MATERIAL AND METHODS
Chemicals
Dexamethasone (micronized) and poloxamer 407 NF were purchased from Spectrum Chemicals.
Acepromazine, ketamine and xylazine were from MWI Veterinary. OTO-104 consisted of a sterile
suspension containing dexamethasone in 16% poloxamer 407 manufactured under GMP conditions.
Animal Studies
All animal studies were conducted in accordance with the policies and recommendations of the United
States Department of Agriculture, the National Institute of Health guidelines for the handling and use of
laboratory animals and received approval from the Institutional Animal Care and Use Committees of
Otonomy Inc and MPI Research Inc.
Pharmacokinetic studies in guinea pigs
Female guinea pigs (Charles River) weighing 200-300g, of approximately 6-8 weeks of age were used (N
= 4 per time point). Prior to any procedure, animals were anesthetized using a combination of xylazine
(10 mg/kg), ketamine (40 mg/kg) and acepromazine (0.75 mg/kg) for up to an hour via the intramuscular
route. If needed, an intraoperative booster was administered intraperitoneally representing one-tenth
of the original dose.
Intratympanic injection – Each animal was positioned so that the head was tilted at an angle to favor
injection towards the round window niche. Briefly, under visualization with an operating microscope, 50
µl of the formulation was injected using a 27G or 30G needle through the tympanic membrane into the
superior posterior quadrant behind which the round window niche is located. Using this approach, the
formulation specifically targeted the round window membrane and minimized the presence of the gel in
other areas. During the procedure and until recovery, animals were placed on a temperature controlled
(40 ⁰C) heating pad. After consciousness was regained, animals were returned to the vivarium.
Perilymph sampling procedure - The skin behind the ear of anesthetized guinea pigs was shaved and
disinfected with povidone-iodine. An incision was then made behind the ear, and muscles were carefully
retracted from over the bulla. A hole was drilled though the bulla using a dental burr so that the middle
ear was exposed and accessible. The cochlea and the round window membrane were visualized under a
stereo surgical microscope. Mucosal tissues of the cochlea were removed and the area washed carefully
to remove any potential drug contaminants. A unique microhole was hand drilled through the bony shell
of the cochlea (otic capsule) adjacent to the round window. Perilymph (5 µl) was then collected using a
microcapillary inserted into the cochlear scala tympani.
Pharmacokinetic studies in sheep
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Female sheep (Buckham Sheep Farm, Kalamazoo, MI) weighing 50-65kg, of approximately 2-4 years of
age were used (N=1, 2 ears per time point). Prior to any procedure, animals were anesthetized using a
combination of xylazine (0.22 mg/kg), glycopyrrolate (0.01 mg/kg) and ketamine (15 mg/kg)
administered IM in addition to isoflurane by inhalation.
Intratympanic injection – Each intubated animal was immobilized and placed laterally in reverse
trendelenburg position, with the rostrum slightly elevated to ensure access to the round window.
Following ear cleaning (using Otocalm and warm saline), and under otoscopic visualization, 600 µl of the
formulation was injected using a 25G or 27G needle through the tympanic membrane into the posterior
inferior quadrant towards the round window niche. After dosing, the animal was left on an incline with
its head up for approximately 30 min to allow the dosing solution to settle into the tympanic cavity.
Procedure was then repeated for the opposite ear.
Perilymph sampling procedure – The animal was intubated and placed in lateral recumbency. A postauricular skin incision was made and the post-auricular vein located and ligated. Cautery was performed
to expose the bulla and temporal bone. The middle ear was accessed using a nitrogen powered drill and
a round-tipped bur. The middle ear ossicles were pushed to the side, with care taken to avoid damaging
the round window membrane. Mucosal tissues of the cochlea were removed and the area washed
carefully to remove any potential drug contaminants. Using a 0.5-1 mm round-tipped burr, a hole was
hand drilled into the basal turn of the cochlea until the bone was thin enough to pierce with a modified
sewing needle. Perilymph (50 µl) was then collected using Hamilton syringe connected to a 28-32G
needle inserted into the cochlear scala tympani.
Acute ototoxicity
Male (n=5 per group) and female (n=5 per group) guinea pigs weighing 200-300g, of approximately 6-8
weeks of age were used. Prior to any procedure, animals were anesthetized using a combination of
xylazine (2.5 or 5 mg/kg) and ketamine (75 mg/kg) for up to an hour via the intramuscular route.
Anesthesia was maintained via isoflurane inhalation during the intratympanic injection procedures.
Intratympanic injection – All animals were injected bilaterally. Each animal was positioned so that the
head was tilted at an angle to favor injection towards the round window niche. Briefly, under
visualization with an operating microscope, 50 µl of the formulation was injected using a 27G or 30G
needle through the tympanic membrane into the superior posterior quadrant behind which the round
window niche is located. During the procedure and until recovery, animals were placed on a
temperature controlled (40 ⁰C) heating pad. After consciousness was regained, animals were returned
to the vivarium.
Auditory Brainstem Responses (ABR) assessment – During the procedure, additional anesthetic (xylazine
and ketamine) was administered if needed to maintain anesthesia depth sufficient to insure
immobilization and relaxation. ABRs were recorded in an electrically and acoustically shielded chamber.
Needle electrodes were placed at the vertex (active) and immediately below the pinna of the test ear
(reference) and contralateral ear (ground). Tucker Davis Technologies (TDT) System III hardware and
SigGen/BioSig software (TDT, Alachua, FL USA) were used to present the stimulus and record the ABR
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responses. Tones were delivered through a Tucker-Davis EC1 driver (aluminum-shielded enclosure made
in house), with the speculum placed just inside the tragus. Acoustic calibration was performed with TDT
software (SigCal) and thresholds are expressed as dB SPL. Stimulus presentation (15 ms tone bursts, with
1 ms rise/fall times) were presented 10 per second. Up to 1024 responses were averaged for each
stimulus level. Responses were collected for stimulus levels in 10 dB steps at higher stimulus levels, with
additional 5 dB steps near threshold. Thresholds were interpolated between the lowest stimulus level
where a response was observed, and 5 dB lower, where no response was observed.
Middle ear assessment – At necropsy, a visual examination of the middle ear was conducted using a
dissecting microscope. The middle ear was opened by removing regions of the temporal bone in order
to reveal the ossicles. A forceps was used to gently apply pressure to the ossicles and mobility was
noted. The presence of fibrous tissue, edema /swelling and erythema (discrete reddened areas with or
without presence of fluids) was scored as follows: (0) not present, (1) minimal, (2) moderate, or (3)
severe.
Plasma collection method - Blood samples (1ml) were collected via the orbital sinus into heparin coated
tubes. No fasting was conducted prior to collection.
Acute dermal toxicity (local tolerance)
Male (n=5) and female (n=5) guinea pigs weighing 200-300g, of approximately 6-8 weeks of age were
used. On the day prior to dosing, the hair from no less than 15% of the total body surface area (BSA) was
clipped from the back of the animal. It was determined by that 1.0 ml of the test article was sufficient to
cover an area of approximately 40 cm2. The following formula was used to calculate the area:
BSA2 = 9*W2/3
where “A” was the estimated area in square centimeters and “W” was the body weight in grams. 20%
OTO-104 was applied by gentle inunction to the animals back with a glass stirring rod, and distributed
evenly over an area starting at the scapular region and continuing caudally over the test site. The test
article was then held in contact with the skin by gauze dressing and nonirritating tape. Following a 24 h
exposure period, the dressing was removed. Observations for clinical signs (morbidity, mortality and
injury) were conducted at approximately 30 min, 2 and 4 h post-dose and once daily for the remainder
of the study. The test site was evaluated at each clinical observation for the presence of reddened areas.
Skin sensitization by Buehler method (Delayed hypersensitivity)
Male and female guinea pigs weighing 200-300g, of approximately 6-8 weeks of age were used. The
vehicle group (n=5 per gender) was administered 0% OTO-104 during the induction phase, and 20%
OTO-104 during the challenge phase. The positive control group (n=5 per gender) received Hexyl
Cinnamic Acid (HCA) during both induction and challenge periods. The test article group (n=10 per
group) received 20% OTO-104 during the induction and challenge phases.
Induction phase - At least 24 h prior to dosing, the left shoulder surface area was shaved from each
animal in an amount to equal approximately one-fourth of the animal’s back and side. On each day of
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dosing (day 1, 8, and 15), each animal received one application to the same shaved left shoulder region.
The amount (0.4 ml) of the vehicle (0% OTO-104), positive control (HCA), or 20% OTO-104 formulation
was placed into a Hill Top Chamber and was secured with 2 to 3 inch 3M Health Care tape, covered with
clear plastic wrap that was folded in thirds, wound around the torso of each animal, and secured with
Vetrap tape. The chamber was then considered applied and the time was documented. After at least 6 h
of exposure, the tape and chambers were removed and the application sites were washed with a Wypall
and tepid tap water. After the last induction, the animals were left untreated for 2 weeks until the
challenge phase.
Challenge phase - All animals exposed during the induction phase were challenged 2 weeks after the last
induction exposure. Approximately 24 h prior to dosing, the left flank surface of the side and back of
each animal was shaved. On the day of dosing (day 29), each animal received one application to the
shaved left flank region. The formulation (0.4 mL) was placed into a Hill Top Chamber and was secured
with 2 to 3 inch 3M Health Care tape, covered with clear plastic wrap that was folded in thirds and
wound around the torso of each animal, and secured with Vetrap tape. At this point, the chamber was
considered applied and the time was documented. After at least 6 h of exposure, the tape and chambers
were removed and the application sites were washed with a Wypall and tepid tap water.
Sensitization scoring - Each site was scored for irritation at approximately 24 and 48 h after patch
removal. At least 8 h prior to the initial skin evaluation, all animals were depilated. The scoring scale was
as follows: (0) no visible change, (0.5) very faint erythema usually patchy, (1) faint erythema usually
confluent, (2) moderate erythema and (3) severe erythema with or without edema. To interpret the
responses, a severity index (SI) and a sensitization index (SII) were developed.
SI = Sum of grades at interval / total number of animals
SII = (number of animals showing a positive response at 24-48h / total number of animals) * 100
Histology
Guinea pigs received vascular perfusion through the heart with an isotonic saline solution followed by
fixative containing 4% paraformaldehyde (PFA) in phosphate buffer. Left and right temporal bones were
removed and trimmed. Cochleae of the left ear were fixed by intrascalar perfusion with 4% PFA. The
temporal bones were then placed in 4% pPFA for approximately 1 hour and transferred to a vial
containing 0.5% PFA in phosophate buffer. The right temporal bones were trimmed, and then each was
placed in a cassette containing of 5% EDTA in phosphate buffer. The cassettes were placed into a PELCO
3451 Microwave System, running constantly for 3-5 days. Ears were then dehydrated in EtOH and
processed in JB-4 Glycol Methacrylate resin, placed into molds containing the resin and polymerized at
~4 ⁰C for ~12h. Blocks containing cochleae were trimmed and five micrometer sections were cut with a
Leica RM2165 Microtome and stained with Paragon stain. Sections were examined under bright field
optics. Middle ear sections were assessed for tissue reaction and inflammatory response. Mild tissue
reaction was defined as small increases in the thickness of the tissue associated with mucosal linings
with some possible vesiculation. Mild – moderate tissue reaction was defined as small regions of new
tissue growth or inflammatory response. Moderate tissue reaction was defined as larger tissue growth
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often associated with an inflammatory response. Moderate to large tissue reaction was defined as
several regions with large amounts of new tissue growth and cells associated with inflammatory
response. Large response was defined as a large tissue growth and inflammatory response associated
with much to all of the middle ear often with new bone growth. Sections with paramodiolar profiles
through the cochlea were assessed for appearance of the organ of Corti, spiral ganglion neurons and
stria vascularis / lateral wall tissues.
The cochleae were carefully removed from left temporal bones and then further dissected, first
removing the bony otic capsule, followed by removal of lateral wall tissues, including spiral ligament and
stria vascularis. The tectorial membrane was then removed and the cochleae placed in a solution of
rhodamine labeled phalloidin, diluted 1:100 in phosphate buffered saline (PBS), for 30-120 mins, in the
dark, followed by two washes in PBS. The cochlear sensory neural epithelium was then dissected away
from the modiolus, starting at the apex, to produce surface preparations of the cochlear spiral. Each
segment was mapped for location and placed on a slide. A quantitative assessment of presence or
absence of hair cells was then carried out, beginning at apex and proceeding to the base, to produce a
cycocochleogram, with presence or absence or hair cells mapped by position along the cochlear spiral.
Analytical method
Determination of dexamethasone concentrations was performed using high pressure liquid
chromatography (HPLC) combined with mass spectrometry detection (MS). The limit of detection of the
method was 1.0 ng/ml. Samples (perilymph, plasma and CSF) were extracted by liquid-liquid extraction
using dichloromethane:hexane:MTBE (1:1:1, v/v/v). The organic portion was then dried and the extracts
reconstituted with a water:methanol solution (1:1, v/v). The samples were analyzed by reversed phase
HPLC (1100 series, Agilent) using an Atlantis dC18 column maintained at 40 ⁰C. The mobile phase was
nebulized using heating nitrogen in a Z-spray source/interface and the ionized compounds detected
using MS/MS (Tandem quadrupole mass spectrometer, Quattro Ultima, Waters). Peak heights of
dexamethasone were determined using MassLynx software (Waters). The calibration curves were
obtained by fitting the peak height ratios of analyte / internal standard and the standard concentrations
to a suitable equation using MassLynx. Sample dexamethasone concentrations were then interpolated
using the equations derived from the calibration curves.
Data Analysis
Results are presented as mean + standard error to the mean (SEM) whenever possible. Pharmacokinetic
parameters are calculated using conventional non-compartmental pharmacokinetic methods. A metric
to estimate the time during which dexamethasone concentrations remained above therapeutic levels in
the perilymph (0.04 µg/ml) was developed and termed Terminal Exposure Time (TET). A non linear
regression approach was taken to derive TET, using the exponential decay equation:
Y = span * e(-k.x) + plateau
Where Y is the concentration of dexamethasone in the perilymph, span the difference between Y0 and
plateau, k is the rate constant equal to the reciprocal of the time, and x is the time.
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