Veterinary Dermatology 2004, 15, 294 303

Investigation on the clinical efficacy and safety of 0.1% tacrolimus

ointment (Protopic) in canine atopic dermatitis: a randomized,
double-blinded, placebo-controlled, cross-over study

Blackwell Publishing, Ltd.

R. MARSELLA, C. F. NICKLIN, S. SAGLIO and J. LOPEZ

Blanche Saunders Dermatology Laboratory, Department of Small Animal Clinical Sciences, College
of Veterinary Medicine, University of Florida, P.O. Box 100126, Gainesville, FL 326100126, USA
(Received 23 August 2003; accepted 24 December 2003)

Abstract Topical tacrolimus is successfully used in people with atopic dermatitis. Preliminary studies in dogs
with atopic dermatitis using tacrolimus in a compounded lotion formulation indicated that tacrolimus significantly decreased erythema and pruritus according to investigator, but no significant improvement was reported
by the dog owners. The objectives of this study were to evaluate the clinical efficacy and safety of the commercially
available 0.1% tacrolimus ointment (Protopic) in dogs with atopic dermatitis. The study was designed as a
double-blinded, placebo-controlled, cross-over study. Selected dogs were allocated to either tacrolimus or placebo
for 4 weeks. After 4 weeks there was a wash-out period of 2 weeks and treatments were switched. Twelve dogs
completed the study. Clinical signs were scored. Blood samples were collected for complete blood count, chemistry panels and tacrolimus levels at week 0 and 4 of each treatment. Tacrolimus ointment significantly decreased
severity of symptoms for both owners and investigators at the end of the trial. When the same dogs received the
placebo, there were no differences between week 0 and week 4 scores. Dogs with localized disease responded better
than dogs with generalized disease. Tacrolimus was detected in the blood of animals receiving the active ingredient. Levels were below the level of toxicity and no adverse effects were reported in any of the dogs. No changes
in complete blood count and chemistry parameters were detected between groups or within groups. In conclusion,
tacrolimus appears to be a safe alternative treatment in dogs with atopic dermatitis, especially in those with
localized disease.
Keywords: atopic dermatitis, dogs, tacrolimus.

I N TRO D U CT I ON
Tacrolimus (FK-506) is a calcineurin inhibitor currently
approved in the US for use in humans with moderate
to severe atopic dermatitis (AD).1,2 The mechanism of
action of tacrolimus in AD involves T cells, Langerhans cells, mast cells, basophils and keratinocytes.3
Tacrolimus inhibits the T-cell response to antigens and
production of the cytokines responsible for T-cell proliferation, i.e. interleukin (IL)-2. Tacrolimus achieves
this by forming a complex with a cyclophilin-like protein (FKBP12), and this complex, in turn, inhibits the
ability of calcineurin to dephosphorylate the transcription factor required for the activation of IL-2 and IL-4
gene transcription.4 Tacrolimus also inhibits other T-cellderived cytokines, such as IL-3, IL-4, interferon gamma
(IFN-), granulocyte/macrophage colony-stimulating
factor and tumour necrosis factor alpha (TNF-),
that contribute to allergic inflammation.5 In addition

This study was funded by the Morris Animal Foundation.

Correspondence: R. Marsella, Blanche Saunders Dermatology
Laboratory, Department of Small Animal Clinical Sciences,
College of Veterinary Medicine, University of Florida, P.O.
Box 100126, Gainesville, FL 32610 0126, USA. E-mail:
marsellar@mail.vetmed.ufl.edu
294

to lymphocytes, tacrolimus downregulates cytokine

expression in other cells that have tacrolimus-binding
proteins and are important in allergic skin inflammation. These include mast cells, basophils, eosinophils,
keratinocytes and Langerhans cells.6,7 Topical tacrolimus
leads to profound phenotypic and functional alterations of epidermal antigen-presenting dendritic cells in
patients with AD, downregulates the expression of the
high-affinity immunoglobulin (Ig)E receptor (FcRI)
on Langerhans cells and decreases the number of
inflammatory dendritic epidermal cells.8 Tacrolimus
also inhibits the expression of IL-2R (CD25), the costimulatory molecules CD80 (B7.1) and CD40, and
both classes of major histocompatibility complex (MHC).9
Finally, topical tacrolimus significantly inhibits T-cellmediated keratinocyte apoptosis.10 Keratinocyte apoptosis in AD seems to be mediated by IFN- released
from activated T cells and contributes to the severity
of clinical signs.11 Tacrolimus-mediated inhibition of
keratinocyte apoptosis correlates with the clinical
improvement of skin lesions.10
Long-term studies of tacrolimus in humans have
demonstrated that tacrolimus is safe and does not
increase the incidence of secondary skin infections.12
Tacrolimus ointment is not atrophogenic, and is associated with minimal systemic absorption.13 No significant
2004 European Society of Veterinary Dermatology

Tacrolimus for canine atopic dermatitis

changes in laboratory parameters have been reported
with long-term topical tacrolimus therapy.14 The most
common adverse effect is a transient skin burning
sensation and pruritus at the site of application. Both
these sensations resolve spontaneously after a few days
of therapy. Therefore, tacrolimus ointment is a safe and
efficacious treatment for long-term use in paediatric
and adult patients with AD.15
Limited information exists on the use of tacrolimus
in veterinary medicine. Owing to the similarities between
human and canine AD, tacrolimus may be a useful
treatment for canine AD. 16,17 A double-blinded,
placebo-controlled pilot study was conducted in dogs
using a compounded lotion (0.3%), as tacrolimus ointment (Protopic) was not commercially available in
the US at the time of that study.18 In the absence of the
commercial product, a lotion was attempted because of
the ease of application. In that pilot study, tacrolimus was
well tolerated in dogs and minimally absorbed through
canine skin. Tacrolimus significantly decreased clinical
signs of AD according to the investigator. The improvement in clinical signs, however, was not perceived as
significant by the dogs owners. Several factors that
may have underestimated the efficacy of topical tacrolimus in dogs with AD were detected after completion of the study (e.g. scoring system, bias in selection
of severe generalized cases, stability of tacrolimus in a
lotion). Therefore, after the release of the commercial
ointment, the clinical trial was repeated a using the
product practitioners and dermatologists would be
using and altering the scoring system to capture improvement in dogs according to the specific areas of the
body. Currently, there are two commercial tacrolimusbased products (0.1 and 0.03%). Efficacy in humans
appears to be concentration dependent, so it was decided
to use the product with the highest concentration (0.1%).
Therefore, the purpose of this study was to evaluate the
clinical efficacy and safety of 0.1% tacrolimus ointment
in dogs with AD.

MATERIAL S A ND ME T HODS
This study was designed as a double-blinded, placebocontrolled, randomized, cross-over, clinical trial.

Animals
Fourteen dogs were enrolled in the study. Twelve completed the study. One dog dropped out owing to an
inability to remain clear of infections when antibiotics
were discontinued. The second dog dropped out because
of a leg fracture, which required the long-term use of
an E-collar, interfering with the evaluation of pruritus.
These dogs were not included in the statistical analysis.
Inclusion criteria were a diagnosis of AD, which was
based on suggestive history, compatible clinical signs,
and at least three positive reactions on intradermal
skin testing using a panel of 58 allergens. A positive
reaction was considered to be one that scored 2 or
higher on a scale of 04, where 0 was the score given to

295

the negative control (saline) and 4+ was the score of the

positive control (histamine) 15 min after the injections.
Exclusion criteria were the presence of other pruritic
skin diseases (e.g. food allergy and scabies) and the
presence of secondary ear and skin infections (e.g.
staphylococcal pyoderma and Malassezia dermatitis),
as diagnosed by physical examination and cytology of
affected areas. All dogs with nonseasonal clinical signs
underwent food trial for a minimum of 2 months to
rule out the possibility of a food allergy. Other exclusion criteria were the use of topical or oral glucocorticoids and antihistamines in the 2 weeks prior to the
study and injectable glucocorticoids in the 2 months
prior to the study. During the study only heartworm
prevention and flea control (monthly application of
Frontline; Merial Limited, Iselin, NJ, USA) were
allowed. Dogs were bathed only with a nonmedicated
shampoo, as deemed necessary by the owners. They were
asked not to bathe dogs 2 h before or after applying the
ointment. Animals with a pre-existing systemic disease
or animals that could not suspend therapy due to
exceptional severity of AD were excluded. Dogs with
concurrent flea allergy were not excluded, but the allergy
was controlled with strict flea control, as described above.

Experimental design
Selected dogs were divided into two equal groups.
Assignment to treatment was randomized by coin toss,
in that a coin toss prior to enrolling the first dog determined the treatment of that first dog. The remaining
dogs were enrolled in alternating treatments until all of
the dogs were enrolled. This allowed for equal distribution of dogs assigned to each treatment and minimized
order of treatment effects. The principal investigator
was unaware of the treatment assignment throughout
the study.
One group received 0.1% tacrolimus ointment
(Protopic, Fujisawa, Deerfield, IL, USA), once daily
(maximum dose of 0.1 mL/kg/day, equivalent to
0.1 mg/kg/day) on the areas that owners felt were most
pruritic, whereas the other group received the placebo
ointment (vehicle) for 4 weeks. Owners were instructed
to apply ointment once daily to affected areas, rub into
coat thoroughly for good skin contact and distract the
dog until ointment the dries and to use an E-collar if
necessary. They were also instructed to wear gloves and
wash their hands after use. The thickness of the application was determined by the owner. They were given
a maximum volume to apply, and they determined how
this was divided among the affected areas of their dog
each day. Owners of dogs with lesions around the eyes
or on the lips were told not to treat that area. Ointment
was applied to the chin, face folds or perianal region of
dogs affected in those locations. After the first 4 weeks,
there was a wash-out period of 2 weeks, after which the
treatments were reversed, so that each dog received
both treatments and served as its own control.
Doses of tacrolimus were selected based on human
studies and pilot studies performed in dogs with AD by
the investigators (data not shown).18,19 In humans, the

2004 European Society of Veterinary Dermatology, Veterinary Dermatology, 15, 294303

296

R Marsella et al.

maximum daily amount of tacrolimus used is 0.4 and

0.8 mg/kg.20 In pilot studies with dogs, single doses of
0.6 and 0.9 mg/kg (data not shown) were well tolerated
and in a previous clinical trial using dogs, a daily dose
of 0.3 mg/kg for 4 weeks did not cause any adverse
effects.18 The duration of the wash-out period was
determined based on the half-life of tacrolimus in dogs
after systemic administration (9 h).21,22
The placebo ointment was compounded by a local
pharmacy (Westlab Pharmacy, Gainesville, FL, USA).
The pharmacy placed the ointments into coded 60-mL
syringes. Other than the code, the ointments were
indistinguishable. Both the owners and investigators
were blinded to what the syringe contained. The pharmacy maintained the codes until the study was complete.
Gloves were worn when the ointment was applied. The
first (week 0) and last (week 4) ointment applications
were applied by the investigator on clinically affected
areas. At each visit, a physical examination was performed. If lesions were found on dermatological examination, cytology was performed via tape preparation.

If bacteria or yeast were detected, appropriate treatment was prescribed and participation in the study
was delayed. The syringes were all returned at the end
of each treatment period to keep owners from attempting to compare the ointments and to help evaluate
owner compliance. Hair was not clipped in order to
mimic real-life clinical use of this product. All dogs
received both ointments over different 4-week treatment periods.

Clinical evaluation of efficacy

The investigator evaluated the dogs clinical condition
at each visit (weeks 0 and 4 of both treatment periods)
using a modified Canine Atopic Dermatitis Extent and
Severity Index (CADESI, Table 1).23 The owners evaluated pruritus weekly based on a modified CADESI
(Table 2). This scoring system divided the dogs body
into regions and evaluated each for various clinical
signs (or only pruritus in the owners case). Each location was given a score from 0 to 3 (where 0 = absent,
1 = mild, 2 = moderate and 3 = severe) for each clinical

Table 1. Investigator Clinical Score (modified CADESI)

Site/symptom

Erythema

Lichenification

Excoriations

Face
Left
Pinna
Right
Pinna
Neck

Axilla
Sternum
Inguinal
Abdomen
Thorax

Lumbar
Flank
Front Limb

Hind Limb

Front Foot

Hind Foot

Convex
Concave
Convex
Concave

Dorsal
Ventral
Lateral Left
Right
Left
Right
Left
Right
Dorsal
Lateral Left
Right
Dorsal
Left
Right
Left
Medial
Lateral
Right
Medial
Lateral
Left
Medial
Lateral
Right
Medial
Lateral
Left
Ventral
Dorsal
Right
Ventral
Dorsal
Left
Ventral
Dorsal
Right
Ventral
Dorsal

Perineum
Tail

Dorsal
Ventral

Scoring: 0 = absent; 1 = mild; 2 = moderate; 3 = severe.

2004 European Society of Veterinary Dermatology, Veterinary Dermatology, 15, 294 303

Papules

Pruritus

Total score

Tacrolimus for canine atopic dermatitis

Table 2. Owner Clinical Score (modified CADESI)
Site/
symptom

Week 0 Week 1 Week 2

Date:
Date:
Date:

Week 3
Date:

Week 4
Date:

Face
Ear

297

0.3 ng/mL. All values that were below the detection

limit of the assay were reported as 0.15 ng/mL in attempt
to prevent under- or over-estimation of the actual value.

Statistics

Left
Right
Neck (collar region)
Armpits
Chest
Stomach
Groin
Side
Left
Right
Back
Rump
Tail
Anal Region
Front Leg Left
Right
Hind Leg
Left
Right
Front Foot Left
Right
Hind Foot Left
Right
Total SCORE
(For office use only)
Owners please assign a score for each area of the body listed in the table.
Scoring: 0 = absent (no scratching rubbing, chewing or licking
noticed); 1 = mild (scratching, rubbing, chewing or licking for less
than 10% of day); 2 = moderate (scratching, rubbing, chewing or
licking for 50% of day); 3 = severe (scratching, rubbing, chewing or
licking all the time, even at night and during a meal).

sign. No global assessment was performed during this

clinical trial.
Upon evaluation, it was noted if dogs had localized
or generalized disease. Localized disease consisted of
pododermatitis with or without facial dermatitis or
localized ventral abdomen involvement. All other distributions were considered generalized.

Sample collection
Blood samples (35 mL) for the measurement of tacrolimus concentration were collected before (0) and at
2, 4 and 6 h after ointment application at week 0 and
week 4 of each treatment period. The samples were collected via venepuncture using a syringe and needle, and
transferred to an EDTA vacutainer (Becton Dickinson,
Franklin Lakes, NJ, USA). After mixing well, samples
were placed on ice initially and then stored at 20 C
until assayed. Additional blood was collected at 0 h for
complete blood count (CBC) and chemistry panels at
weeks 0 and 4 of each treatment period.

Measurement of whole blood concentrations

of tacrolimus
Tacrolimus concentrations in whole-blood samples
were determined using a commercial tacrolimus enzymelinked immunosorbent assay (ELISA) kit (Pro-Trac II
Tacrolimus ELISA Kit, DiaSorin, Stillwater, MN, USA)18
according to the protocol provided by the manufacturer. Tacrolimus concentration was calculated as
ng/mL of blood. The detection limit of the assay was

Data were analysed using least squares analysis of variance (LS) with all main effects and interactions
included in the model. Differences among treatments
or groups and times were analysed using orthogonal
contrast analysis. If no differences were detected between
periods, indicating that order of treatment did not have
an effect, data were pooled and re-analysed.
Because clinical scores are not normally distributed,
they were rank transformed prior to analysis. Means
and SEM were calculated using the untransformed data.
However, all P-values are representative of the rank
transformed data.
In addition, tests for heterogeneity of regression
were also conducted to evaluate time trends between
treatments. The analysis was performed at the highest
significant order of regression.
All analyses were performed using the statistical
software package SAS System for Windows version 8.2
(SAS Institute, Cary, NC, USA). A value of P < 0.05
was considered significant. All data is presented as
mean SEM, unless otherwise indicated.

R E SU LT S
Animals
Of the 12 dogs that completed the study, 8 were spayed
females, 3 neutered males and 1 intact male. The average
age was 4.2 years (range = 28.5 years). The breeds represented included Jack Russell terrier (n = 1), beagle
(n = 1), Lhasa Apso (n = 1), schnauzer (n = 1), boxer
(n = 1), poodle (n = 1), German shepherd (n = 1),
English bulldog (n = 1), Boston terrier (n = 2) and
two mixed-breed dogs. The average weight was 16.06 kg
(range = 5.0038.86 kg). The actual dose applied ranged
from 0.05 to 0.12 mL/kg with an average dose of
0.10 mL/kg. Six dogs had localized disease and six
dogs had generalized disease (Table 3). No adverse effects
were reported by owners.

Table 3. Summary of dogs who completed this study

Dog

Breed

Sex

Age
(year)

Onset
(year)

Distribution

1
2
3
4
5
6
7
8
9
10
11
12

Jack Russell terrier

beagle
cockapoo
Lhasa Apso
mixed
schnauzer
boxer
poodle
German shepherd
Boston terrier
Boston terrier
English bulldog

FS
FS
MC
FS
FS
FS
FS
FS
M
FS
MC
MC

2.5
4
3
6.5
2
7
4.5
4
3.5
8.5
2.5
2.5

1.5
<1
1
2
2
1.5
1
unknown
unknown
1.5
1
unknown

Generalized
Generalized
Localized
Generalized
Generalized
Generalized
Localized
Localized
Localized
Localized
Generalized
Localized

2004 European Society of Veterinary Dermatology, Veterinary Dermatology, 15, 294303

298

R Marsella et al.

Figure 1. Mean clinical score as determined by the investigator.

Error bars indicate mean SEM. *Significant difference (P 0.05)
from week 0 within the group.

Period effect
No differences were detected between periods for all
variables, indicating that order of treatment did not
have an effect, so data were pooled and reanalysed.

Clinical score
Investigator score (Fig. 1) Scores were first analysed
considering all 12 dogs as a group and then further
analysed to evaluate whether dogs with localized disease responded more favourably to topical tacrolimus
than dogs with generalized disease. When all dogs were
considered, within the tacrolimus group, scores decreased
significantly after 4 weeks of treatment (P = 0.0019),
whereas no significant differences were detected within
the placebo group. Investigator scores improved by
> 50% in 7 of 12 dogs (58%) during tacrolimus treatment
(2 generalized and 5 localized) and 3 of 12 dogs (25%)
during placebo (3 generalized).
When scores of dogs receiving tacrolimus were evaluated according to the extent of the disease, the dogs
with generalized disease had significantly higher scores
(P = 0.03) at week 0 than the localized dogs. By week 4,
the dogs with generalized disease still had significantly
higher scores (P = 0.002) than dogs with localized disease. In both groups, scores decreased from week 0 to
week 4 (P 0.03). In the generalized group, scores
decreased by an average of 24%, whereas in the localized
group, scores decreased by an average of 60%.
Owner score (Fig. 2) When all 12 dogs were considered, owner scores significantly decreased starting at
week 3 of tacrolimus treatment (P 0.01). The scores
decreased after 2 weeks of treatment but the decrease
was not statistically significant (P = 0.056). There were
no statistically significant differences in owner scores
within the placebo group. Owner scores improved by
> 50% (week 0 minus week 4 only) in 5 of 12 dogs
(41%) during tacrolimus treatment (1 generalized and
4 localized) and 2 of 12 dogs (16%) during placebo
treatment (1 generalized and 1 localized). Of the 5

Figure 2. Mean clinical score as determined by the owners. Error

bars indicate mean SEM. *Significant difference (P 0.05) from
week 0 within the group.

tacrolimus-treated dogs with > 50% improvement according to owners, 3 were also included in the 7 dogs with
> 50% improvement according to the investigator.
When the dogs receiving tacrolimus were divided
according to the extent of their disease, there were no
differences in owner scores between dogs with localized and dogs with generalized disease at week 0. By
week 1 and through the rest of the study, the dogs with
localized disease had significantly lower scores than the
generalized dogs (P 0.01). Within the localized group,
week 4 scores were significantly lower than week 0 scores
(P < 0.0001). Owner scores in the localized group decreased
an average of 58%. Owner scores in the generalized
group decreased an average of 19%, but this was not
statistically significant.
Tacrolimus blood levels (Fig. 3) Over all times, the tacrolimus group had significantly (P = 0.03) higher blood
levels than the placebo group (0.77 and 0.46 ng/mL,
respectively). The tacrolimus group had significantly
(P = 0.001) higher levels of tacrolimus in the blood
than the placebo group at week 4, 6 h post application
(1.4 and 0.53 ng/mL, respectively). Within the tacrolimus
group, blood levels at week 0, 2 and 6 h (0.50 and
0.65 ng/mL, respectively) were significantly (P 0.02)
lower than at week 4, 2 and 6 h (1.1 and 1.4 ng/mL, respectively). At week 4, the blood levels at 6 h (1.4 ng/mL)
were significantly higher (P 0.03) than both 0 and 4 h
samples (0.66 and 0.84 ng/mL, respectively). There were
no statistically significant differences within the placebo
group.

CBC and chemistry panels

No significant changes were noted in CBC and chemistry values.

D IS C U S S IO N
In this double-blinded, placebo-controlled, cross-over
study, the clinical efficacy and safety of commercially
available 0.1% tacrolimus ointment (Protopic) was

2004 European Society of Veterinary Dermatology, Veterinary Dermatology, 15, 294 303

Tacrolimus for canine atopic dermatitis

299

Figure 3. Mean tacrolimus concentration in

the blood. Error bars indicate mean SEM.
*Significant difference (P 0.05) from week
0 within the group. Significant difference
from hour 6 within the group. Significant
difference between treatments. indicates the
detection limit of the assay.

evaluated. This study found that tacrolimus ointment

significantly decreased clinical signs over a 4-week period
according to both investigators and owners. The decrease
in clinical signs was seen as early as 2 weeks after the
start of treatment and became statistically significant
after 3 weeks of daily application.
Very little information is available concerning the
use of tacrolimus in veterinary dermatology. The
results of this study are more encouraging than those
previously reported using a 0.3% tacrolimus lotion for
canine AD.18 In that pilot study, tacrolimus was well
tolerated and minimally absorbed, and although it significantly decreased clinical signs of AD according to
the investigator, a significant improvement was not
perceived by the owners. The difference in these results
might be due to the increased number of dogs enrolled
in the study, the different formulation of tacrolimus, a
different population of dogs and/or an improved scoring system that better reflected the extent of each dogs
condition and improvement. The previous scoring system generalized the evaluation over the entire dog and
did not, therefore, account for regional disease. In
addition, most of the dogs enrolled in the previous study
had generalized disease. This greatly underestimated
the efficacy of treatment as only a small amount of
lotion was allowed on a daily basis. Owners were asked
to apply the lotion to the areas that were the most
pruritic. However, in dogs with generalized disease, the
improvement of the treated areas may not been sufficient to change the overall clinical score and owner
perception of pruritus. There were also concerns by the
compounding pharmacy regarding the stability of tacrolimus in a lotion formulation, as tacrolimus is not
water soluble. It is important to note, however, that a
tacrolimus lotion had been reported effective in human
patients that could not tolerate the vehicle of the commercial ointment24 and tacrolimus was detected in the
blood of the treated dogs.18
The results of this study are more similar to reports
in the human literature in which tacrolimus was reported

to clearly improve clinical signs within the first few

weeks of therapy.25 Tacrolimus has been used in several
long-term clinical trials on patients with moderate to
severe AD and has been demonstrated to be effective,
safe and well tolerated even in paediatric patients with
facial lesions.2628 Tacrolimus significantly improved
the quality of life of both adult and paediatric patients
with 25 and, similar to this study, the absorption of
tacrolimus after topical application was minimal and
no changes in chemistry parameters were detected.29
In this study, investigator scores (CADESI) improved
by > 50% in 58% of dogs during tacrolimus treatment
and in 25% of dogs during placebo. The average owner
score improved by > 50% in 41% of dogs during tacrolimus treatment and in 16% of dogs during placebo.
These results are not as superb as those reported in the
human literature in which an improvement of 50%
was observed in 19.8, 61.6 and 72.7% of patients receiving placebo, 0.03% tacrolimus ointment and 0.1%
tacrolimus ointment, respectively.30 This difference may
be due, in part, to the frequency of application of the
ointment, which was once daily in our study and twice
daily in the human study. In addition, a different scoring system was used in the human clinical trial (Eczema
Area and Severity Index or EASI score). The CADESI
was developed in an attempt to apply similar criteria
to animals but within the limitations of veterinary
medicine.23,31
In this study, dogs with localized disease appeared to
respond better than those with generalized disease.
Investigator scores decreased an average of 24% in the
generalized group, whereas scores in the localized group
decreased an average of 60%. Owner scores decreased
an average of 58% in the localized group, whereas owner
scores in the generalized group decreased an average of
19% (not statistically significant). It is likely that the
reason for which the improvement was more pronounced in the dogs with localized disease is in the
total daily amount of tacrolimus allowed. The dose and
frequency of application were calculated conservatively

2004 European Society of Veterinary Dermatology, Veterinary Dermatology, 15, 294303

300

R Marsella et al.

compared with human studies.19 Doses three times as

high as those used in this study did not cause adverse
effects in dogs as a single dose (unpublished data). It
may have been safe to use higher doses or to use more
frequent application. It is reasonable to speculate
that an increase in dose and/or frequency may further
improve clinical response as in humans the clinical
improvement appears to be dose and concentration
dependent. An increase in the dose and/or frequency of
application would also significantly increase the cost of
this therapy.
The per cent improvement (60%) observed in patients
with localized disease in this study is similar to the
improvement in clinical scores reported with cyclosporin
treatment and with topical triamcinolone therapy.
Olivry et al.31 evaluated the efficacy of two cyclosporin
dosages (2.5 and 5 mg/kg) in dogs with AD in a multicentre, randomized, controlled trial. After 6 weeks,
scores of lesion severity were reduced from baseline by
an average of 34, 41 and 67% in dogs treated with placebo, cyclosporin at the low dose, and cyclosporin at
the high dose, respectively. As far treatment success with
topical triamcinolone, this was evident in 67% of the
triamcinolone-treated dogs (mean improvement, 1.98)
and 24% of the vehicle-treated dogs (mean improvement, 0.29). Treatment success was defined as improvement of at least two of six grades in overall clinical
score. The authors concluded that triamcinolone used
as a spray solution at a concentration (0.015%) approximately one-sixth the concentration of triamcinolone
topical preparations currently available for veterinary
use is effective for short-term alleviation of allergic
pruritus in dogs. Adverse effects reported in that study
were few and mild.32
Tacrolimus is an expensive drug. However, it is less
expensive than oral daily treatment with cyclosporin.
The monthly cost of tacrolimus treatment for a 25 lb
dog at 0.1 mg/kg is approximately US $120 (0.1 mL/kg,
2.2 mg/day, 66 mg of tacrolimus in 1 month = approximately one 60 g 0.1% tube, which costs US $120). The
monthly cost of cyclosporin therapy at 5 mg/kg is
approximately US $210 (Neoral liquid, 50 mL bottle,
110 mg/mL, $320 per bottle). Based on these calculations, tacrolimus may be an appealing treatment option
for dogs with localized disease or in cases in which systemic use of a calcineurin inhibitor might be a concern.
This study was a placebo-controlled study, therefore,
the efficacy of tacrolimus was only compared with that
of the vehicle. There was no direct comparison with the
efficacy of topical glucocorticoids. In human literature,
0.1% tacrolimus was similar to a moderately potent
corticosteroid (0.1% hydrocortisone-17-butyrate) in the
treatment of adult patients with moderate-to-severe
AD.33 The mean per cent decrease in a modified EASI
(mEASI) score was 47.0, 36.5 and 36.1% for patients
who received 0.03% tacrolimus, 0.1% tacrolimus and
0.1% hydrocortisone butyrate, respectively. Tacrolimus
at 0.1% was significantly more effective than 0.03%
tacrolimus. Laboratory parameters showed no treatment differences and no marked changes over time. In

another study comparing the cost-effectiveness of highpotency topical corticosteroids with tacrolimus ointment
for the treatment of moderate-to-severe AD, tacrolimus
ointment was found to be more cost-effective than glucocorticoids administered in 2-week treatment cycles
and similar in cost-effectiveness to 4-week cycles of highpotency glucocorticoids.34 In veterinary dermatology,
tacrolimus used daily would be more expensive than
high-potency glucocorticoids. A monthly treatment
with Genesis (16 oz. bottle, 0.015% triamcinolone
acetonide per mL) would be approximately US $20.
Tapering the tacrolimus dose or frequency of application was not addressed in this study, but it is reasonable
to consider this option in the future, especially based
on anecdotal reports of success in clinical cases.
Development of secondary skin infections is always
a concern when immunomodulatory therapy is used.
In this study, none of the dogs developed cutaneous
bacterial infections while on tacrolimus treatment. The
use of topical tacrolimus has been associated with minimal risk of skin infections in the human literature and
that has been considered a major advantage over the
use of glucocorticoids. In one study, the incidence of all
cutaneous infections in patients was 18.0, 24.8 and
17.7% for adult patients and 20.9, 19.6 and 23.6% for
paediatric patients treated with the vehicle, 0.03 and
0.1% tacrolimus ointment, respectively.12 The incidence
of any individual cutaneous infection was not significantly higher in the tacrolimus group than in the vehicle
group, with the exception of folliculitis in adults, which
was more common in patients receiving tacrolimus. In
two open studies with long-term use of 0.1% tacrolimus
ointment (up to 1 year), there was no evidence of
increased risk for cutaneous infections, based on the
incidence of adverse events, incidence by cumulative
length of exposure or hazard rates. Furthermore, it was
demonstrated that tacrolimus decreases colonization of
Staphylococcus on lesional skin in humans after 3 weeks
of therapy.35 Because tacrolimus has no antistaphylococcal activity in vitro, these findings are consistent
with the theory that the inflammatory skin condition
in AD is a predisposing factor for colonization with
S. aureus. Therefore, it is conceivable that the decrease in
S. aureus colonization was a consequence of an improvement in the skin surface due to the anti-inflammatory
effect of the drug, and possibly due to the emollient
effect of the vehicle. Another possible explanation is
that tacrolimus selectively inhibits the inflammatory
dendritic epidermal cells (IDEC) which are recruited
ex novo in sites of atopic inflammation without altering
the residential Langherans cells, responsible for the local
immune defence.8 The selective effect of tacrolimus
on IDEC sparing other dendritic cells differentiates it
from cyclosporin, which significantly inhibits epidermal
Langerhans cells.3638 In a recent study on the use
cyclosporin for canine AD, the incidence of cutaneous
infections was comparable with glucocorticoids.39
One additional advantage of topical tacrolimus over
glucocorticoids is the absence of atrophogenic properties, which is desirable especially in cases of long-term

2004 European Society of Veterinary Dermatology, Veterinary Dermatology, 15, 294 303

Tacrolimus for canine atopic dermatitis

treatment. By contrast, it is important to mention that
application of an ointment may not be ideal in dogs,
especially in cases of pododermatitis. Several dog owners
in our trial complained of greasiness and dirt trapped
in the hair by the ointment.
Calcineurin inhibitors have been of tremendous benefit in the therapy of AD in both human and veterinary
dermatology. Other topical compounds with mechanisms of action similar to tacrolimus are being developed and tested. Some of these compounds may have
a potential benefit in dermatology. Rapamycin is one
of them. Rapamycin impairs antigen uptake of human
dendritic cells, which seems to be, at least in part, mediated by the FKBP immunophilins.40 Pimecrolimus is
another calcineurin inhibitor already on the market
with a label for mild to moderate AD.41,42 Although its
potency is less than tacrolimus, it is available in a lotion
formulation (Elidel, Novartis), which would be more
suitable than an ointment for use in dogs. No information is currently available on the use of pimecrolimus in
canine AD. The quest for alternative safe and effective
treatments for canine AD continues and additional
clinical trials will be necessary.
Based on the results of this double-blinded, placebocontrolled clinical trial, it is reasonable to conclude
that 0.1% tacrolimus ointment is safe and minimally
absorbed in canine patients with AD. It is well tolerated and significantly decreased severity of clinical
signs of AD according to both owners and the investigator. Clinical improvement was more pronounced in
dogs with localized disease.

REFEREN CE S
1. Lazarous, MC, Kerdel, FA. Topical tacrolimus Protopic.
Drugs of Today 2002 38: 7 15.
2. Rustin M. Tacrolimus ointment for the management of
atopic dermatitis. Hospital Medicine 2003; 64: 21417.
3. Gewirtz AT, Sitaraman SV. Tacrolimus Fujisawa. Current Opinion in Investigational Drugs 2002; 3: 130711.
4. Schreiber SL, Crabtree GR. The mechanism of action of
cyclosporine A and FK-506. Immunology Today 1992;
12: 136 42.
5. Sakuma S, Higashi Y, Sato N et al. Tacrolimus suppressed the production of cytokines involved in atopic
dermatitis by direct stimulation of human PBMC system.
(Comparison with steroids.) International Immunopharmacology 2001; 1: 1219 26.
6. de Paulis A, Stellato C, Cirillo R et al. Anti-inflammatory
effect of FK-506 on human skin mast cells. Journal of
Investigative Dermatology 1992; 99: 723 8.
7. Thomson AW, Nalesnik M, Abu-Elmagd K et al. The
influence of FK-506 on T lymphocytes, Langerhans
cells, and the expression of cytokine receptors and adhesion molecules in psoriatic skin: a preliminary study.
Transplantation Proceedings 1991; 23: 3330 1.
8. Wollenberg. A, Sharma S, von Bubnoff D et al. Topical
tacrolimus (FK506) leads to profound phenotypic and
functional alterations of epidermal antigen-presenting
dendritic cells in atopic dermatitis. Journal of Allergy
and Clinical Immunology 2001; 107: 519 25.

301

9. Panhans-Gross A, Novak N, Kraft S et al. Human

epidermal Langerhans cells are targets for the immunosuppressive macrolide tacrolimus (FK506). Journal of
Allergy and Clinical Immunology 2001; 107: 34552.
10. Trautmann A, Akdis M, Schmid-Grendelmeier P et al.
Targeting keratinocyte apoptosis in the treatment of
atopic dermatitis and allergic contact dermatitis. Journal
of Allergy and Clinical Immunology 2001; 108: 83946.
11. Trautmann A, Akdis M, Klunker S et al. Role of apoptosis in atopic dermatitis. International Archives of
Allergy and Immunology 2001; 124: 2302.
12. Fleischer AB Jr, Ling M, Eichenfield L et al. Tacrolimus
ointment for the treatment of atopic dermatitis is not
associated with an increase in cutaneous infections.
Journal of the American Academy of Dermatology 2002;
47: 56270.
13. Rico MJ, Lawrence I. Tacrolimus ointment for the treatment of atopic dermatitis: clinical and pharmacologic
effects. Allergy and Asthma Proceedings 2002; 23: 191
7.
14. Gupta AK, Adamiak A, Chow M. Tacrolimus. A review
of its use for the management of dermatoses. Journal of
the European Academy of Dermatology and Venereology
2002; 16: 10014.
15. Allen BR. Tacrolimus ointment: its place in the therapy
of atopic dermatitis. Journal of Allergy and Clinical
Immunology 2002; 109: 4013.
16. Marsella R, Olivry T. Animal models of atopic dermatitis.
Clinics in Dermatology 2003; 21: 12233.
17. Marsella R, Olivry T. The ACVD task force on canine
atopic dermatitis: mediators of cutaneous inflammation.
Veterinary Immunology and Immunopathology 2001;
81: 20513.
18. Marsella R, Nicklin CF. Double blinded-placebo controlled, cross-over study to evaluate the efficacy and safety
of 0.3% topical tacrolimus in canine atopic dermatitis.
Veterinary Dermatology 2002; 13: 20310.
19. Nakagawa H, Etoh T, Ishibashi Y et al. Tacrolimus ointment for atopic dermatitis. Lancet 1994; 344: 883.
20. Boguniewicz M, Fiedler VC, Raimer S et al. A randomized,
vehicle controlled trial of tacrolimus ointment for the
treatment of atopic dermatitis in children. Journal of
Allergy and Clinical Immunology 1998; 102: 63744.
21. Takada K, Katayama N, Kiriyama A, Usuda H. Distribution characteristics of immunosuppressants FK506 and
cyclosporin A in the blood compartment. Biopharmaceutics and Drug Disposition 1993; 14: 65971.
22. Venkataramanan R, Warty VS, Zemaitis MA et al. Biopharmaceutical aspects of FK-506. Transplantation
Proceedings 1987; 19: 305.
23. Olivry T, Rivierre C, Jackson HA et al. Cyclosporine
decreases skin lesions and pruritus in dogs with atopic
dermatitis: a blinded randomized prednisolone-controlled
trial. Veterinary Dermatology 2002; 13: 7787.
24. Sugiura H, Uehara M, Hoshino N et al. An open study
of a lotion formulation to improve tolerance of tacrolimus
in facial atopic dermatitis. British Journal of Dermatology
2001; 145: 7958.
25. Drake L, Prendergast M, Maher R et al. The impact of
tacrolimus ointment on health-related quality of life of
adult and pediatric patients with atopic dermatitis.
Journal of the American Academy of Dermatology 2001;
44: S6572.
26. Kang S, Lucky AW, Pariser D et al. Long-term safety
and efficacy of tacrolimus ointment for the treatment of

2004 European Society of Veterinary Dermatology, Veterinary Dermatology, 15, 294303

302

27.

28.

29.

30.

31.

32.

33.

34.

R Marsella et al.

atopic dermatitis in children. Journal of the American

Academy of Dermatology 2001; 44: S58 64.
Kang S, Paller A, Soter N et al. Safe treatment of head/
neck AD with tacrolimus ointment. Journal of Dermatological Treatment 2003; 14: 86 94.
Reitamo S, Wollenberg A, Schopf E et al. Safety and efficacy of 1 year of tacrolimus ointment monotherapy in
adults with atopic dermatitis. Archives of Dermatology
2000; 136: 999 1006.
Soter NA, Fleischer AB Jr, Webster GF et al. Tacrolimus
ointment for the treatment of atopic dermatitis in adult
patients: part II, safety. Journal of the American Academy
of Dermatology 2001; 44: S39 46.
Hanifin JM, Ling MR, Langley R et al. Tacrolimus ointment for the treatment of atopic dermatitis in adult
patients: part I, efficacy. Journal of the American
Academy of Dermatology 2001; 44: S28 38.
Olivry T, Steffan J, Fisch RD et al. Randomized controlled trial of the efficacy of cyclosporine in the treatment
of atopic dermatitis in dogs. Journal of the American
Veterinary Medical Association 2002; 221: 370 7.
Deboer DJ, Schafer JH, Salsbury CS et al. Multiplecenter study of reduced-concentration triamcinolone
topical solution for the treatment of dogs with known or
suspected allergic pruritus. American Journal of Veterinary Research 2002; 63: 408 13.
Reitamo S, Rustin M, Ruzicka T et al. Efficacy and
safety of tacrolimus ointment compared with that of
hydrocortisone butyrate ointment in adult patients with
atopic dermatitis. Journal of Allergy and Clinical Immunology 2002; 109: 547 55.
Ellis CN, Drake LA, Prendergast MM et al. Costeffectiveness analysis of tacrolimus ointment versus

35.

36.

37.

38.

39.

40.

41.

42.

high-potency topical corticosteroids in adults with moderate to severe atopic dermatitis. Journal of the American
Academy of Dermatology 2003; 48: 55363.
Constance C, Pournaras JL, Saurat JH. Staphylococcal
colonization in atopic dermatitis treatment with topical
tacrolimus (FK506). Journal of Investigative Dermatology 2001; 116: 4801.
Petzelbauer P, Wolff K. Effects of cyclosporin A on resident and passenger immune cells of normal human skin
and UV-induced erythema reactions. British Journal of
Dermatology 1992; 127: 5605.
Dupuy P, Bagot M, Michel L et al. Cyclosporin A inhibits the antigen-presenting functions of freshly isolated
human Langerhans cells in vitro. Journal of Investigative
Dermatology 1991; 96: 40813.
Haftek M, Urabe A, Kanitakis J et al. Cyclosporin A
inhibits DNA synthesis by epidermal Langerhans cells.
Regional Immunology 199091; 3: 23641.
Steffan J, Alexander D, Brovedani F et al. Comparison of
cyclosporine A with methylprednisolone for treatment of
canine atopic dermatitis: a parallel, blinded, randomized
controlled trial. Veterinary Dermatology 2003; 14: 1122.
Monti P, Mercalli A, Leone BE et al. Rapamycin impairs
antigen uptake of human dendritic cells. Transplantation
2003; 75: 13745.
Nghiem P, Pearson G, Langley RG. Tacrolimus and
pimecrolimus. From clever prokaryotes to inhibiting calcineurin and treating atopic dermatitis. Journal of the
American Academy of Dermatology 2002; 46: 228 41.
Meurer M, Folster-Holst R, Wozel G et al. Pimecrolimus
cream in the long-term management of atopic dermatitis
in adults: a six-month study. Dermatology 2002; 205:
2717.

Rsum Le tacrolimus topique est utilis avec succs chez lhomme pour le traitement de la dermatite atopique
(AD). Des tudes prliminaires chez les chiens AD utilisant le tacrolimus sous forme liquide ont montr que
le tacrolimus permettait une diminution significative de lrythme et du prurit selon linvestigateur, mais aucune
amlioration na t observe par le propritaire. Les buts de cette tude taient dvaluer lefficacit clinique et
la tolrance dune prsentation commerciale de 0.1% de tacrolimus (Protopic) chez des chiens prsentant une
AD.
Ltude tait en double aveugle, contrle par placbo, en cross-over. Les chiens slectionns ont reu soit le
traitement avec le tacrolimus soit le placbo pendant 4 semaines. Aprs 4 semaines, une priode de 2 semaines
sans traitement tait ralise et les traitements taient changs. Douze chiens ont t inclus. Les signes cliniques
ont t scors. Des prlvements sanguins ont t raliss pour numration-formule, biochimie et dtermination
des niveaus sanguins de tacrolimus avant et la fin de chaque priode de traitement.
La pommade au tacrolimus a permis une diminution significaitve de la svrit des lsions pour les propritaires
et les investigateurs. Lorsque les chiens taient traits avec le placbo, aucune diffrence na t note entre les
scores avant et aprs traitement. Les chiens dermatose localise ont mieux rpondu que ceux prsentant une
dermatose gnralise. Le tacrolimus a t dtect dans le sang des animaux traits avec le principe actif. Les
niveaux dcels taient infrieurs ceux potentiellement toxiques et aucun effet secondaire na t not chez les
chiens traits. Aucune modification des donnes de la NF ou de la biochimie na t observe. En conclusion, le
tacrolimus apparat comme une alternative sre et efficace comme traitement de lAD, en particulier chez les
chiens dermatose localise.
Resumen El tacrolimus tpico se utiliza con xito en personas con dermatitis atpica (AD). Los estudios preliminares en perros con AD utilizando tacrolimus en una formulacin en forma de locin compuesta indicaron
que el tacrolimus disminua significativamente el eritema y prurito de acuerdo con el investigador, pero los propietarios no apreciaron una mejora significativa. Los objetivos del presente estudio fueron evaluar la eficacia clnica
y la seguridad del ungento de tacrolimus comercial al 0,1% (Protopic) en perros con AD.
El trabajo fue diseado como un estudio cruzado doble ciego, con control placebo. A los perros seleccionados
se les administr tacrolimus o placebo durante 4 semanas. Despus de 4 semanas se estableci un periodo de retirada
de 2 semanas y se intercambiaron los tratamientos. Doce perros completaron el estudio. Se puntuaron los
2004 European Society of Veterinary Dermatology, Veterinary Dermatology, 15, 294 303

Tacrolimus for canine atopic dermatitis

303

sntomas clnicos. Se tomaron muestras de sangre para realizar un recuento completo (CBC), paneles
bioqumicos y los niveles de tacrolimus en las semanas 0 y 4 de cada tratamiento.
El ungento de tacrolimus disminuy significativamente los sntomas tanto segn los propietarios como para
los investigadores al final de la prueba. Cuando los mismos perros recibieron el placebo, no existan diferencias
entre las puntuaciones de las semanas 0 y 4. Los perros con enfermedad localizada respondieron mejor que los
perros con enfermedad generalizada. Se detect tacrolimus en sangre de animales que reciban el producto activo.
Los niveles se encontraban por debajo el nivel de toxicidad y no se observaron efectos adversos en ninguno de
los perros. No se detectaron cambios en el CBC y parmetros bioqumicos entre grupos o dentro de los mismos.
En conclusin, el tacrolimus parece ser una alternativa segura como tratamiento de perros con AD, especialmente
en aquellos con enfermedad localizada.
Zusammenfassung Topisches Tacrolimus wurde bei Menschen mit atopischer Dermatitis (AD) erfolgreich
angewandt. Einleitende Studien mit Tacrolimus als Lotion bei Hunden mit atopischer Dermatitis zeigten, dass
Tacrolimus nach Einschtzung der Investigatoren signifikant Erythem und Pruritus verminderte, von den
Besitzern wurde jedoch keine signifikante Verbesserung berichtet. Ziel dieser Studie war es, die klinische Wirksamkeit und Vertrglichkeit einer kommerziell verfgbaren 0,1%-igen Tacrolimus-Salbe (Protopic) bei Hunden
mit AD zu prfen. Studiendesign war doppelt verblindet, placebo-kontrolliert und cross-over. Die ausgewhlten
Hunde wurden fr vier Wochen entweder der Tacrolimus-oder der Placebogruppe zugeteilt. Nach 4 Wochen gab
es einen Wash-out-Zeitraum von 2 Wochen und dann wurden die Behandlungen gewechselt. Zwlf Hunde
vollendeten die Studie. Die klinischen Anzeichen wurden bewertet. Blutproben fr Blutbild, Blutchemie und
Tacrolimusspiegel wurden an Woche 0 und 4 jeder Behandlung entnommen.
Tacrolimus-Salbe verringerte zum Ende der Untersuchung signifikant den Grad der Symptome sowohl fr die
Besitzer als auch fr die Investigatoren. Wenn dieselben Hunde Placebo erhielten, gab es keine Unterschiede
zwischen der Bewertung von Woche 0 und 4. Hunde mit lokalisierter Erkrankung reagierten besser als Hunde
mit generalisierter Erkrankung. Im Blut der Hunde, die den aktiven Wirkstoff erhielten, wurde Tacrolimus
nachgewiesen. Wirkstoffspiegel waren unterhalb der Toxizittsgrenze und Nebenwirkungen wurden bei keinem
der Hunde berichtet. Vernderungen des Blutbildes und der Blutchemie wurden zwischen den Gruppen und
innerhalb der Gruppen nicht nachgewiesen. Folglich scheint Tacrolimus eine sichere Alternative zur Behandlung
von Hunden mit AD, besonders solcher mit lokalisierter Erkrankung zu sein.

2004 European Society of Veterinary Dermatology, Veterinary Dermatology, 15, 294303