Highlights
· Similar to that in human medicine, HER2 is a promising therapeutic target for canine
malignant tumors.
· Lapatinib tosilate hydrate is a molecular targeted drug against the HER family including
HER2; it has a safe and proven anti-tumor effect in humans.
· A dose-escalation study and long-term administration test of lapatinib were performed in
six healthy dogs to determine the MTD and safety of lapatinib in veterinary medicine.
· Oral administration of 35 mg/kg/day lapatinib with meals was found safe for treating
cMGTs.
· Dose-limiting toxicities of lapatinib were weight loss and ALP elevation.
Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor and a member
of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. Its overexpression is
reported to correlate with the factors influencing the malignancy of human breast cancer,
such as proliferation, metastasis, and drug resistance [1, 2]. Human breast cancer is clinically
classified into intrinsic subtypes according to the expression status of hormone receptors
and HER2 and the proliferation index [3, 4]. The treatment for human breast cancer is selected
based on the subtype, and HER2-targeted therapy is administered to patients with
HER2-positive cancer. The first-line chemotherapy for these patients is trastuzumab
(Herceptin®; Chugai Pharmaceutical Co., Ltd., Tokyo, Japan), a humanized
monoclonal antibody that targets human HER2. Once cancer reaches an advanced stage or is
unresponsive to trastuzumab therapy, combination therapy with capecitabine and lapatinib is
considered in human breast cancer. Lapatinib is a low-molecular-weight compound targeting
epidermal growth factor receptor (EGFR) and HER2 [2]
and is reported to be effective against advanced HER2-positive breast cancer [5].
In veterinary medicine, canine mammary gland tumors (cMGTs) are among the most prevalent
tumors in female dogs, and the first choice of treatment of these tumors in the early
clinical stage is surgical excision [6]. For animals
with distant metastasis, conventional cytocidal anticancer agents, such as carboplatin and
doxorubicin, are commonly applied. These agents are widely used for neoplasms in dogs and
prolong the mean survival time after the surgical operation in cases of advanced cMGTs (228
days in cases in which surgery has been performed along with adjuvant chemotherapy and 194
days in cases in which surgery alone has been performed) [7]. However, these agents have side effects, such as myelosuppression and
cardiotoxicity, which sometimes limit their usage and necessitate the development of a new
treatment for cMGTs.
HER2 overexpression has been reported in 18–46% of cMGTs [8]. Therefore, the HER2-targeting agent, lapatinib, might be the new candidate
agent against cMGTs. According to the datasheet of lapatinib, two series of safety tests in
dogs have been conducted: single- and multiple-dose toxicity tests. In the single-dose
toxicity test, doses of 10, 60, and 360 mg/kg were used in healthy dogs and only mild
diarrhea was reported as a side effect. On the other hand, in the multiple-dose toxicity
test, doses of 10, 40, and 160 mg/kg/day were used for 13 weeks and for 39 weeks, and some
side effects including skin inflammation and hepatotoxicity were observed at doses higher
than 40 mg/kg/day concluding NOAEL as 10 mg/kg/day in either time period [9]. In our preliminary study, no adverse effect was
observed at a dose of 20 mg/kg (unpublished data). On the basis of these findings, we
conducted the dose-escalation toxicity test starting from a dose of 30 mg/kg to determine
the maximum tolerated dose (MTD) of lapatinib in dogs.
Healthy Beagle dogs were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Three male
and three female dogs were included in this study. All the dogs were 18 months old and their
mean body weight was 9.2 ± 0.74 kg (range: 7.9–10.1 kg). They were fed with Urban Life
Junior dry food (Royal Canin, Dubai, United Arab Emirates), whose quantity was decided
according to the animals’ body weights, and had ad libitum access to fresh
water. The room temperature was maintained at around 25°C and the humidity was approximately
60%. All animal experiments were approved by the Animal Use Committee of the Faculty of
Agriculture at The University of Tokyo, and we followed the guidelines of The University of
Tokyo for the care and use of animals (approval number: P18-048).
Lapatinib tosilate hydrate (Tykerb®) was purchased from Novartis (Basel,
Switzerland). Lapatinib was encapsulated into capsules (Matsuya, Osaka, Japan) for each
dose. The capsules were administered once a day with food, per os. The general physical
condition of the animals, including the amount of food intake and the presence of vomiting
and diarrhea, was evaluated twice a day. The dogs underwent physical examinations including
heart and respiration rate measurement and auscultation once a week, body weight measurement
and blood examinations every 2 weeks, and chest X-ray before and after the examination. The
blood examination included complete blood count, coagulation test, and biochemistry,
including determination of albumin, alkaline phosphate (ALP), alanine aminotransferase
(ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), total bilirubin
(T-BIL), blood urea nitrogen (BUN), creatinine, glucose, and electrolyte levels (DRI-CHEM
7000V, Fujifilm Corp., Tokyo, Japan). Side effects were evaluated according to the
Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events
(VCOG-CTCAE) ver. 1.1, and some representative side effects are shown in Supplementary Table
1 [10].
Two studies were planned: a dose-escalation study and long-term administration test. In the
dose-escalation study, starting from an initial dose of 30 mg/kg/day, the dose was increased
by 5 mg/kg/day every 2 weeks until a side effect more severe than VCOG-CTCAE grade 3
appeared. The maximum dose that did not result in a grade 3 side effect was considered the
MTD and proceeded to the next examination. In the long-term administration test, we
administered the MTD for 8 weeks after an interval of 8 weeks from previous study. Similar
to many protocols with conventional antitumor drugs, administration for 8 weeks was
considered as one term. The side effect was determined as described above, and the
appearance of grade 3 side effects was set as the endpoint of the study. All dogs were
confirmed to show no clinical abnormalities and normal values in all examinations at the
start of both studies.
The dose-escalation study was conducted to determine the proper dose of lapatinib for dogs.
Table 1 shows the side effects observed with
the administration of 30–40 mg/kg/day lapatinib. One dog developed grade 3 toxicity when
lapatinib was administered at a dose of 40 mg/kg/day. The dose-limiting toxicity was a grade
3 adverse effect, which was defined as a weight loss of >15%. In this dog, the percentage
weight decreased to 84.4%. At doses of 30 and 35 mg/kg/day, no side effects of severity
higher than grade 3 were observed.
Table 1.
Adverse events observed in the dose-escalation study
| Adverse event |
30 mg/kg/day |
35 mg/kg/day |
40 mg/kg/day |
| No. of dogs showing adverse events |
CTCAE grade |
No. of dogs showing adverse events |
CTCAE grade |
No. of dogs showing adverse events |
CTCAE grade |
| Constitutional clinical sign |
Weight loss |
4/6 |
Grade 1 |
2/6 |
Grade 1 |
2/5 |
Grade 1 |
| 1/6 |
Grade 2 |
2/6 |
Grade 2 |
1/5 |
Grade 2 |
|
|
|
|
1/5 |
Grade 3 |
| Dermatologic/Skin |
Erythema |
- |
|
- |
|
3/5 |
Grade 1 |
| Gastrointestinal |
Loose stool |
1/6 |
* |
1/6 |
* |
1/5 |
* |
| Metabolic/Laboratory |
ALP |
5/6 |
Grade 1 |
2/6 |
Grade 1 |
5/5 |
Grade 2 |
| 1/6 |
Grade 2 |
4/6 |
Grade 2 |
|
|
| ALT |
3/6 |
Grade 1 |
2/6 |
Grade 1 |
3/5 |
Grade 1 |
|
|
1/6 |
Grade 2 |
|
|
| T-BIL |
- |
|
1/6 |
Grade 1 |
2/5 |
Grade 1 |
|
|
1/5 |
Grade 2 |
| GGT |
1/6 |
* |
- |
|
2/5 |
* |
*: “loose stool” and “GGT” were not graded because they are not included in the Common
Terminology Criteria for Adverse Events (CTCAE) . -: no abnormalities or adverse events
were observed. ALP, alkaline phosphatase; ALT, alanine aminotransferase; T-BIL, total
bilirubin; GGT, γ-glutamyl transpeptidase.
Based on the results of the dose-escalation study, the long-term administration test was
performed with a dosage of 35 mg/kg/day. The side effects observed during the long-term
administration test are shown in Table 2. As the
reference range of ALP in dogs is 20 to 156 IU/l, the magnitude of a grade 3 side effect in
terms of the ALP level was calculated and defined as 780 to 3120 IU/l in this study
(Supplementary Table 1). After lapatinib administration for 8 weeks, three of the six dogs
showed high ALP levels (814, 967, and 1316 IU/l), which were above the lower limit of the
range defined. None of the other parameters exceeded the toxicity level beyond grade 3.
Table 2.
Adverse events observed in the long-term administration test
| Adverse event |
35 mg/kg/day |
| Week 0 |
Week 2 |
Week 4 |
Week 6 |
Week 8 |
| No. of dogs showing adverse events |
CTCAE grade |
No. of dogs showing adverse events |
CTCAE grade |
No. of dogs showing adverse events |
CTCAE grade |
No. of dogs showing adverse events |
CTCAE grade |
No. of dogs showing adverse events |
CTCAE grade |
| Dermatologic/Skin |
Erythema |
- |
|
- |
|
3/6 |
Grade 1 |
3/6 |
Grade 1 |
3/6 |
Grade 1 |
| Gastrointestinal |
Loose stool |
- |
|
1/6 |
* |
1/6 |
* |
1/6 |
* |
1/6 |
* |
| Metabolic/ Laboratory |
ALP |
- |
|
2/6 |
Grade 1 |
6/6 |
Grade 2 |
6/6 |
Grade 2 |
3/6 |
Grade 2 |
|
|
4/6 |
Grade 2 |
3/6 |
Grade 3 |
| ALT |
- |
|
2/6 |
Grade 1 |
4/6 |
Grade 1 |
3/6 |
Grade 1 |
1/6 |
Grade 1 |
|
|
1/6 |
Grade 2 |
1/6 |
Grade 2 |
| T-BIL |
- |
|
1/6 |
Grade 1 |
1/6 |
Grade 1 |
3/6 |
Grade 1 |
3/6 |
Grade 1 |
| 2/6 |
Grade 2 |
| GGT |
- |
|
- |
|
1/6 |
* |
3/6 |
* |
2/6 |
* |
*: "loose stool" and "GGT" were not graded because they are not included in the Common
Terminology Criteria for Adverse Events (CTCAE) . -: no abnormalities or adverse events
were observed. ALP, alkaline phosphatase; ALT, alanine aminotransferase; T-BIL, total
bilirubin; GGT, γ-glutamyl transpeptidase.
Since HER2 expression has been reported in some canine tumors including mammary gland
tumors, lapatinib can be considered a useful treatment option [8, 11,12,13]. Furthermore, HER2
inhibition was reported to be effective in canine mammary cell lines [14]. However, while several safety studies with limited dosages [9] have been performed in dogs, no practical protocols
have been established.
In this study, the MTD of lapatinib for dogs was found to be 35 mg/kg/day within 8 weeks.
In humans, lapatinib is administered at the dose of 1,250 mg/head/day, which equals to 25
mg/kg for a person weighing 50 kg, with capecitabine. The MTD defined in this study is a
clinically applicable dose in dogs, and higher than the extrapolated dose applied in human
medicine. A limitation of this study is that it only examined the safety of lapatinib use,
and not the effect on canine tumors. Further studies are needed to define a clinically
useful protocol for the drug.
In this study, weight loss and a high ALP level were the markers of dose-limiting toxicity.
Similar to that in humans, lapatinib can cause hepatobiliary toxicity, although the values
reported to be affected differ between dogs and humans: ALT, AST, and T-BIL levels have been
reported to increase in humans, but the parameter most affected in this study was ALP. The
reason for weight loss was not clear in this study. Because appetite and diet were unchanged
during the study, the causative factor might be an intestinal tract disorder. Determination
of the detailed pharmacodynamics of lapatinib and mechanisms of adverse effects in dogs
might be necessary for further investigations.
Among antitumor agents many frequently used drugs cause bone marrow suppression. At the
same time, molecular targeted agents are not commonly used in veterinary hospitals, possibly
because of limited information and costs. However, the results of this study show that
lapatinib can be used relatively safely even in dogs, without any adverse myelosuppressive
effect. To determine the possibility of using the drug with conventional antitumor drugs,
further safety studies are required.
Drug pharmacokinetics are reported to change depending on the fat intake in humans; a high
fat intake is associated with a high area under the concentration-time curve (AUC) and
maximum blood concentration [5]. In human medicine, to
avoid pharmacokinetic changes, lapatinib is encouraged to be administered between meals.
However, in veterinary medicine, administration of oral medication to dogs is often
difficult at home, especially without any treats. To establish a possible medication
protocol, we decided to administer the drug with meals. At the same time, assuming that
various types of diet are given to dogs at home, in this study, we provided the dogs with
comprehensive nutrition, and the diet chosen had a rather high fat content (less than 16% of
total fat). If the drug is administered between meals or with low-fat meals, the MTD might
be higher. In conclusion, oral administration of lapatinib at a dose of 35 mg/kg/day with
meals within 8 weeks was found to be safe for dogs. This finding might be helpful for a
clinical evaluation of its antitumor effect and adverse effects in tumor-bearing dogs.
