Background
Hearing loss is the third leading chronic disorder and exceeds the number of persons with either diabetes or visual loss combined. Unfortunately, the existing strategies for hearing loss reduction are inadequate. In the U.S., the incidence of hearing loss is increasing and affects over 30% of the general population. The onset begins in children and young adults and can be induced by loud sounds, drugs, or infections. In addition, hearing loss is often progressive leading to greater disability year after year.

We are born with a relatively small number of auditory hair cells (about 15,000 per cochlea) and these have to last a lifetime. The very low number of auditory hair cells present in the cochlea is one of the major reasons hearing loss is so common. Once the cells in a particular area of the cochlea are lost, hearing at that frequency is also lost. There is little in the way of compensatory hearing offered by neighboring hair cells as they are tuned to respond to a particular frequency. It is the irreversible injury or loss of auditory hair cells, supporting cells, and neurons that cause the majority of hearing loss.

The following table indicates maximal Sound Pressure Levels (SPLs), durations and sources of sound before the inner ear is at risk of permanent hearing loss:

SPL	Duration	Source	Industry
140 dB	< 1 min	firearms, jet planes	military
130 dB	> 1 min	jackhammers	construction/mining
120 dB	> 5 min	amplified car stereo	recreational
110 db	> 15 min	rock concerts, planes	musicians
100 dB	> 1 hr	woodshops, chainsaws	logging
90 dB	> 4 hr	motorcycles, lawnmowers	recreational
85 dB	> 8 hr	interior plane cabins	aviation

Biology of hair cell death
Irreversible hair cell death is elicited by metabolic or biochemical changes in the hair cells that involve reactive oxygen species or ROS. This process is illustrated in the following figure which appeared in a book chapter, co-authored by Dr. Kil.

Noise and drug-induced hair cell injury and death.

A. Shows the formation of damaging ROS such superoxide (O2- ) and hydroxyl anion (OH-).

B. Shows some of the internal antioxidant mechanisms that hair cells employ to control ROS induced damage including reduced glutathione and antioxidant enzymes.

C. ROS can irreversibly damage cell membranes, mitochondria, nuclear DNA and proteins and signal the cell to die through apoptosis.

SPI is focused on the development of drugs to prevent hearing loss and restore hearing in our three product pipelines: Otoprotection, Chemoprotection, and Regeneration.


Otoprotection
Otoprotection studies in animals by a number of established academic labs have validated that the inner ear can be protected from the irreversible effects of noise damage by the systemic administration of pharmacologic agents or drugs. SPI-1005, a proprietary oral formulation of ebselen, is a small molecule mimic and inducer of glutathione peroxidase (GPx). GPx is the dominant catalytic antioxidant enzyme in the cochlea and is critical for hearing health. GPx reduces reactive oxygen species (ROS) by the binding of free radicals to its Selenium moiety. By reacting with glutathione, GPx limits free radical toxicity, exhibiting strong activity against peroxynitrite (ONOO-). In addition, ebselen has been shown to reduce cytochrome-C release from mitochondria and nuclear damage during lipid peroxidation, attenuating neuronal apoptosis associated with oxidative stress.

Our published results with SPI-1005 (Lynch et al., 2004, 2005, Kil et al., 2007) have been replicated and extended by independent researchers (Pourbahkt et al., 2003, Yamasoba et al., 2005, Park et al., 2006). SPI-1005 has been evaluated in Phase I studies and found to have a favorable toxicity and pharmacokinetic profile. Sound Pharmaceuticals is now testing SPI-1005 in Phase II trials with the Navy/Marine Corps.


Chemoprotection
Several classes of therapeutic agents including cancer chemotherapy drugs and aminoglycoside antibiotics can cause oxidative stress in the inner ear resulting in irreversible cell death. In particular, the platinum containing chemotherapies (cisplatin, carboplatin) are well noted and studied for their ototoxicity.

Hearing loss due to ototoxic medications is sensorineural and usually presents initially at the high frequencies and subsequently progresses to the lower frequencies. Impaired hearing as a result of ototoxicity can be temporary, but usually is permanent. The incidence of cisplatin or carboplatin-induced hearing loss ranges up to 62% in early clinical reports and as high as 84-100% in pediatric patients. Clinical symptoms of ototoxicity are associated with hearing loss, tinnitus, and difficulty understanding speech.

Our lead product candidate, SPI-3005, ebselen has been shown to protect against chemotherapy induced hearing loss and neurotoxicity in several animal studies (Lynch et al., 2005a). Significant chemoprotection was achieved without interfering with the tumor activity of the cisplatin in rat and mouse tumor models (Lynch et al., 2005b). Sound Pharmaceuticals has recently received FDA notification of allowance to proceed with Phase II clinical testing of a proprietary oral formulation of ebselen, in cancer patients receiving cisplatin or carboplatin as part of their chemotherapy. SPI expects to develop its proprietary formulations into oral and injectable drugs to protect against drug induced hearing loss.


Regeneration
For those patients who already have substantial hearing loss, SPI is developing drugs aimed to restore hearing. SPI is currently optimizing compounds that antagonize specific cell cycle proteins resulting in new cell division or proliferation. In mice deficient in p27Kip1, a growth inhibitory protein, normally non-dividing epithelial cells within the inner ear are now able to divide. More importantly, these newly dividing cells have the capacity to become replacement auditory hair cells, supporting cells, and neurons.

National Institute's Director of Deafness and other Communication Disorders commends our novel efforts in sensorineural regeneration to the US Congress...
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Validation of p27Kip1 as a target for inducing supporting cell proliferation and hair cell regeneration in the organ of Corti was further substantiated by two independent research teams. One lead by Dr. Kil while at the University of Washington and Otogene, the other by Dr. Neil Segil at the House Ear Institute in Los Angeles, CA.

Hair cell regeneration in the organ of Corti of p27 -/- and +/- mice. Viewed as a whole mount (A) or cross section (B) shows OHCs and IHCs and the intervening tunnel of Corti (*). (C, E, G) Proliferating cells were identified using BrdU immunoperoxidase. (C) p27-/- has BrdU+ cells, while (E) p27+/- and (G) p27+/+ show absolutely no proliferation. (D, F, H) Treated with amikacin an aminoglycoside antibiotic that kills hair cells and then stained with BrdU immunoperoxidase. (D) p27-/- has multiple BrdU+ cells including three BrdU+ Dieter’s cells (arrow heads) and one BrdU+ IHC (arrow). (F) p27+/- has BrdU+ cells after amikacin treatment indicating that ototoxic drugs induce cell proliferation and regeneration when p27 is decreased. Note two BrdU+ OHCs (arrows) and a single BrdU-positive supporting cell (arrowhead). (H) p27+/+ is completely devoid of BrdU+ cells following amikacin treatment. Note two dead OHCs (arrows) and three BrdU- supporting cells (arrowheads).

See the paper describing the cochlear phenotype in the p27 knockout as published in the Proceedings of the National Academy of Sciences:

Gene disruption of p27Kip1 allows cell proliferation in the postnatal and adult organ of Corti

SPI has developed and is now optimizing RNAi based antagonists of p27 in a proprietary formulation that is capable of inducing hair cell regeneration within the damaged mammalian cochlea. Our current focus is to continue to optimize the delivery and proliferative effect of formulated p27 RNAi.


Hair cell regeneration in the organ of Corti of p27 -/- and +/- mice.

Viewed as a whole mount (a) or cross section (b) shows Outer Hair Cells and Inner Hair Cells and the intervening tunnel of Corti (*). (c, e, g) Proliferating cells were identified using antibody detection of the DNA proliferation marker Bromo-deoxyUridine (BrdU). (c) p27-/- has BrdU+ cells, while (e) p27+/- and (g) p27+/+ show no proliferation. (d, f, h) Treated with amikacin an aminoglycoside antibiotic that kills hair cells and then stained for the presence of BrdU. (d) p27-/- has multiple BrdU+ cells including three BrdU+ Dieter’s cells (arrow heads) and one BrdU+ Inner Hair Cell (arrow). (f) p27+/- has BrdU+ cells after amikacin treatment indicating that ototoxic drugs induce cell proliferation and regeneration when p27 is decreased. Note two BrdU+ Outer Hair Cells (arrows) and a single BrdU-positive supporting cell (arrowhead). (h) p27+/+ is completely devoid of BrdU+ cells following amikacin treatment. Note two dead OHCs (arrows) and three BrdU- supporting cells (arrowheads).