Clinical evaluation of a long lasting topical vehicle, TRI-726, containing
ketoconazole 0.15% and hydrocortisone 1% in canine malassezia otitis externa
Gotthelf, L.N.*, Alur, H.H. †
*Animal Hospital of Montgomery
†TriLogic Pharma, LLC
Introduction
Vehicles used for medication delivery in the treatment of ear infections in dogs range
from water, alcohols, oils, lanolin, and waxes. Topical ear treatment formulas usually
have drugs either suspended or dissolved in liquids and are applied as drops into the
affected vertical ear canal. When applied to an infected ear canal, most of the
medication is in contact with the affected epithelium for only a short time as gravity
moves the material deeper into the horizontal ear canal and away from the infected
vertical ear canal.
Liquid ear medication may be mechanically removed from the ear canal by head
shaking. Ear wicks or ear sponges expand in the presence of liquid medication to mold
to the ear canal when hydrated to allow liquid medications to maintain contact with the
affected ear canal epithelium, but even ear wicks can be shaken out. Some of the fatty
or waxy materials with medication imbedded that are used in the dog’s ear canals have
to be removed periodically. Waxes and fats when packed into the ear canal become
occlusive and their presence prevents the removal of cytotoxic enzymes elaborated
from some bacterial and yeast ear infections. Some ear packings contain lanolin, which
may be a sensitizer in some allergic patients.
TRI-726 is a viscous liquid and undergoes solution to gel (sol-to-gel) transition using
body temperature as the trigger. This property renders the product to be easily and
directly placed in the ear, where it transforms to a gel following application. By the
process of “reverse thermal gelation”, the viscosity of TRI-726 increases 2 – 3x of its
viscosity at room temperature. Once in the gel form TRI-726 forms an emulsion on the
entire ear canal epithelial surface. TRI-726 loaded with medications binds to the ear
canal epithelium and slowly releases the medication from micelles into an aqueous
layer, providing a constant source of fresh medication over a long period of time from
one single application.
The purpose of this study was to evaluate the efficacy of TRI-726, loaded with
ketoconazole and hydrocortisone in treating clinical cases of Malassezia otitis externa in
dogs.
Materials and Methods
Materials
TRI-726 consists of a combination of tri-block copolymers and a natural polysaccharide.
For this study, samples of TRI-726 containing ketoconazole (0.15%) and hydrocortisone
(1%) were prepared under cGMP conditions and were supplied in a 3 mL, luer-lock
polypropylene syringes. Flexible applicator tips were provided separately with the
samples.
Animals and Inclusion Criteria
Seventeen (17) adult dogs of various breeds and sexes were recruited from the
Montgomery, Alabama, locale (table 1). Selection criteria excluded any dog that was on
any type of skin, ear, or allergy medication within the last 2 weeks including any
systemic antihistamine, antifungal, antibiotic, steroid or any type of topical ear cleaner or
medication. On the first examination and evaluation, a video otoscopic examination
was done on each candidate to make sure that there was exudate in the ear(s). A
stained, microscopic ear swab cytology was done on each affected ear to determine if
there was a Malassezia infection and to grade the number of yeasts per high power field
based on a grading scale of 0 to 4.
In addition to yeast numbers, clients were asked at the initial evaluation to grade the
current severity of their dogs pruritus based on a grading scale of 0 to 4. Grades 3 and
4 were considered severely pruritic requiring that something be done to help. Grades 0
to 2 were considered only mildly pruritic, which was tolerable.
For inclusion in the study, the dogs had to satisfy at least one of the 2 criteria
1. A Malassezia Count Grade of 2,3 or 4
2. Yeast presence with a pruritus Grade of 3 or 4.
Statistical Analysis
Yeast and pruritis scores at days 0, 7 and 14 were analyzed by one-way ANOVA
initially, followed by a Tukey HSD post-hoc test to analyze the difference in the mean
yeast and pruritis scores. p-values of < 0.05 were considered significant.
Study Protocol
Day 0
Dogs’ ears were assessed for eligibility for otitis by video otoscopic examination,
followed by ear cytology to confirm a Malassezia yeast infection (Grade 2, 3, & 4).
Owners were shown how to evaluate their dogs’ otic pruritus with the “itchy ear” scale.
The severity of otic pruritus based solely on the clients’ observations was charted on
Day 0 using the “itchy ear” scale.
If all study requirements were met, the dog was enrolled in the study and underwent the
following procedure. Wax from the ear(s) was thoroughly flushed using dioctyl sodium
sulfosuccinate and warm water. Ear(s) canal(s) was then dried using suction. Before the
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application of the test product, the supplied applicator tip was attached to the luer lock
syringe containing the test material. The applicator tip was gently inserted into the ear
canal(s) as deep as was comfortable for the patient. The semi-solid medication was
instilled in the ear by slowly pushing the plunger. At the same time, the
applicator/syringe was slowly removed as injection was made. The test material was
allowed to gel in the ear with increased temperature by restraining the head for 2
minutes.
Day 7
Ear(s) was rechecked and Malassezia count and owner’s pruritus score were recorded.
Day 14
Ear(s) was rechecked and Malassezia count and owner’s pruritus score were recorded.
A Malassezia count of 0 or 1 (< 10 per HPF) on follow-up (days 7 and 14) was
considered a successful outcome. A pruritis score of 0, 1 or 2 was considered as mild,
while a score of 3 or 4 was considered as severe on follow-up visits (days 7 and 14).
Results
Malassezia Counts
All 17 dogs (23 infected ears) completed the three evaluations during this clinical trial.
On the first day, the Malassezia counts were: Grade 4 (15 ears), Grade 3 (6 ears),
Grade 2 (2 ears) (table 2). The data from the second evaluation 7 days post infusion
indicated that the Malassezia counts changed significantly from the initial visit. Grade 4
(3 ears), Grade 3 (1 ear), Grade 2 (1 ear), Grade 1 (2 ears), and Grade 0 (16 ears)
(table 2).
On day 14 post infusion, the Malassezia counts were Grade 4 (7 ears), Grade 3 (2
ears), Grade 2 (0 ears), Grade 1 (7ears), and Grade 0 (7 ears) (table 2).
Pruritus Scores
Pruritus scores on the first day were: Grade 4 (22 ears) and Grade 3 (1 ear) (table 3).
By day 7, the pruritus scores were: Grade 4(1 ear), Grade 3 (3 ears), Grade 2 (13 ears),
Grade 1 (3 ears), and Grade 0 (3 ears) (table 3).
On Day 14, the pruritus scores were: Grade 4 (3 ears), Grade 3 (1 ear), Grade 2 (5
ears), Grade 1 (11 ears), Grade 0 (3 ears) (table 3).
Of the 23 ears treated, 18 of 23 ears(78%) had a Malassezia count of 0 or 1 and were
considered successfully treated at one week after treatment with a single application of
TRI-726 with ketoconazole(0.15%) and hydrocortisone(1%).The mean Malassezia
score at day 7 was statistically significantly lower than the mean score at day 0 (table
4).
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At two week after treatment (day 14), 14 of 23 ears (61%) continued to have a
Malassezia count of 0 or 1 and were considered successfully treated with a single
application of TRI-726 with ketoconazole (0.15%) and hydrocortisone (1%). The mean
Malassezia score at day 14 was statistically significantly lower than the mean score at
day 0, but not statistically significantly different than the mean score at day 7 (table 4).
Pruritis scores at the start of the study showed that 22/23 (97%) ears were very pruritic
(itch scores of 3 and 4), which was dramatically reduced to 17% with a single infusion of
TRI-726 with ketoconazole (0.15%) and hydrocortisone (1%) at one week after
treatment. The mean pruritis score at day 7 was statistically significantly lower than the
mean score at day 0 (table 5).
Pruritis scores at day 14 continued to be lower (17% with itch scores of 3 and 4) after
single application of TRI-726 with ketoconazole (0.15%) and hydrocortisone (1%). The
mean itch score at day 14 was statistically significantly lower than the mean pruritis
score at day 0, but not statistically significantly different than the mean score at day 7
(table 5).
Discussion
Otitis externa (OE) is the inflammation of the external ear canal and can be triggered by
many different causes . The most common clinical sign associated with otitis externa in
dogs is pruritus of the ears. In dogs, underlying primary problems may include atopy,
cutaneous adverse food reactions, seborrhea inducing diseases, ear mites, ear canal
conformation, or irritation from foreign bodies such as parts of plants, shrubs or trees
that have gained access to the ear canal. Bacteria and yeast colonization resulting in
infection constitute perpetuating causes of otitis externa.
Ear cleaning in this study was not done with patients sedated or under video otoscopic
guidance. The protocol for this study was to put a detergent ear cleaner, DSS (dioctyl
sodium sulfosuccinate), in the ear for 5 minutes and then to flush the ears with water
under pressure. Then the ear canals were suctioned and a cotton tipped applicator was
used in the ear canal to determine if any liquid was remaining prior to treatment.
The initial reduction in yeast numbers could be accounted for by the ear cleaning
procedure. A good, thorough ear cleaning can help to remove yeasts and waxy
material containing inflammatory compounds. The majority of the patients showed
excellent decreases in the Malassezia scores between day 0 and day 7. However, when
looking at the data from the day 14 evaluation and comparing that to the initial data,
there were still 14 ears that showed a Grade 0 or a Grade 1 Malassezia count. That
should not be attributed to mere ear cleaning, but to the presence of the anti-yeast
medication remaining in the ear canal for a long period of time. In fact, there were 2
dogs that did not show a reduction in the Malassezia count until the day 14 evaluation,
even though their ears were cleaned and dried before test material application. Three
ears showed more Malassezia in their ears at the end of the study than at the
beginning. This could be from incomplete ear cleaning, shaking out the material before
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it completely gelled, or from ongoing allergic dermatitis. In these ears, however, the
pruritus scores did improve at day 14 even though the Malassezia scores did not.
Clients assessing their dogs’ pruritus scores in this study were very happy with the
comfort that their dogs displayed after the TRI-726 treatment. Most, but not all, of the
dogs in this study had a decrease in the Malassezia numbers, but almost all of the dogs
in this study were very much more comfortable by day 14 at the end of the study than at
the beginning. We would like to attribute this effect to a continuous level of
hydrocortisone on the inflamed ear canal epithelium. Infusion of the TRI-726 vehicle
matrix binds to the cleaned ear canal and releases the hydrocortisone from the gel
matrix slowly over time. The use of a more potent, potentially systemically absorbed
topical steroid in the ear may not be necessary when continuously exposed to
hydrocortisone provided in the TRI-726 gel matrix.
It can be concluded from the above study that TRI-726 with ketoconazole and
hydrocortisone was not only successful in reducing the yeast count and pruritis scores
with one dose but also was effective in keeping the Malassezia counts reduced for 14
days. Ketoconazole and hydrocortisone in TRI-726 were effective at reducing
Malassezia and severe pruritus with significant relief from itching. This could be a direct
effect of the hydrocortisone or it could be from the reduction in the pruritus from the
reduction of the Malassezia yeast numbers in the ears.
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GRADING SCALE FOR MALASSEZIA MICROSCOPIC EVALUATION
0
No Malassezia seen
1
A few Malassezia yeasts seen (1 to 10 yeasts per high power field (HPF)).
2
Between 11 and 30 yeasts per HPF.
3
> 31 yeasts counted per HPF.
4
Teeming with yeasts everywhere on the HPF (too numerous to count).
Table 1: Breed Statistics
Breed
COCKER
PITT BULL
POODLE
SHIH TZU
LAB
LHASA APSO
LAB/COON
MALTESE
GOLDEN RET
DACHSHUND
SHARPEI
LABRADOODL
SHEP/ROTT
Number
2
2
1
1
3
1
1
1
1
1
1
1
1
OWNER EAR GRADING SCALE FOR PRURITUS (“ITCHY EAR” SCALE)
4
Dog shows pain, itches, scratches, & rubs ear(s) almost constantly throughout
the day and night.
3
Dog itches, scratches, or rubs ear(s) several times through most of the day and
night.
2
Dog itches, scratches, or rubs the ear(s) often through the day but is
comfortable at night.
1
Dog itches, scratches, or rubs the ear(s) occasionally through the day but is
comfortable at night.
0
Dog does not bother its ear(s) at all.
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Table 2: Distribution of Malassezia counts.
Malassezia Counts (number of ears)
Day
0, 1
2
3
4
0
0
2
6
15
7
18
1
1
3
14
14
0
2
7
Table 3: Distribution of Pruritis scores.
Pruritis Score (number of ears)
Day
0, 1 or 2
3
4
0
0
1
22
7
19
3
1
14
19
1
3
Table 4: Outcome of the study.
Outcome (Mean  standard deviation)
Day
Malassezia Count
Success Rate (%)
0
3.57 ± 0.66
--7
0.83 ± 1.47*
78
, ns
14
1.74 ± 1.74*
61
*p < 0.05 when compared with the day 0 count.
ns
p > 0.05 when compared with the day 7 count.
ns = not statistically significantly different.
Table 5: Outcome of the study.
Outcome (Mean  standard deviation)
Day
Pruritis Score
Success rate (%)
0
3.78 ± 0.85
--7
1.83 ± 0.98*
83
, ns
14
1.57 ± 1.20*
83
*p < 0.05 when compared with the day 0 count.
ns
p > 0.05 when compared with the day 7 count.
ns = not statistically significantly different.
Abstract
TRI-726 is a patented inert vehicle for drug delivery in both human and veterinary
applications. TRI-726 rapidly changes from solution to gel with increased temperature.
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When compounded with various pharmaceutical agents, the gel form of TRI-726
remains present on/in tissues for up to 15 days. This clinical trial tested the long acting
properties of a compounded preparation of TRI-726 containing ketoconazole (0.15%)
and hydrocortisone (1%) in Malassezia otitis externa cases presented to a general
veterinary practice. The affected ears were first flushed and dried, then infused with the
test material and allowed to gel for 2 minutes. Study dogs were evaluated weekly based
on the change in the number of Malassezia yeasts seen on otic cytology over a fourteen
day period from the initial infusion. Otic pruritis scores were reported by the owners at
the initial infusion, then weekly over the same fourteen day period. Of the 23 ears
treated, 18/23 ears (78%) were successfully treated with a single infusion seven days
after treatment initiation and 14/23 ears (61%) continued to be successfully treated at
fourteen days. Itching scores reported by the owners showed a reduction from 22/23
dogs itching initially (96%) to only 4/23 dogs (17%) itching at both seven and fourteen
days. The data from this small clinical trial shows that a single infusion of this topical
formulation is beneficial in treating Malassezia otitis externa and provides comfort from
pruritus for up to fourteen days.
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This study was funded by TriLogic Pharma, LLC
Gotthelf, L. N. is a minority stockholder in TriLogic Pharma, LLC
Alur, H. H. is the Chief Scientific Officer at TriLogic Pharma, LLC
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