Administration of Miltefosine and Meglumine Antimoniate in Healthy ...

Toxicologic Pathology, 000: 1-6, 2009
Copyright # 2009 by The Author(s)
ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1177/0192623309344088
Toxicol Pathol OnlineFirst, published on August 18, 2009 as doi:10.1177/0192623309344088
Administration of Miltefosine and Meglumine Antimoniate
in Healthy Dogs: Clinicopathological Evaluation of the Impact
on the Kidneys
PAOLO BIANCIARDI, 1 CLAUDIO BROVIDA, 2 MARIALUISA VALENTE, 3 LUCA ARESU, 4 LAURA CAVICCHIOLI, 4 CLAUDIA VISCHER, 5
LUCIE GIROUD, 5 AND MASSIMO CASTAGNARO 4
1 20147 Milano, Italy
2 ANUBI Ospedale per Animali da Compagnia, 10024 Moncalieri, Torino, Italy
3 Department of Medical-Diagnostic Sciences and Special Therapies, University of Padua, Medical School,
35121 Padova, Italy
4 Dipartimento di Sanità Pubblica Patologia Comparata e Igiene Veterinaria, Facoltà di Medicina Veterinaria,
Università degli Studi di Padova, 35020 Legnaro, Padova, Italy
5 Virbac S.A., Carros, France
ABSTRACT
In canine leishmaniosis (CanL), kidneys are affected in virtually all dogs. Treatment of CanL is limited in Europe to meglumine antimoniate and
miltefosine. This study evaluated the pharmacological, toxicological, and pathological effects of both drugs in healthy beagle dogs. Four male and
four female dogs were divided into two groups. The animals in Group 1 were administered an oral solution of 2% of miltefosine at 2 mg/kg b.w. once
a day, for twenty-eight days. The animals in Group 2 were administered a preparation of meglumine antimoniate at 100 mg/kg b.w. subcutaneously
once a day for twenty-eight days. After treatment, all dogs were followed-up for a further twenty-eight days. Dogs were observed daily and clinically
examined ten times throughout the study. On days -1 and 55 a renal biopsy was performed on all dogs and analyzed by light microscopy, immunofluorescence,
and electron microscopy. All the examinations failed to demonstrate any lesions in the miltefosine-treated dogs. Conversely, all the
meglumine antimoniate–treated dogs demonstrated severe tubular damage, characterized by tubular cell necrosis and apoptosis. In conclusion,
although no clinical signs of renal disease were evident, the use of meglumine antimoniate in the pharmacological treatment approach of CanLaffected
dogs should be carefully considered.
Keywords: Leishmania; kidney; dogs; miltefosine; meglumine antimoniate; pharmacological effect.
INTRODUCTION
Canine leishmaniosis (CanL) is a multisystemic disease with
variable clinical signs (Baneth 2006; Ciaramella et al. 1997) . The
majority of affected dogs present with poor body condition, generalized
muscular atrophy, lymphadenomegaly, and excessive
skin scaling. Kidneys are affected in virtually all dogs with
CanL, and renal disease might be the only apparent abnormality
in infected dogs (Baneth and Shaw 2002; Costa et al. 2003).
Until recently, the most commonly used drug for the treatment
of CanL has been the pentavalent antimony meglumine
antimoniate (N-methylglucamine antimoniate, Glucantime,
Mérial), alone or in combination with allopurinol (Miró,
Cardoso, et al. 2008; Nieto et al. 1992, Noli and Auxilia 2005).
In 2007, the first oral therapy for CanL based on miltefosine
became available in some EU countries. Miltefosine (1-Ohexadecylphosphocholine,
Milteforan, Virbac), an alkylphosphocholine
and a membrane-active synthetic ether–lipid
Address correspondence to: Paolo Bianciardi, DVM, Dip.EVPC, via
dell’Usignolo 1, Milano, Italy; e-mail: paolobianci@libero.it.
Financial Disclosure: All expenses required for trial (availablity of dogs,
materials for procedures and laboratory analyses) were financed by Virbac SA.
Abbreviations: LA, Luca Aresu; LC, Laura Cavicchioli; MC, Massimo
Castagnaro.
1
analogue originally developed for the treatment of cutaneous
metastasis from mammary carcinomas (Hilgard et al. 1993),
has proved to be an effective treatment for both human cutaneous
and visceral leishmaniosis (Jha et al. 1999; Pearson
2003; Sundar et al. 1998; Sundar et al. 1999; Verma and Dey
2004) and later on for canine visceral leishmaniosis (Mateo
et al. 2009; Miró, Oliva, et al. 2009).
Although the treatment of CanL is currently limited in some
EU countries mainly to meglumine antimoniate and miltefosine,
the information on the impact of these drugs on the
clinical and kidney status of healthy dogs is limited.
Since the evaluation of the impact of these drugs on the kidneys
in CanL affected dogs is not practical owing to the influence
of underlying renal disease, the aim of this study was to
evaluate the pharmacologic, toxicologic, and pathologic effects
of a standard treatment with both drugs in healthy beagle dogs.
Animals
MATERIALS AND METHODS
Four healthy male and four healthy female beagle dogs from
Harlan (Netherlands) were used. The animals were seven to
nine months of age and weighed from 7 to 9 kg at the start

2 BIANCIARDI ET AL. TOXICOLOGIC PATHOLOGY
Treatment Groups
of the study. They were housed in individual cages in an environmentally
controlled room with a twelve-hour light cycle, a
temperature of 18 C + 3 C and 55% + 10% relative humidity.
They were fed daily with dry pet food (Virbac Vet Complex)
according to their individual weight. Drinking water
was available ad libitum. All procedures and maintenance of
animals were in accordance with internal procedure and the
principles of humane treatment as elaborated by the National
Institutes of Health. All dogs were identified with a code tattooed
on the inside of the ear flap.
Study Design
The dogs were divided into two randomized groups
(Table 1). The animals in Group 1 were administered an oral
solution of 2% of miltefosine (Milteforan, Virbac) at a dose
rate of 2 mg/kg b.w. once a day, for twenty-eight consecutive
days (1 mL/10 kg/day). The oral solution was poured onto the
dogs’ food at mealtime. The animals in Group 2 were administered
an injectable preparation of meglumine antimoniate (Glucantime,
Mérial) at a dose rate of 100 mg/kg b.w.
subcutaneously once a day for twenty-eight consecutive days
(3.3 mL/10 kg/day).
Dogs were observed daily and clinically examined ten times
throughout the study. At the initial pre-inclusion visit, day -4,
dogs were clinically examined and randomized. On the same
day, the animals were weighed, and this procedure was
repeated weekly until the end of the study (to adjust the drug
dose rates according to body weight). Dogs underwent weekly
complete clinical examinations on days -1, 6, 13, 21, 27, 34, 41,
48, and 55. On days -1, 27, and 55, a blood sample was taken
from the jugular vein. Furthermore, a urine sample was collected
from the bladder, by cystocentesis, prior to the biopsy
procedure. The analyses performed included routine hematology,
biochemistry, and protein electrophoresis on the blood and
complete urinalysis. All the samples were analyzed by the same
laboratory (Vébiotel Laboratoire de Biologie Vétérinaire,
Arcueil, France). On days -1 and 55, an ultrasound-guided
renal biopsy was performed under anaesthesia on all dogs
(Table 2). After the clinical evaluation, an intravenous catheter
(22G) was placed in the left or right cephalic vein and connected
to an infusion line, for fluid therapy (normal saline solution,
0.9% NaCl at the rate of 10 mL/kg/hour) and anaesthesia
induction. Anaesthesia was performed using an association of
tiletamine chloride and zolazepam chloride (Zoletil 50, Virbac),
at the dose of 0.15 mL/kg injected intravenously. The dog
TABLE 1.—Identification of dogs in each treatment group.
Group 1: Miltefosine (Milteforan, Virbac: 1 mL/10 kg orally
Group 2 Meglumine antimoniate (Glucantime, Mérial: 3.3 mL/10 kg s.c.
once a day for twenty-eight days)
once a day for twenty-eight days)
H7E 1023 Female H7I 1417 Female
H7I 1401 Female H7G 1329 Female
H7F 1144 Male H7F 1076 Male
H7G 1342 Male H7E 0886 Male
TABLE 2.—Study protocol.
Day Actions
D -4 Weight - clinical examination
Randomization
D -1 Clinical examination
Blood sampling
Urine sampling
Kidney biopsy
D0 to D27 1st treatment with miltefosine with the food (Group 1)
1st treatment with meglumine antimoniate by s.c.
injection (Group 2)
Daily observation
D6; D13; D21 Weight - clinical examination
D27 Weight - clinical examination
Blood sampling
Urine sampling
D28 to D54 Daily observation
D34; D41; D48 Weight - clinical examination
D55 Weight - clinical examination
Blood sampling
Urine sampling
Kidney biopsy
was positioned in right lateral recumbency for the first biopsy
that was performed on the left kidney on day -1, and vice versa
on the right kidney, with the dog recumbent on the left side on
day 55. This approach was chosen to avoid that the second
biopsy would be performed on scarred tissue following the previous
procedure. For both procedures, the haircoat was clipped
and the skin area prepared aseptically.
The biopsies were ultrasound guided (GE, mod Logic E,
with microconvex 5–9 Mhz probe) and performed using an
18G True-cut disposable biopsy needle driven by a gun, with a
spring trigger system (Magnum Bard). Based on an immediate
evaluation by the pathologist, using a stereomicroscope, one or
two biopsies were taken from the renal caudal pole of each dog.
To avoid clot formation in the renal pelvis, fluid therapy was
maintained for about thirty minutes after each biopsy until the
dogs woke up, whereas the femoral pulse and the capillary
refill time (CRT) were continuously monitored. Before discharge,
each dog was checked by ultrasound for signs of
hemorrhage at the biopsy site.
Once the dogs were completely awake and clinically normal,
they were taken to their individual cages. For the handling
of the biopsy specimens, a standard protocol was followed.
Renal biopsies were divided into three parts for light microscopy,
immunofluorescence, and electron microscopy

Vol. 000, No. 00, 2009 MILTEFOSINE AND GLUCANTIME IN DOGS 3
purposes. Renal biopsies were evaluated at the Department of
Public Health, Comparative Pathology and Veterinary
Hygiene–Anatomical Pathology Section of Padua University.
Light Microscopy
For light microscopy, biopsies were fixed in formalin,
embedded in paraffin, and serially sectioned at 4-mm intervals.
Alternate sections were stained with: hematoxylin-eosin (HE),
periodic acid-Schiff (PAS), acid-fuchsin orange G (AFOG),
Masson’s Trichrome, Periodic acid-Schiff methanamine
(PASM), and Miller’s elastin stain. The evaluations were performed
blinded by three independent pathologists (LA, LC,
MC). Discordant data were reevaluated by the investigators,
and a consensus evaluation was used for the definitive analysis.
Immunofluorescence
For direct immunofluorescence examination, fresh unfixed
renal biopsies were embedded in optimal cutting temperature
(OCT) compound, snap-frozen in liquid nitrogen, and stored
at –80 C. Subsequently, 5-mm-thick sections were fixed with
acetone for fifteen minutes. After washing with phosphate buffered
saline (PBS) (two passages), slides were incubated with
FITC-labelled anti-goat IgA, IgG, IgM and complement C3
antibodies (Bethyl Laboratories, Inc., Montgomery, TX, USA).
Primary antibodies were omitted as negative controls and substituted
with PBS.
Electron Microscopy
Renal tissue specimens were fixed in Karnovsky fixative,
stored in 0.1 M sodium cacodylate (pH 7.2), and postfixed in
2% 0s0 4, buffered with 0.1 M sodium cacodylate (pH 7.2).
After rinsing twice for thirty minutes each time with distilled
water, tissue specimens were stained with 2% uranyl acetate
twice for two hours each time at room temperature, dehydrated
in a graded series of acetone, and embedded in Durcopan ACM
(Fluka Ag., Buchs, Switzerland). Ultrathin sections (50 nm)
were stained with lead citrate and examined with a LEO electron
microscope (Carl Zeiss electron microscopes, NY).
Baseline Characteristics
RESULTS
At the pre-inclusion visit (day -4) and at the day -1 visit, all
dogs were clinically healthy. The blood analyses performed on
all dogs on day -1 did not show any significant abnormalities.
The urinalysis performed on day -1 indicated an abnormal
urine protein/urine creatinine (Up/Uc) ratio in only one dog
from Group 2 (Up/Uc ratio ¼ 1.74).
Body weight
The dogs in Group 1 had a weight gain of 0.5 to 0.9 kg
between day -4 and day 55. Three of the four dogs in Group
2 had a weight gain of 0.15 to 0.7 kg during the same period.
One dog from Group 2 (H7E 0886) had a weight loss of
0.15 kg between Day -4 and Day 55.
Clinical Results
Globally, all the animals remained clinically healthy
throughout the study period.
Blood Results
The blood examinations performed on day 27 did not show
any relevant abnormality. The hematological and biochemical
results were within the normal range for all dogs. At day 55,
one dog in Group 2 (H7E 0886) showed an increased urea value
(0.8g/L; cutoff level: 0.6 g/L).
Urine Results
The urinalysis performed on day 27 indicated increased
urine protein levels in four dogs (two dogs in each group), with
all other parameters remaining within normal reference ranges.
At the examination performed on day 55, three out of the four
dogs still showed increased urine protein levels, without any
other detected abnormalities.
Light Microscopy
Sampling for light microscopy was adequate in all biopsies,
with a mean glomerular count of 17.4 (range, ten to thirty-six
glomeruli). Light microscopy evaluation of the renal biopsies
showed no alterations in the eight dogs at baseline (day -1).
After fifty-five days, the two groups of dogs treated with the
two compounds presented different histology features. The
four dogs treated with miltefosine showed normal glomeruli
and minimal vacuolization of the proximal tubular epithelial
cells with a scattered distribution. The vascular compartment
was within normal limits. The four dogs treated with meglumine
antimoniate showed a normal glomerular pattern. The
predominant findings were restricted to the tubular compartment
and were similar in all dogs treated with this drug. Diffusely,
tubular cells showed marked swelling with pale
cytoplasm, and in multifocal areas, coagulative necrosis
features were evident. Sloughed tubular cells were present in
tubular lumina, and individual tubular cell necrosis with picnotic
nuclei was characteristic in these tubules (Figure 1). Some
tubular cells underwent apoptosis with phagocytosis of apoptotic
bodies by neighboring epithelial cells (Figures 2 and 3). No
tubular casts or crystalline deposits were evident. The luminal
borders were markedly irregular, with simplified cells alternating
with enlarged, hypereosinophilic cells. Focal interstitial
edema with minimum mononuclear inflammatory infiltrates
without tubulitis was evident. Vessels were normal.
Immunofluorescence
Immunofluorescent reactions for IgG, IgM, IgA and C3
were negative in the renal biopsies at baseline (day -1) and at
the control time (day 55) in both groups.

4 BIANCIARDI ET AL. TOXICOLOGIC PATHOLOGY
FIGURE 1.—Sloughed tubular cells are present in tubular lumina, and
individual tubular cell necrosis with pyknotic nuclei are evident.
400 ; hematoxylin and eosin.
Electron Microscopy
Electron microscopy confirmed the severe tubular damage
seen in the meglumine antimoniate–treated dogs. Proximal
tubules exhibited epithelial simplification with reduced organellar
content, loss or attenuation of brush border, cellular
detachment from the tubular basement membrane, apical blebbing,
widened intercellular spaces, individual cell necrosis, and
focal shedding of cytoplasmic debris into the tubular lumen. No
lesions were detected at the ultrastructural level in the
miltefosine-treated dogs.
DISCUSSION
The kidneys are affected in virtually all dogs with CanL, and
renal disease might be the only apparent abnormality in
infected dogs. Renal disease can progress from asymptomatic
proteinuria to the nephrotic syndrome or chronic renal failure
with glomerulonephritis, tubulointerstitial nephritis, and amyloidosis
(Koutinas et al. 1999). Glomerular lesions developing
in dogs during infection with Leishmania organisms are associated
with the presence of immune complexes (Nieto et al.
1992) and have been classified histologically as mesangial glomerulonephritis,
membranous glomerulonephritis, membranoproliferative
glomerulonephritis, and focal segmental
glomerulosclerosis (Aresu, Pregel, et al. 2008). Tubulointerstitial
nephritis has been reported with different incidences by different
authors in CanL-affected dogs, but it has never been
observed as a solitary lesion (Aresu, Rastaldi, et al. 2008). The
occurrence of tubulointerstitial lesions is generally considered
as a consequence of the progression of immune-mediated glomerular
lesions, which are classically primary lesions (Zatelli
et al. 2003).
Despite the high prevalence of kidney damage, increase of
serum creatinine and urea resulting from primary kidney failure
is evident only when the majority of nephrons are lost, which
happens rather late during disease progression (Baneth et al.
2008).
FIGURES 2 AND 3.—Diffuse desquamation of apoptotic tubular epithelial
cells into the lumen, several with pyknotic nuclei. Granular casts
are evident with necrotic cell debris. 400 ; hematoxylin and eosin.
Treatment with antimonials has sometimes been pointed out
as being responsible for the deterioration of renal conditions of
already affected kidneys in leishmaniotic dogs. Nevertheless,
the information available on the toxicology, pharmacokinetics,
and pharmacodynamics of this drug in the dog is scarce. A specific
study with meglumine antimoniate has shown that the
pharmacokinetic behavior of this drug in healthy dogs differs
considerably from that described in man (Tassi et al. 1994).
This and similar studies (Belloli et al. 1995) have shown that
the subcutaneous route of administration is the most suitable
to maintain the serum level of antimony (Sb) over time. After
subcutaneous administration, the availability of the drug was
close to 100%, and the maximum concentration of antimony was
achieved within three to five hours, with an immediate decline in
the drug concentration, which reached values close to the detection
limit only eighteen hours after injection. The half-life was of
121 + 6 minutes (Tassi et al. 1994). Meglumine antimoniate has
been reported to be rapidly eliminated by the kidneys in healthy
dogs, mainly by glomerular filtration. After subcutaneous injection,
more than 80% of the drug is eliminated through the kidneys
(Belloli et al. 1995; Tassi et al. 1994).

Vol. 000, No. 00, 2009 MILTEFOSINE AND GLUCANTIME IN DOGS 5
Monitoring of Sb urinary excretion indicates that the kidneys
are the almost exclusive route of elimination of meglumine
antimoniate (Hantson et al. 2000); however, no specific
guidelines exist for dosage adjustment in renal failure. Even
if nephrotoxicity has rarely been related to this treatment in
humans, some cases of acute renal failure as a result of acute
tubular necrosis, followed by death after receiving Glucantime,
have been reported in human patients (Rodrigues et al. 1999).
In animals, renal function was assessed in rats treated with
the pentavalent antimonials, Glucantime (meglumine antimoniate,
Rhodia) or Pentostam (sodium stibogluconate, Wellcome).
When administered at a dose rate of 30 mg of Sb
(Glucantime or Pentostam) per 100 g of body weight per day
for thirty days, renal functional changes were observed consisting
of disturbances in urine concentrating capacity, suggesting
an interference of the drugs in the action of antidiuretic
hormone on the distal tubules and collecting ducts. The disturbance
in urine concentration was reversible after a seven-day
period without the drugs being administered. No significant
histopathological alterations were observed in the kidneys of
the rats treated with the drugs. Only the rats treated with a high
dose of Pentostam (200 mg/100 g body weight/day) demonstrated
functional and histopathological alterations of acute
tubular necrosis (Veiga et al. 1990)
Adverse effects of antimonial therapy in humans include
lethargy, anorexia, pneumonia, nausea, urticaria, fever, vomiting,
abscess formation, cardiotoxicity, hepatotoxicity, and
nephrotoxicity (Davidson 1998; Ikeda-Garcia et al. 2007).
In dogs, the most commonly observed adverse effects associated
with meglumine antimoniate are apathy, anorexia,
vomiting, diarrhea, and pain at the site of injection (Baneth and
Shaw 2002); however, the frequency and severity of these
adverse effects are unknown (Bravo et al. 1993; Noli 1999).
Miltefosine has only recently become available for the treatment
of CanL, therefore very little is known of its effect on the
kidney of leishmaniotic dogs in the field. Several pharmacokinetic
studies have been performed with miltefosine, in laboratory
animals and in the target species; miltefosine was also
assayed in dog plasma, urine, and feces by HPLC-MSMS analyses
(Virbac, data on file Study Nos. F-107.010000-60008;
F-107.010000-60010; F-107.010000-60027).
In rats and dogs, miltefosine showed an absolute bioavailability
of 82% and 94%, respectively, with the time for
maximum concentration values (T max) ranging from four to
forty-eight hours. In dogs, after repeated oral administration
of miltefosine in food for twenty-eight days, plasma clearance
was determined to be 3.40 + 0.447 mL/kg/hr, corresponding to
an overall body extraction ratio of about 0.06% for a 10-kg dog.
This finding suggests that, in dogs, the metabolic efficiency to
transform miltefosine into its different metabolites is low and
that there is no hepatic first-pass metabolism.
The terminal elimination half-life in rats was approximately
eighty hours (3.3 days), and 153 hours (153 + 13.7 hr) in dogs,
equivalent to 6.3 days. This long terminal half-life can be
explained by the low plasma clearance of miltefosine. Considering
this terminal half-life in dogs, a steady state can be
expected after about three to four weeks of repeated daily
administration of miltefosine.
In dogs, repeated administration of 2 mg/kg/day of miltefosine
for twenty-eight days led to an accumulation index of
7.65 + 1.99. The accumulation index is the ratio between the
amount of miltefosine (AUC0-24hr) reached at a steady state and
that obtained after the first administration of miltefosine. After
the last administration, the Cmax was 32582 + 4030 ng/mL
with a mean Tmax of 5.0 + 2.0 hours.
These data indicate an increase of plasma miltefosine concentration
within the first two weeks of treatment, reaching a
‘‘steady state’’ until the end of treatment (twenty-eight days).
At the end of treatment, there is a slow and linear decrease
of plasma miltefosine with complete elimination in a further
four weeks. Miltefosine is widely distributed in body organs,
undergoes a slow metabolic breakdown, and is metabolized
in the liver into choline and choline-containing metabolites
(Berman 2005).
Miltefosine is slowly excreted in the feces. The mean fecal
clearance observed was low (0.32 + 0.13 mL/kg/hr), which is
very similar to the bile flow rate in dogs, suggesting that miltefosine
fecal clearance is actually a biliary clearance. The contribution
of fecal clearance to total body clearance was 10% +
4.86%, meaning that the elimination of miltefosine is likely to
undergo an extensive, but slow, metabolism.
As fecal clearance represents about 10% of total clearance,
it can be concluded that only about 10% of the administered
dose is eliminated as its parent drug in the feces (i.e., about
200 mg/kg/day).
The miltefosine urinary concentration was rather low and
below the limit of quantification (20 ng/mL of miltefosine)
after three days. The contribution of renal clearance to total
body clearance was considered as negligible (about 0.03%).
These results suggest that urine is therefore only a minor route
of miltefosine parent drug elimination.
As in human beings, side effects induced by miltefosine in
dogs are mostly gastrointestinal, such as occasional vomiting
and diarrhea. They are dose-dependent and at the recommended
dose rate (2 mg/kg/day) are generally mild and transient,
disappearing without therapy after the end of treatment
period.
Our results confirm that the impact of miltefosine on kidney
functions and on the clinical status of dogs is limited. The only
abnormal clinical value was limited to an abnormal Up/Uc ratio
in one dog from the meglumine antimoniate–treated group
(Up/Uc ratio ¼ 1.74) at the pre-inclusion visit, which can be
explained by blood contamination of the urine samples during
cystocentesis. In the miltefosine-treated group, light microscopy
examinations demonstrated normal glomeruli and minimal
vacuolization of the proximal tubular epithelial cells with
scattered distribution. Immunofluorescent examinations failed
to detect any immune deposits, and electron microscopy examinations
did not demonstrate any lesions. Although there is a
lack of clinical evidence of kidney involvement, all the
meglumine antimoniate–treated dogs demonstrated morphological
changes consistent with severe tubular damage. In light

6 BIANCIARDI ET AL. TOXICOLOGIC PATHOLOGY
microscopy examinations, diffuse marked swelling with pale
cytoplasm of tubular cells and multifocal areas of coagulative
necrosis were seen. Sloughed tubular cells and individual
tubular cell necrosis with picnotic nuclei were also evident in
these tubules. Ultrastructural examination confirmed the
damage seen at the light microscopy level. This severe damage
was evident four weeks after the end of the therapy without
signs of regeneration of the damaged tissues.
In conclusion, although in both groups of treated animals no
clinical signs of renal disease were evident, in light of our morphological
results on renal biopsies, the pharmacological treatment
approach of CanL-affected dogs should be carefully
evaluated.
REFERENCES
Aresu, L., Pregel, P., Bollo, E., Palmerini, D., Sereno, A., and Valenza, F.
(2008) Immunofluorescence staining for the detection of immunoglobulins
and complement (C3) in dogs with renal disease. Vet Rec 163,
679–82.
Aresu, L., Rastaldi, M. P., Pregel, P., Valenza, F., Radaelli, E., Scanziani, E.,
and Castagnaro, M. (2008) Dog as model for down-expression of
E-cadherin and beta-catenin in tubular epithelial cells in renal fibrosis.
Virchows Arch 453, 617–25.
Baneth, G. (2006). Leishmaniases. In Infectious diseases of the dog and cat
(3rd ed.), ed. C. E. Green, 685–98. Philadelphia: Saunders.
Baneth, G., Koutinas, A. F., Solano-Gallego, L., Bourdeau, P., and Ferrer, L.
(2008). Canine leishmaniosis–new concepts and insights on an expanding
zoonosis: part one. Trends Parasitol 24, 324–30.
Baneth, G., and Shaw, S. E. (2002). Chemotherapy of canine leishmaniosis. Vet
Parasitol 106, 315–24.
Belloli, C., Ceci, L., Carli, S., Tassi, P., Montesissa, C., De Natale, G., Marcotrigiano,
G., and Ormas, P. (1995). Disposition of antimony and aminosidine
in dogs after administration separately and together: implications
for therapy of leishmaniasis. Res Vet Sci 58, 123–27.
Berman, J. (2005) Miltefosine to treat leishmaniasis, Expert Opin Pharmacother
6, 1381–88.
Bravo, L., Frank, L. A., and Brenneman, K. A. (1993). Canine leishmaniasis in
the United States. Compendium on Continuing Education for the Practicing
Veterinarian 15, 699–708.
Ciaramella, P., Oliva, G., Luna, R. D., Gradoni, L., Ambrosio, R., Cortese, L.,
Scalone, A., and Persechino, A. (1997). A retrospective clinical study of
canine leishmaniosis in 150 dogs naturally infected by Leishmania infantum.
Vet Rec 141, 539–43.
Costa, F. A., Goto, H., Saldanha, L. C., Silva, S. M., Sinhorini, I. L., Silva,
T. C., Guerra, J. L. (2003). Histopathologic patterns of nephropathy in
naturally acquired canine visceral leishmaniasis. Vet Pathol 40, 677–84.
Davidson, R. N. (1998). Practical guide for treatment of leishmaniasis. Drugs.
56, 1009–18.
Hantson, P., Luyasu, S., Haufroid, V., and Lambert, M. (2000). Antimony
excretion in a patient with renal impairment during meglumine antimoniate
therapy. Pharmacotherapy 20, 1141–43.
Hilgard, P., Klenner, T., Stekar, J., and Unger, C. (1993). Alkylphosphocholines:
a new class of membrane active anticancer agents. Cancer Chemother
Pharmacol 32, 90–95.
Ikeda-Garcia, F. A., Lopes, R. S., Ciarlini, P. C., Marques, F. J., Lima, V. M.,
Perri, S. H., and Feitosa, M. M. (2007). Evaluation of renal and hepatic
functions in dogs naturally infected by visceral leishmaniasis submitted
to treatment with meglumine antimoniate. Res Vet Sci 83, 105–8.
Jha, T. K., Sunder, S., Thakur, C. P., Bachmann, P., Karbwang, J., Fischer, C.,
Voss, A., and Berman, J. (1999). Miltefosine, an oral agent, for the treatment
of Indian visceral leishmaniasis. N Engl J Med 341, 1795–1800.
Koutinas, A. F., Polizopoulou, Z. S., Saridomichelakis, M. N., Argyriadis, D.,
Fytianou, A., and Plevraki, K. G. (1999). Clinical considerations on
canine visceral leishmaniasis in Greece: A retrospective study of 158
cases (1989–1996). J Am Anim Hosp Assoc 35, 376–83.
Mateo, M., Maynard, L., Vischer, C., Bianciardi, P., and Miró, G. (2009) Comparative
study on the short term efficacy and adverse effects of miltefosine
and meglumine antimoniate in dogs with natural leishmaniosis.
Parasitol Res 105, 155–62.
Miró, G., Oliva, G., Cruz, I., Canavate, C., Mortarino, M., Vischer, C., and
Bianciardi, P. (2008). Multi-centric and controlled clinical field study
to evaluate the efficacy and safety of the combination of miltefosine and
allopurinol in the treatment of canine leishmaniosis. Vet Dermatol 19
(Suppl 1), 7–8.
Miró, G., Cardoso, L., Pennisi, M. G., Oliva, G., and Baneth, G. (2008). Canine
leishmaniosis–new concepts and insights on an expanding zoonosis: Part
two. Trends Parasitol 24, 371–77.
Nieto, C. G., Navarrete, I., Habela, M. A., Serrano, F., and Redondo, E. (1992).
Pathological changes in kidneys of dogs with natural Leishmania infection.
Vet Parasitol 45, 33–47.
Noli, C. (1999). Leishmaniosis canina. Waltham Focus 9, 16–24.
Noli, C., and Auxilia, S. T. (2005). Treatment of canine Old World visceral
leishmaniasis: A systematic review. Vet Dermatol 16, 213–32.
Pearson, R. D. (2003). Development status of miltefosine as first oral drug in
visceral and cutaneous leishmaniasis. Curr Infect Dis Rep. 5, 41–42.
Rodrigues, M. L., Costa, R. S., Souza, C. S., Foss, N. T., and Roselino, A. M.
(1999). Nephrotoxicity attributed to meglumine antimoniate (Glucantime)
in the treatment of generalized cutaneous leishmaniasis. Rev Inst
Med Trop Sao Paulo 41, 33–37.
Sundar, S., Gupta, L. B., Makharia, M. K., Singh, M. K., Voss, A., Resenkaimer,
F., Engel, J., Murray, H. W. (1999). Oral treatment of visceral leishmaniasis
with miltefosine. Ann Trop Med Parasitol 93, 589–97.
Sundar, S., Rosenkaimer, F., Makharia, M. K., Goyel, A. K., Mandal, A. K.,
Voss, A., Hilgard, P., and Murray, H. W. (1998). Trial of oral miltefosine
for visceral leishmaniasis. Lancet 352, 1821–23.
Tassi, P., Ormas, P., Madonna, M., Carli, S., Belloli, C., De Natale, G.,
Ceci, L., and Marcotrigiano, G. O. (1994). Pharmacokinetics of
N-methylglucamine antimoniate after intravenous, intramuscular and
subcutaneous administration in the dog. Res Vet Sci 56, 144–50.
Verma, N. K., and Dey, C. S. (2004) Possible Mechanism of Miltefosine-
Mediated Death of Leishmania donovani. Antimicrob Agents Chemother
48, 3010–15.
Veiga, J. P., Khanam, R., Rosa, T. T., Junqueira Júnior, L. F., Brant, P. C.,
Raick, A. N., Friedman, H., and Marsden, P. D. (1990). Pentavalent antimonial
nephrotoxicity in the rat. Rev Inst Med Trop Sao Paulo 32, 304–9.
Virbac Data On File, European Registration Dossier: Study No. F-107.010000-
60008.
Virbac Data On File, European Registration Dossier: Study No. F-107.000000-
40010.
Virbac Data On File, European Registration Dossier: Study No. F-107.000000-
40027 [Toutain, P. L., (2006) Expert Report: Study of the pharmacokinetics
of miltefosine in plasma, urine and faeces of dogs after oral
repeated administrations of miltefosine in food for 28 days: Pharmacokinetic
Analysis Report].
Zatelli, A., Borgarelli, M., Santilli, R., Bonfanti, U., Nigrisoli, E., Zanatta, R.,
Tarducci, A., and Guarraci, A. (2003). Glomerular lesions in dogs
infected with Leishmania organisms. Am J Vet Res 64, 558–61.
For reprints and permissions queries, please visit SAGE’s Web site at http://www.sagepub.com/journalsPermissions.nav.