 2, under the leadership of partner MHH.
The single center, randomized, placebo-controlled, double-blind phase II clinical trial “ACEMg mediated hearing preservation in cochlear implant patients receiving different electrode lengths” was designed to evaluate the effect of β-carotene (vitamin A), ascorbic acid (vitamin C), trolox (vitamin E) and the vasodilator magnesium (Mg), or ACEMg, on the residual hearing of 140 CI-patients. The primary measure was based upon the reduction in post-operative low frequency air conduction pure tone thresholds compared to pre-operative thresholds in ACEMg treated patients compared to those of a placebo-group. Additionally, the effect of different electrode lengths (20, 24, 28mm) was analyzed. Study visits were scheduled before surgery, at first fitting (4 weeks after surgery) and 3, 6, 9 and 12 months after first fitting. The primary endpoint was the air conduction hearing loss at 500Hz 3 months after first fitting. Additionally, speech recognition tests, hearing aid benefit in the implanted ear and electrophysiological measurements of implant function were assessed.
In order to describe the main results obtained, we will consider the following:
1. Course of PROHEARING clinical trial
2. Patient recruitment
3. Patient follow up
4. Descriptive analysis
5. Safety data
6. Conclusion

1. Course of the clinical trial
After obtaining all necessary regulatory approvals (BfArM (German competent authority) and responsible ethics committee) the clinical trial was launched on December 16th, 2013.
Initially, the clinical trial was conducted with chewing tablets administered twice daily for 105 days/patient. The technology was available, but not tested in a larger patient population. During the clinical testing the clinical site got feedback regarding compliance problems (in particular: taste of the tablets). As a consequence it was decided to issue a new application form for the ingredients. In early 2014 BfArM was approached to discuss whether it is possible under certain circumstances to change the study medication during a clinical trial. BfArM had no objection to change to another formulation providing twice daily comparable amounts of vitamins and magnesium were given to the patient. During the meeting it was discussed to use a liquid formulation, i.e. a powder which was to be resolved with water. HHSE went forward to develop this formulation (HHSE identified a European manufacturer to enable direct release for a clinical trial in Europe; subcontractor: Patheon). Patheon then started to develop a powder formulation. During stability studies it came out that it was actually very difficult to develop a stable powder containing the required vitamins and magnesium. Therefore, Patheon proposed to find another formulation. The best suitable approach (also most cost-effective) was to move forward with softgel capsules. Patheon started to manufacture batches and put medication on long term stability studies.
In February 2015 the new batch of softgel IMP was produced by Patheon. Batch records began to be available from Patheon in March 2015. On April 1st, the EC issued its letter approving a one-year extension of the grant, if the PROHEARING consortium could meet two requirements by June 30th, 2015: (1) Written approval by BfArM to use the softgel capsules as the study medication in the clinical trial, and (2) approval of the continuation of the trial by BfArM and responsible Ethics Committee. The quality documents were ready for submission to BfArM on May 16th, 2015, and the entire IMPD, including the revised Investigators Brochure (IB) was submitted to BfArM on May 21st, 2015. BfArM approved the study amendment including the IMPD on June 26th, 2015, and the project was therefore prolonged by the EC until June 30th, 2016. In May 2015 recruit-ment had to be stopped because of end of stability of the study drug on June 30th, 2015. Despite the prolongation of the grant period the problems partner HHSE were facing did not allow a timely procurement of study medication, thus the trial needed to be halted.
According to the agreed work packages, partner HHSE has been responsible for providing study medication to WP2. Due to the unclear financial situation of HHSE, invoices issued by the manufacture of the new study medication (Patheon, UK) could not be paid by HHSE. As a result Patheon held back the new study medication which basically was available in early 2015. Thus, the required secondary labelling (to be executed by Theorem GmbH in Bad Soden) could not be initiated. Therefore, a smooth transition from the former chewing tablets (which ran out of stability) to the more convenient softgel capsules was not anymore possible.
As a usual procedure during clinical trials the clinical study protocol was amended several times to adjust the protocol to practical experiences in the daily management of patients recruited to the trial. In total three substantial amendments were approved by the competent authority (BfArM) and the responsible Ethics Committee, resp., and have been implemented. The third substantial amendment was necessary because of the above described change of the formulation of the study medication (from chewing tablet to softgel capsule). Prior to the submission of this substantial amendment, this topic was discussed with the BfArM during a scientific advice meeting on May 22nd, 2014. The following table summarizes the regulatory activities (Table 1-WP2):
Table 1-WP2: Summary of regulatory activities.
Date Regulatory activity Status
19 February 2014 Modification of patient information Approved
30 April 2014 Substantial amendment No 1: Modification of study protocol to increase patient recruitment Approved
29 August 2014 Substantial amendment No 2: Modification of study protocol to increase patient recruitment Approved
10 October 2014 Extension of stability of study medication Approved
14 January 2015 Development safety update report (DSUR) No 1 Accepted
21 May 2015 Substantial amendment No 3: change of formulation of study medication and sample size Approved
26 June 2015 Notification of study interruption Accepted

Regarding data management parallel to continuously providing the electronic data capture platform three amendments to the eCRF were implemented (July 2014, October 2014, and May 2015) in relation to the clinical trial protocol amendments.
2. Patient recruitment
After the initiation of the clinical trial at MHH on December 16th, 2013, patient screening and recruitment started. So far, 722 patients were screened, from which 51 were recruited to the trial (Table 2-WP2).
Table 2-WP2: Summary of recruitment per month.
(* stop of recruitment due to lack of study medication)
Date Recruitment Date Recruitment
1/2015 5
1/2014 0 2/2015 5
2/2014 0 3/2015 3
3/2014 1 4/2015 5
4/2014 4 5*-12/2015 0/month
5/2014 1 1-6/2016 0/month
8/2014 0 Total 51
9/2014 3
10/2014 7
11/2014 3
12/2014 3

Recruitment was initially slow and several modifications to the clinical trial protocol were implemented to facilitate the clinical trial procedures and to enable successful patient recruitment. In 2014, 33 patients (2.75/month) were recruited, in 2015 18 patients (4.5/month; January-April). Patients had to be supplied with the IMP for three month post cochlear implant surgery.
Several procedural changes of the patient recruitment processes were implemented as reported in the consolidated response to the external review carried out for PROHEARING in late 2014. The goal was initially to recruit between 5-10 patients per month. The clinical team was very active in screening every patient suitable for recruitment in terms of inclusion/exclusion criteria. As seen in table 2 above in 3 out of 4 months of 2015 5 patients were recruited. For May and June 2015 even more patients (8 per month) were identified during screening and expected to be recruited. The clinical team had implemented professional screening, recruitment and follow-up processes very efficiently. For the department head, Prof. Dr. Thomas Lenarz, a smooth recruitment process has been the highest priority within the department’s overall scientific program.
3. Patient follow up
Within the first three years of the project all 51 PROHEARING-patients were recruited. From project month 37 to 48 those patients were followed up, visiting our clinic one month after surgery for first fitting and 3, 6, 9 and 12 months after first fitting. During those study visits tests according to the study protocol (table 3) were performed.
Table 3-WP2: post implantation study visit procedures.

4 weeks post-op (first fitting)
Air-conducted hearing test and bone-conducted threshold
Speech tests in quiet and in noise
Technical check of the implant
Vitamin E blood level
Nijmegen cochlear implant patient quality of life questionnaire
3, 6, 9 and 12 months post- first fitting (follow up)
Speech tests in quiet and in noise
Air-conducted and bone-conducted threshold, (aided threshold )
Technical check of the implant
Only month 6 post FF
Vitamin E blood level
Only 3 and 12 months post FF
Nijmegen cochlear implant patient quality of life questionnaire
At each study visit hearing tests were performed to follow up the development of the residual hearing after surgery (table 4-WP2).
Table 4-WP2: hearing tests performed from 4 weeks post-op until 12 months post first-fitting
Test Condition
Pure tone, air conduction Unaided
Pure tone, air conduction Unaided-contralateral
Pure tone, bone conduction Unaided
Pure tone, bone conduction Unaided-contralateral
OLSA training
OLSA in noise EAS or ES + residual hearing
OLSA in noise ES and ipsilateral closed ear canal
OLSA in noise AS if activated
Warble ES (only 3 months post first fitting)
Warble AS if available (only 3 months post first fitting)
EAS: electric-acoustic stimulation; ES: electric stimulation; AS: acoustic stimulation
OLSA: Oldenburger Satztest/Oldenburger sentence test

4. Descriptive analysis
Descriptive analysis has been conducted on a preliminary data set (state: march 2016), which hasn´t been fully monitored. Therefore caution has to be taken in interpreting the descriptive results and drawing valid conclusions. A final report according ICH-E13 including a detailed analysis of the unblinded full data set will be available after finalizing the study. Table 5-WP2 gives an overview on the descriptive analysis taken. For details we refer to deliverable 2.6.

Table 5-WP2: List of Analysis.
Group Detail
1 Descriptive Analysis of Baseline a Sex
b Age
c Planned ear
d Planned length of electrode
e Treated ear
f Used electrode length
g Surgeon
2 General medical history a Rotatory Vertigo
b Familiar deafness
c Infectious disease
d Renal disease
e Kidney stones
f Cervical spine disease
g Iron-storage disease (thalassemia, hemochromatosis, sideroblastic anemia)
h Thyroid disease
i Pregnancy or lactation
j Lactose intolerance
k Heavy smoking (= 20 cigarettes per day)
3 Medical history of implanted ear a Hearing loss (implanted ear)
b Years since hearing loss in implanted ear occurred
c Years since hearing loss in implanted ear (categorized)
d Severity of hearing loss (implanted ear)
e Hearing aid (implanted ear)
f Years since hearing aid in implanted ear
g Years since hearing aid in implanted ear (categorized)
h Tinnitus (implanted ear)
i Years since tinnitus in implanted ear
j Years since tinnitus in implanted ear (categorized)
k If tinnitus, intensity (implanted ear)
l Previous ear operations (implanted ear)
m Years since operation in implanted ear
n Years since operation in implanted ear (categorized)
o If previous ear operation, what operation? (implanted ear)
4 Medical history of not implanted ear a Hearing loss (not implanted ear)
b Years since hearing loss in not implanted ear
c Years since hearing loss in not implanted ear (categorized)
d Severity of hearing loss (not implanted ear)
e Hearing aid (not implanted ear)
f Years since hearing aid in not implanted ear
g Years since hearing aid in not implanted ear (categorized)
h Tinnitus (not implanted ear)
i Years since tinnitus in not implanted ear
j Years since tinnitus in not implanted ear (categorized)
k If tinnitus, intensity (not implanted ear)
l Previous ear operations (not implanted ear)
m Years since operation in not implanted ear
n If previous ear operation, what operation? (not implanted ear)
5 Aetiology of implanted ear a Congenital
b Hereditary
c Ototoxic
d Meningitis
e Meningitis ototoxic
f Mastoiditis
g Otosclerosis
h Measles
i Mumps
j Viral
k Trauma
l Unknown
m Other
n If other, specification
6 Aetiology of not implanted ear a Congenital
b Hereditary
c Ototoxic
d Meningitis
e Meningitis ototoxic
f Mastoiditis
g Otosclerosis
h Measles
i Mumps
j Viral
k Trauma
l Unknown
m Other
n If other, specification
7 Descriptive analysis of residual hearing at baseline with imputed values if dezibel limit is reached
a air conducted audiometry at 125 Hz pre-operative with imputed values (105) if dezibel limit (95) is reached
b air conducted audiometry at 250 Hz pre-operative with imputed values (110) if dezibel limit (100) is reached
c air conducted audiometry at 500 and 750 Hz and 1, 1,5, 2, 3, 4, 6 kHz pre-operative with imputed values (120) if dezibel limit (110) is reached
d air conducted audiometry at 8 kHz pre-operative with imputed values (100) if dezibel limit (90) is reached
e bone conducted audiometry at 125 Hz pre-operative with imputed values (45) if dezibel limit (35) is reached
f bone conducted audiometry at 250 Hz pre-operative with imputed values (65) if dezibel limit (55) is reached
g bone conducted audiometry at 500 Hz pre-operative with imputed values (70) if dezibel limit (60) is reached
h bone conducted audiometry at 750 Hz pre-operative with imputed values (80) if dezibel limit (70) is reached
i bone conducted audiometry at 1 kHz pre-operative with imputed values (85) if dezibel limit (75) is reached
j bone conducted audiometry at 1,5, 2 and 3 kHz pre-operative with imputed values (90) if dezibel limit (80) is reached
k bone conducted audiometry at 4 kHz pre-operative with imputed values (80) if dezibel limit (70) is reached
l bone conducted audiometry at 6 kHz pre-operative with imputed values (75) if dezibel limit (65) is reached
m bone conducted audiometry at 8 kHz pre-operative with imputed values (70) if dezibel limit (60) is reached
8 Measurability of primary endpoint
a Measurability of air conducted audiometry at 500 Hz pre-operative and 3 months post first fitting
b Descriptive analysis of the primary endpoint without imputation of not measurable values
Change of residual hearing at 500 Hz air conducted audiometry 3 months post fitting compared to baseline
c Descriptive analysis of the primary endpoint with imputation of not measurable values
Change of residual hearing at 500 Hz air conducted audiometry 3 months post fitting compared to baseline

5. Safety data
A detailed safety report was submitted as deliverable 2.5. in project month 42. In short, up to date, eleven Serious Adverse Events (SAE) case reports with a total of fourteen SAE terms have been communicated to the sponsor of the PROHEARING trial. The number and type of SAEs that occurred within the PROHEARING trial are within the expected range of the study population. No clinically important new safety information has evolved from PROHEARING so far. Thus, the overall safety assessment as outlined in the study protocol has not changed. No changes to the PROHEARING protocol are deemed necessary for safety reasons.
6. Conclusion
Descriptive analysis of the overall ITT patient population (n=51) shows a mean hearing loss of 28.16 (±16.67) dB for 500 Hz air conducted audiometry at 3 months post first fitting compared to preoperatively hearing. Final data analysis will be reported at https://www.clinicaltrialsregister.eu/ EudraCT Number: 2012-005002-22). Work package 2 did sent all deliverables due within the 3rd reporting period. Unfortunately milestones MS 8 “Patient recruitment for Clinical Trial completed” and MS15 “Clinical proof of ACEMg mediated hearing preservation” are not fulfilled since recruitment had to be stopped in project month 34.

Work package 3.-
Partner 5 (KI) has addressed a set of tasks designed to investigate the efficacy of ACEMg as a food supplement to prevent or attenuate hearing impairment caused by ageing and acoustic overstimulation, or a combination thereof. The approach has been to use different animal models in order both to provide further experimental evidence to support future clinical studies, and to characterize mechanisms related to the protective effects. Two different mouse strains were used to mimic early onset of age-related hearing loss (ARHL) as well as late onset ARHL. The DBA/2J mice, which express hearing impairment at an early stage, were put on an ACEMg supplemented diet at four weeks of age and functionally monitored throughout the experiment (up to 14 weeks of age). Late onset ARHL was studied using the C57Bl/6J mouse strain. Also these animals were put on an ACEMg supplemented diet at four weeks of age and then monitored up to the 28th week age when hearing impairment is clearly observed). To test the possibly additive or synergistic effect of noise on ARHL, a subgroup of the C57Bl/6J mice was also exposed to acoustic overstimulation. Finally, the efficacy of different ACEMg dosages to prevent noise-induced hearing loss was tested in an animal model based on normal hearing rats (Sprague Dawley). The young adult rats were put on a diet supplemented with ACEMg and then, 10 days later, exposed to noise known to cause a permanent hearing loss of up to 35 dB. The animals were monitored for four weeks post exposure. To ascertain an appropriate uptake of the active components, blood samples taken at the end of the experimental sessions of the different models. For example, in the study of early onset ARHL (DBA/2J mice), the plasma levels of Vitamin C and Vitamin E were increased by 112% and 91%, respectively, clearly demonstrating that the animals responded to the food supplementation.
In the experiments designed to address early onset ARHL (DBA/2J mice; task 1) the animals were followed from 4 to 14 weeks of age up, during which time period hearing thresholds increased (i.e. indicating a progressing hearing loss) as expected. However, despite a significant increase in the plasma levels of at least vitamins C and E, hearing was not better in the animals on the supplemented diet compared to animals on control diet, which could suggest that the ACEMg supplement diet did not prevent early onset ARHL, at least in the DBA/2J strain. The results were somewhat unexpected and also contradictory to observations made by other partners and several explanations were discussed. One possible reason for the lack of an effect could have been that the ACEMg treatment, starting at 4 weeks of age, was initiated too late and that degenerative changes had already progressed too far. As four weeks of age if the minimum age for delivery of mice at the KI animal department, it was discussed to start feeding pregnant DBA/2J mice with ACEMg supplemented food. It was, however, decided to try this approach only when a positive ACEMg effect had been shown using the other animal models. Another possible cause of the negative findings could be changes occurring in the food due to the irradiation process prior to the experiments. The regulations at Karolinska Institutet require that all animal food supplies have to be irradiated before being brought in to the animal facility. To compensate for such a possible effect, it was decided to increase the concentration of vitamins A, C and E in the subsequent experiments. Thus, in order to prevent the risk of significant reduction of the final antioxidant concentration due to degradation, the vitamin concentrations were increased by 50% compared to the original formula.
To test the effects of ACEMg on subjects suffering from hearing loss starting later in life, C57Bl/6J mice expressing a late onset of ARHL were studied (task 2). These animals were studied for 24 weeks (4 to 28 weeks of age), during which time they were feed an ACEMg supplemented diet (or control diet). As acoustic overstimulation is expected to augment the progressive functional impairment related to the ageing process, the experiments were designed to test also for the effect of noise exposure. Subgroups of animals (with and without an ACEMg supplemented diet) were thus exposed to noise at the age of 5 weeks. As expected in this strain, hearing (auditory brainstem, ABR) thresholds increased over time. The noise exposed animals displayed increased (20-30 dB) thresholds after the exposure. There was, however, no significant difference between the ACEMg and control groups (neither for the exposed or non-exposed animals). Thus, the ACEMg supplemented diet did not prevent ARHL in strain used in the present experiment (using irradiated food). The control groups given a normal, i.e. a non-supplemented, diet were further studied in order to obtain additional information on the mechanisms underlying ARHL.
The effects of different ACEMg dosages were further tested in a rat model (Sprague Dawley; task 3). Normal hearing subjects were thus put on a supplemented diet (0.5X to 2X re. the original formula) for 10 days prior to noise exposure and until sacrifice. Controls included both rats given the same type of food without the ACEMg supplementation but also the regular (slightly different) rat diet used at the KI animal department. Functional tests were performed prior the noise exposure (4 kHz at 110 dB SPL for 4 hours) and at three time points post-exposure (24 hours, 1 week and 4 weeks). Again, plasma concentrations of vitamins C and E were significantly increased in animals on the ACEMg diet. At four weeks after the noise exposure, there was a remaining, permanent threshold shift of around 20 dB. There were, however, no statistical differences between any of the groups, suggesting no functional effect of ACEMg (irradiated food). As Partner 1 (UCLM) started to report positive effects of ACEMg in Work Package 4, it was hypothesized that the lack of protective effects of ACEMg on the progression of hearing loss in Work Package 3 was indeed caused by the irradiation procedure. Partner 1 and Partner 4 have jointly tried to address this issue by comparing irradiated and non-irradiated food (task 4). This work has been performed at UCLM (Partner 1) as the Spanish animal department presently does not require irradiation of animal food. In these experiments a rodent model of noise-induced hearing loss us used to test the otoprotective properties of a non-irradiated ACEMg supplemented diet by comparing with non-irradiated food. As it was shown that the ACEMg dose efficacy is narrow, the comparison was made at 2X and 2.5X for non-irradiated and irradiated ACEMg food, respectively. Rats were exposed to intense noise (118 dB SPL for 4 hours per day during 4 days). In animals that were fed a non-irradiated 2X ACEMg diet (starting 10 days before noise exposure and until the end of the noise exposure protocol) the threshold shift caused by noise exposure was significantly reduced. In contrast, in animals fed an irradiated 2.5X ACEMg diet there was no positive effect on the threshold shifts following noise exposure. Based on our results it is concluded that irradiation significantly reduces the otoprotective effects of ACEMg. One hypothesis, supported by some reports in the literature, is that the irradiation process may actually have resulted in the formation of free radicals in the chow. This would obviously eliminate the possible positive effect of the ACEMg diet. The finding itself is, however, very interesting as it implies that irradiation of food supplies, expected to improve the safety and extend the food shelf life by reducing or eliminating microorganisms and insects, could actually be harmful. It should be noted that food irradiation is used in several countries, and the method is approved by the FDA. No conclusive evidence has been presented that that food irradiation would cause any significant harm in humans.
The fact that vitamins do not appear to be degraded in the irradiated food, as indicated by the plasma level, does not necessarily mean that they are active as antioxidants. It is possible that the irradiation process impairs antioxidant efficacy or affects other compounds within the food in a negative way. One hypothesis, supported by some reports in the literature, is that the irradiation process may actually have resulted in the formation of free radicals in the chow. This would obviously eliminate possible positive effects of the ACEMg diet. Obviously, we can at this point only speculate on the underlying mechanisms but based on our results it is thus concluded that irradiation significantly reduces the otoprotective effects of ACEMg.

Work package 4.-
Partner 1 addressed tasks aimed at unravelling possible mechanisms of ACEMg protection against cellular stress in the inner ear. In a first step, a rat model of noise-induced hearing loss (NIHL), complementing those used by Partner 5, was optimized. For this purpose, young adult (3 months old) Wistar rats were exposed to broadband noise (0.5-32 kHz, 118 dB SPL) for 4 hours a day during 4 consecutive days. Animals were tested for brainstem auditory evoked potentials (auditory brainstem response, ABR) immediately prior to the exposure protocol and 1 day, four days and 10 days after completion of the noise exposure protocol. Either 1 day, 4 days or 10 days after completion of the noise stimulation protocol, in a group of animals cochlear tissues were subject to quantitative real time polymerase chain reaction (qRT-PCR) to test the expression timeline of genes related to antioxidation (genes for the antioxidant enzymes catalase, SOD-1 and GPx1) and to apoptotic death mechanisms (antiapoptotic: Bcl-2 and proapoptotic, Bax and Casp3). In another group from each time point, cochlear tissue from experimental animals was processed for immunocytochemical detection of antioxidant enzyme proteins with antibodies against catalase, SOD-1 or Bcl-2, using an enzyme-based detection system (avidin/byotinilated peroxidase method, ABC). Surface preparations were stained for actin with fluorescently (rhodamin) labelled phalloidin, in order to test loss of cell integrity in the auditory receptor.
This noise stimulation protocol leads to a permanent auditory threshold shift as a consequence of NIHL, giving rise to a consistent and reproducible model in the context of the needs of this project. In parallel to increased ABR thresholds, there is damage to the auditory receptor, with significant loss of receptor hair cells (mostly outer hair cells, OHC).
We have shown in this model that cellular and molecular mechanisms of NIHL include changes in the expression of antioxidant enzymes and apoptosis genes. From a mechanistic standpoint, NIHL involves increase in levels of antioxidant enzymes. Genes coding for three main antioxidant enzymes, catalase, GPx1 and SOD1, detected by RT-qPCR, are upregulated after noise exposure. Catalase is variably upregulated, at the limit of statistical significance, shortly after noise exposure (1 day), returning to normal expression values after 10 days. In contrast, GPx1 and SOD 1 are upregulated later, with expression values significantly higher than normal at 10 days after noise exposure.
Changes in the expression of the corresponding proteins are being analyzed by immunocytochemistry. Catalase immunoreactivity staining is increased in cells of the auditory receptor 1 day after noise exposure. Ten days after exposure, staining is less intense. Cells in the receptor lamina and primary sensory neurons in the spiral ganglion, both show transiently increased levels of catalase. These results may be interpreted as an attempt of the cell populations of the auditory receptor to restore the balance of free radicals, likely altered by noise overstimulation. Therefore, these results lend further support to the hypothesis that oxidative stress is involved in the intimate mechanism of NIHL, strengthening the way to the therapeutic exploitation of this mechanism.
In order to further explore mechanism of cell damage in NIHL, we tested apoptotic cell death in the auditory receptor after noise exposure. Results with qRT-PCR suggest noise-induced disregulation of relevant apoptotic genes. The details and implications of this finding in relation to otoprotection are currently being worked out.
The otoprotective mechanisms of ACEMg were tested in the experimental model outlined above. Using this model, in the context of task 4.1 of this work package, we have shown that oral ACEMg protects significantly against NIHL and relevant cellular and molecular mechanisms have been worked out.
Animal chowder supplemented with ACEMg was obtained according to needs through the participating partner HHSE, working with Harlan Laboratories. For our purposes, concentrations were Vitamin A: (as beta-carotene 20%) 2.1 g/kg of chow, Vitamin C: (as ascorbyl-2-plyphosphate 35%) 20.58 g/kg of chow, Vitamin E: (as DL-alpha tocopheryl acetate, 500 IU/g) 15.52 g/kg of chow, Mg++: (as magnesium citrate), 26.96 g/kg of chow. Concentrations correspond to 2X the concentration of a previously tested ACEMg Enhanced Rodent Diet.
Rats were fed “ad libitum” with supplemented chow starting 10 days prior to application of the noise stimulation protocol described above, continuing through the four days of noise exposure, up to a total of 15 days. Plasma concentrations (not shown) were tested. Matched controls were fed identically but they were not overstimulated with damaging noise. Noise-exposed animals and their controls were blinded to the experimenter.
Available results from ABRs, show a statistically significant shift in auditory thresholds towards lower, more physiological levels. Although a return to completely normal, control thresholds were not seen, threshold recovery was particularly notorious in the 2 to 4 kHz range This result lends support to the hypothesis of otoprotective mechanism attributable to the ACEMg supplement.
In the next step, we generated and tested an animal model (Wistar rat) of age-related hearing loss (ARHL). Results clearly indicate that the Wistar rat is a suitable, easily accesible animal model of ARHL. In rats 12-14 months old auditory thresholds are significantly increased relative to younger ages, with values of 61.88 ± 6.89 to 68.13 ± 8.84 dB at the low and high ends of the tested frequency spectrum, respectively. Such an increase is even more evident in rats aged 18-20 months, with auditory thresholds ranging rom 74.38 ± 2.77 to 77.50 ± 2.67 dB depending on frequency. Threshold shifts in the older rats range from 24.38 ± 1.93 to 33.75 ± 3.40 dB for the 12- to 14-month-old group and from 30.63 ± 5.14 to 46.25 ± 1.95 dB for the 18 to 20-month-old group, compared with 6-8 month old rats ). Threshold shifts are significantly different statistically among all age groups, supporting progressive on going degradation of hearing thresholds during the aging process. Such degradation is more pronounced for higher frequency thresholds, a characteristic of ARHL.
The next aim of our work in the context of task 4.2 was to test in our Wistar rat model of ARHL, whether repeated temporary auditory threshold shifts (TTS) produced by short duration, long-term noise overexposure, accelerates and worsens ARHL. For this, we generated and tested an animal model (Wistar rat) combining temporary threshold shift (TTS) and ARHL. The rationale behind this was threefold: i) reproduce in an animal model the association in humans between life-long exposure to environmental noise and ARHL, as the latter seldom or never appears alone, ii) shorten the duration of experimental approaches by accelerating ARHL, iii) set the foundations to analyze whether experimental ARHL combined with noise exposure could be reduced by an oral combination of the free radical scavengers vitamin A, C and E combined with vasodilator Mg++ (ACEMg). We report.that TTS accelerates ARHL. First of all, TTS caused after one session of a noise exposure protocol (white noise at an intensity of 110 dB SPL, starting at three months. Animals are exposed for 1 hour a day during 5 days, allowing 2 days for recovery) is characterized by a dramatic increase in auditory thresholds throughout the tested frequency range, recorded 1 day after the noise exposure protocol. Normal auditory thresholds ranging approximately from 43 to 35 dB SPL, depending on stimulus frequency shift to above 70 dB SPL the day after the beginning of the exposure. Three days after the end of the exposure, however, thresholds have returned to values not significantly different from those of Wistar rats with normal hearing.
The effects of persistent TTS starting at 3 months in auditory aging in the Wistar rat are that, in the beginning, TTS-exposed animals have, on average, auditory thresholds undistinguishable from those of rats of similar age not exposed to TTS noise, supporting initial reversibility of noise damage at young ages. At 6-8 months of age, however, whereas average threshold shifts in normal animals are similar to those at 3 months, in the TTS rats there is a already a clear cut increase in auditory thresholds. Average threshold shifts after TTS in 6-8 month-old rats, following the short-duration, long-term noise overstimulation are much higher than in rats of the same age, not exposed to TTS . At 12-14 months, average threshold values are significantly higher in both non- TTS exposed and in TTS-exposed rats, strongly. Average thresholds in noise-exposed animals are significantly higher than in non-exposed animals (Fig. 5B, D), suggesting acceleration and potentiation of ARHL by noise producing TTS.
As a conclusion to the grant commitment expressed in task 4.2 we then tested otoprotection from ARHL and the combined effects of noise producing TTS and ARHL by vitamins A, C, E and Mg++ (ACEMg). We tested otoprotective effects of ACEMg in rats with ARHL accelerated by noise exposure, according to the animal models described above, and possible mechanisms. At 3 months of age, rats with normal hearing thresholds determined by auditory evoked potentials recordings (ABR) are randomly assigned to treatment (“Enhanced Diet”, ED group) or control groups (“Normal Diet”, ND group). Test treatment consists of free access to chow appropriately supplemented with vitamin A, C, E and Mg++. (ACEMg), according to dosage previously determined. Control treatment is regular rat chow. Treatment starts 7 days before the first noise exposure and is continued throughout the duration of the experiments. ABRs are performed during and after treatment, and the inner ears and brains from experimental animals processed for quantitative real time PCR and immunohistochemistry for oxidative stress and inflammation and apoptosis markers (see D4.1).
Rats treated orally with ACEMg in the daily chow (ED) have ABR recordings closer to normal than those fed with regular chow (ND). Their auditory thresholds improved. Although thresholds following accelerated ARHL are still higher both in ND and ED rats at 6-8 and 12-14 months than in 3 month-old controls, thresholds in ED animals are lower, particularly in the low to mid frequency range. Compared to controls, threshold shift in the ND rats ranged from 16.25 ± 2.27 to 29.38 ± 3.05 dB at 6-8 months and from 25.00 ± 2.04 to 40.00 ± 2.04 dB at 12-14 months (Fig. 6C). Threshold shift in the ED rats ranged from 7.50 ± 3.13 to 28.88±2.82 dB at 6-8 months and from 17.50 ± 4.33 to 37.50 ± 3.23 dB at 12-14 months of age (Fig. 6D).
As reported in detail in interim reports, ACEMg protects against permanent noise induced hearing los (NIHL) in the Wistar rat and other species. We have shown that this correlates, among others, with dynamic changes in the expression of antioxidant enzyme and apoptosis genes in the organ of Corti. One day after completing a PTS noise exposure protocol, gene expression levels for antioxidant enzymes in the organ of Corti, measured by quantitative RT-PCR, in particular catalase (Cat) and glutathione peroxidase (GPx1), are more elevated in animals treated orally with ACEMg (Enriched Diet, ED) than in untreated, noise-exposed control animals. Actually, GPx1 and Cat gene expression levels at one day after finishing noise exposure resemble more high expression levels detected 10 days after noise exposure in untreated animals. We speculate that an antioxidant response naturally triggered by damaging noise exposure, suggested by elevated enzyme gene expression levels at 10 days after noise exposure in control untreated animals, is put much forward in time by oral ACEMg treatment, with elevated Cat and GPx1 gene expression levels in treated animals one day after noise exposure comparable to those found in untreated animals 10 days following noise exposure. At this time after noise-exposure in animals treated with ACEMg, increased expression levels of Cat and SOD1 are comparable to those found in untreated animals 10 days after noise exposure, whereas Gpx1 expression levels in ACEMg treated animals fall to baseline values. In parallel with this, changes in the expression of apoptosis genes in the organ of Corti also take place with ACEMg treatment. Whereas expression levels of proapoptotic genes (Bax and Caspase 3, Casp 3) are not significantly affected by treatment, the antiapoptotic Bcl-2 gene increases its expression in ACEMg treated animals at 10 days after noise-exposure almost twofold relative to levels in untreated animals. These changes in gene expression correlate with changes in the intensity of immunolabeling for the corresponding proteins. These changes may be part of the protective mechanism triggered by antioxidants. We are now analyzing whether these or other potentially protective cellular responses to ACEMg are present in ARHL in combination with TTS. Once tissue analysis is completed we will readily report on this aspect.
In summary, foreground knowledge on the effects and mechanisms of ACEMg otoprotection against the combined effects of noise and ARHL have been gained through the completion of tasks 4.1 and 4.2. We believe that these results show success of ProHearing WP4 and its commitment of providing knowledge of translational value towards the final goals of ProHearing. The final stages of the analysis of results are being completed and new scientific publications are in progress.
Work package 5.-
WP5 deliverables are dependent on the completion of tasks from other Work Packages. As stated for each WP5 deliverable, they will be completed after the WP2, WP3 and WP4 deliverables are completed, to accomplish the following:
• Pre- and post-operative hearing conservation therapy for cochlear implant
patients is dependent on WP2.
• Lifetime hearing conservation therapy with appropriate dosing for age and stress is dependent on WP3.
• New products to treat hearing impairment are dependent on WP4.
WP2 deliverables have not been completed and WP3 deliverables were almost completed. WP4 deliverables should provide significant justification for modifying WP5 deliverables. Therefore, WP5 deliverables will be completed after the WP2, WP3 and WP4 deliverables are completed, to accomplish the items listed above.
Significantly, it is expected that the deviations should enable some profitable commercial revenue from sales of ACEMg to address the ARHL and NIHL markets to be allocated to fund continued research, including WP2 and WP3 research.

Potential Impact:

For the potential impact of the results, see “Description of the main S&T results/foreground” under “Work Package 5”.
Regarding dissemination activities, they were aimed at two targets, namely the scientific community in the field of hearing research and neuroscience and the general public/civil society. Scientific results were initially presented at national and international meetings, notably the Association for Research in Otolaryngology meetings in the years 2014 to 2016. The numerous abstracts corresponding to presentations at meetings are not listed for the sake of brevity. Presentations at scientific meetings have resulted to date in 11 scientific publications in specialized peer reviewed journals. Several other publications are currently undergoing review or the manuscripts being prepared for submission. One book was published in direct connection with the project, containing state-of-the-art, fully updated information on free radicals and antioxidation mechanisms in the ear. The book, with contributions from researchers participating in ProHearing will become a very useful reference in the field. As far as dissemination among the general public is concerned, reports in national and local newspapers as well as press releases from participating institutions, published in paper or electronic format, have drawn attention on the relevance of the research, reaching a considerably wide audience.
As far as exploitation of results is concerned, two patents have been filed in the U.S. as EU regulations do not allow protection of the type of IP generated through ProHearing in Europe. One patent protects a method to treat noise-induced hearing loss, whereas a second one protects a method to treat age-related hearing loss.

List of Websites:
www.pro-hearing.eu
Coordinator; JoseManuel.Juiz@uclm.es
Jose M. Juiz MD, PhD
Universidad de Castilla-La Mancha
Instituto de Investigación en Discapacidades Neurológicas-IDINE. Campus Albacete