Research in Hearing Protection

Oricula Therapeutics evolved from converging research into hearing loss and compounds that have potential to prevent it. Oricula’s scientists, working at the University of Washington, have studied compounds causing hearing loss in humans.

Understanding antibiotic-induced hearing loss 

It has been long understood that the aminoglyosides class of antibiotics induce hearing loss by causing the death of sound-sensing mechanosensory hair cells in the inner ear; howver, the exact mechanism of this toxicity is not known. The inner ear of mammals is located in the hardest bone of the body and it is extremely difficult to directly observe the effects of various chemicals on the hair cells of the cochlea. Therefore, our early work focused on the lateral line hair cells of zebrafish.

Work by us and many colleagues worldwide has shown that zebrafish hair cells have many properties in common with the inner ear hair cells of humans and other mammals. We have shown that they share common genes with mammals and are affected by the same chemical compounds. Since the zebrafish lateral line hair cells are on the outside of the fish, they are exposed to the chemical milieu of the swimming fish. Also, using a variety of flourescent dyes, the health of the hair cells is directly observable.

Identifying compounds with potential to protect hair cells

Using zebrafish, we we able to accomplish two key things: demonstrate that known ototoxic chemicals killed lateral line hair cells and identify compounds that protected hair cells from damage. After screening libraries of thousands of compounds, we identified one (we called ORC-0001) that was effective at protecting zebrafish lateral hair cells from the toxic effects of aminoglycosides (see Zebrafish screening).

Testing in mammals

The next step in creating a drug that could protect human hearing from the toxicity of aminoglycosides was to show that our compound (ORC-0001) was effective in a mammalian model. We chose to test this in rats using an assay called auditory brainstem response (ABR). ABR is used both clinically in humans and in animals as a way to determine the threshold sound intensity necessary to produce a response in the brain using sound of varying frequencies. Treatment of rats for ten days with kanamycin, amikacin, or other aminoglycosides causes a loss of hearing as demonstrated by higher thresholds to evoke an ABR response. Morphologically, we were also able to show that the aminoglycoside treatment caused hair cell death in the cochlea of the rats. Concurrently administered ORC-0001 was effective at eliminating most of the hearing loss and hair cell death (see Mammalian Efficacy.)

Protecting the findings

Patents for Method of Use and Composition of Matter are exclusively licensed from University of Washington by Oricula Therapeutics.

Zebrafish: A Novel Screening Method


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Zebrafish: Counting hair cells

Work by us and colleagues worldwide has shown that zebrafish hair cells have many properties in common with the inner ear hair cells of humans and other mammals. We have shown that they express common genes and are affected by the same chemical compounds as mammals. Since the zebrafish lateral line hair cells are on the outside of the fish, they are readily accessible to visualization and manipulation. Using a variety of fluorescent dyes, the health of hair cells is directly observable. The pictures (left) show a normal zebrafish with the fluorescent dye staining the hair cells of the lateral line. Normal hair cells appear in stereotyped locations. Treatment with chemicals that kill hair prevents fluorescent staining. Here, a five-day-old zebrafish larva is exposed to a concentration of 200 micromolar neomycin and exhibits almost total loss of hair cells.


Screening hair cell reaction to chemicals

Five-day-old zebrafish larvae can be placed in wells of a 96-well microplate. Each well contains normal zebrafish media, with additional compounds to test for potential toxic damage. In this way, hundreds or thousands of chemicals can be screened in a short time. Our early work demonstrated many important findings:

  • Low False Negative Rate: Screening libraries of FDA-approved drugs revealed that most known ototoxants caused hair cell death.

  • Low False Positive Rate: Screening libraries of FDA-approved drugs identified only a few drugs that were not known to be ototoxins caused hair cell death; however, many of these had case reports of hearing loss side effects.

  • Dose Response Curve: Known ototoxins, such as aminoglycosides antibiotics, showed a clear dose response curve of increasing hair cell death with increased dose of toxant.

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Hair cells exposed to aminoglycosides

In the graph to the left, hair cell survival is plotted against the concentration of neomycin. Five-day-old zebrafish larvae are exposed to neomycin, and after one hour of incubation, removed, washed, and stained to count the surviving hair cells. The variability of the assay, shown by the standard deviation (SD), is very low.


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Identifying hair cell protectants

Using the same techniques, it is possible to assess the combination of a hair cell toxin (neomycin) with a chemical to protect hair cells against the toxin. We screened a library of more than 10,000 chemicals looking for any that would protect the hair cells. Hits were examined in great detail with a full dose response curve to determine the range of dose that was effective and the HC50 (the dose that protected 50 percent of the hair cells). We also examined the effectiveness of the protectant against a range of doses of the toxin. The graph (left) shows the result of using various concentrations of our best protectant to protect against the full range of effective neomycin concentrations. The compound ORC-0001 (aka BPN-0001) was the most effective of the 10,000 chemicals in protecting zebrafish hair cells exposed to neomycin.

Mammalian Efficacy


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ABR testing in rats

We used the Auditory Brainstem Response (ABR) assay in rats to show that ORC-0001 will prevent hearing damage in mammals. The ABR measures inner ear and brain electrical potentials using surface electrodes in response to sound stimuli of different frequencies and intensities. The ABR assay is the same as that used in non-human primates and humans in the lab or the clinic and correlates highly with audiometric results. These measures could be used for evaluating Phase II clinical trials and will likely serve as a FDA-approved endpoint for product registration.

Chronic treatment of rats with aminoglycoside antibiotics, such as kanamycin or amikacin, results in an elevation of ABR thresholds, typically most pronounced at higher frequencies. Co-administration of ORC-0001 led to robust protection across all frequencies tested. Subsequently, we tested the lead compounds coming out for the SAR program to determine which were effective in protecting hearing in rats and which had the greatest therapeutic index. ORC-13661 was selected as the most effective at protecting hearing, demonstrating 100 percent hearing protection in rats treated with subcutaneous amikacin at a dose of 320 mg/kg/day for ten days. (This data has not yet been published in a refereed journal.)  Early safety and toxicology tests show it to have a wide therapeutic index.