Background
Cytotoxic chemotherapy still forms the basis of systemic therapy for many cancers. Treatment plans typically consist of repeated cycles of chemotherapy at as high a dose as possible, without causing unacceptable toxicity, the maximum tolerated dose (MTD). Maximum drug doses are determined in dose escalating phase I clinical trials until reaching an appropriate balance between efficacy and toxicity [
1]. In contrast, preclinical studies usually employ doses based on convention within a research group, on published studies that may or may not have reported optimisation experiments or on quick optimisation steps in which the methods are varied [
2,
3]. In addition, while in clinical studies further dose escalation is allowed by extensive supportive care measures such as intravenous hydration, anti-emetics, antihistamines and corticosteroids; this is usually lacking in animal studies [
2]. Together, this may result in both the use of sub-therapeutic dosages and excess use of animals.
There is a clear dose-response relationship between chemotherapy and tumour regression in preclinical studies [
4] and in the clinical setting [
5]. Furthermore, there are many secondary antitumour effects that depend on chemotherapy dose, for example immune stimulatory potential of dendritic cells [
6]; production of IL-17 by peripheral blood and splenic CD4
+ T cells [
7]; antiangiogenic effects [
8,
9] or depletion of regulatory T cells [
10‐
13]. Therefore, the dose used in preclinical studies may significantly affect translation into clinical trials [
14]. Human MTDs are often well predicted by animal studies. A meta-analysis of the preclinical and subsequent clinical development phases of 25 cancer drugs showed that rodent toxicology generally provided a safe and reliable way of assessing starting dosages in humans and adequately predicted potential side effects [
15]. It is reasonable then that preclinical studies should make use of MTD regimes. However, to our knowledge, there has not been a systematic study done to determine the MTD for chemotherapeutics from each of the canonical classes. As part of the clinical development pathway LD50 values (median lethal dose) have often been determined, giving some indication of where the MTD will be. However, many of these studies were done decades ago in mouse strains that are often no longer used in cancer research, while the tolerability to chemotherapy varies considerably between mouse strains [
16‐
18]. This compromises the extrapolation of those MTDs to currently standard mouse strains. We therefore aimed to create a murine cancer chemotherapy MTD resource in BALB/c and C57BL/6 mice, which would both reduce individual dose optimising investigations and allow standardized dosing strategies in preclinical cancer research. We chose weight loss and clinical score (Additional file
1: Table S2) as endpoints, because in previous murine studies weight loss was by far the most common dose-limiting toxicity (81%), followed by clinical signs, such as neurotoxicity or diarrhoea [
15]. This allowed us a straightforward way of assessing toxicity that we think is universally relevant. We also tested whether the single-dose MTD could be readily extrapolated to repeated cycles and whether there were differences in MTDs between mouse strains. Lastly, because the corticosteroid dexamethasone and the 5-hydroxytryptamine 3 (5-HT
3) receptor antagonist ondansetron are commonly administered in conjunction with chemotherapy to reduce nausea and anorexia in patients, we determined the effect of these drugs on the MTD in mice.
Discussion
Chemotherapy administration to patients is governed by strict guidelines relating to dosage and scheduling as determined in dose-optimising phase I studies. Yet these same standards are often not applied to in vivo preclinical studies [
2,
3]. When we searched the literature for chemotherapy dosages in order to inform related studies, we found that unlike the clinical situation, dosages varied widely between studies. For example, doxorubicin is reportedly used at dosages varying between 2 and 3 mg/kg [
22,
23] and 10–12 mg/kg [
24,
25]. In addition, in some studies, doxorubicin is given intratumourally [
26‐
28], which may provide interesting mechanistic information, but does compromise translatability. A further discrepancy is with the definition of low and high dose chemotherapy. Low-dose cyclophosphamide has often been studied in regards to its capacity to deplete regulatory T cell (Treg) [
29]. However, the concentrations used are quite varied, with doses ranging from 30 to 200 mg/kg, even though it has been shown that specific depletion of Tregs occurs at 20 mg/kg but not at 200 mg/kg [
30]. Thus this unclear definition of ‘low-dose’ could lead to misinterpretations of preclinical findings, and thus hamper translation of these studies into the clinic.
This study was undertaken to provide some guidance on the MTD of chemotherapy in mice for future studies. We chose to use practical measurements that can be applied easily to any research setting, and which have been validated in previous studies [
15]. By following this strategy, we found the MTD of some drugs to be quite different from commonly used dosages in the literature. The largest differences were seen with docetaxel and gemcitabine and to a lesser extent with 5-FU and irinotecan. Docetaxel had an MTD of 130 mg/kg, far higher than the commonly used dose of 16 to 33 mg/kg found in the literature [
31‐
33]. The MTD of gemcitabine was 700 mg/kg, five-fold higher than those used by others (and our own group previously [
34]) with the most common dose being 120 mg/kg [
35‐
37]. It should also be noted however that many similar MTDs were found between this study and others. Cisplatin is commonly dosed at 5–6 mg/kg [
34,
38,
39], which is concordant with the MTD of 6 mg/kg found in this study. Similarly, vinorelbine is often administered at 10 mg/kg [
40‐
42], the same dose reported here.
We anticipate that other research groups may want to use the MTDs described here as a starting point for their own studies, potentially reducing the number of animals needed to optimize protocols. However, there are some limitations to our studies. Firstly, although we found that the MTD for both cisplatin and vinorelbine were similar for C57BL/6 J and BALB/c mice, the BALB/c mice did show slightly more weight loss for the tested chemotherapeutics. This suggests that care should be taken when transposing dosages between strains, including immunodeficient strains. Secondly, we determined the MTD when given as a single dose. Although we found that multiple cycles of vinorelbine at single-dose MTD was very well tolerated, this was not the case for cisplatin. A dose reduction was needed to maintain the weights of the animals when giving multiple dosages. This suggests that some further optimization steps will be needed when using the dosages as described, depending on the required scheduling regimen and the mouse strain used and possibly the emetogenicity of the chemotherapeutic [
43]. Furthermore, small differences between research centres may affect either the response to or toxicity from chemotherapy. These include temperature [
44], time of dosing [
45], sex [
46], microbiome [
47], and age [
48] which should all be considered before beginning experimental studies.
Of interest, some of the MTDs that we determined are very close to or sometimes even over the reported LD50 [
49]. Explanations for this could be related to the above-mentioned factors, and particularly with the mouse strain used for determining the LD50 [
49]. For example, the LD50 for irinotecan was originally determined in ICR mice, whereas we used BALB/c mice, which also in the primary paper reportedly could tolerate higher dosages of irinotecan, similar to C57BL/6 mice [
16]. Similarly, we found that cisplatin could be safely dosed at 6 mg/kg, and that 1/3 mice lost more than 20% weight after 8 mg/kg, while the reported LD50 of cisplatin is 6.6 mg/kg [
49]. However, this LD50 is based on studies in DBA mice [
50]; in other strains, dosages as high as 18 mg/kg have been reported [
51]. These data underscore the importance of considering mouse background when interpreting preclinical chemotherapy dosages.
The final aim of this study was to investigate the effect of two common supportive care agents, ondansetron and dexamethasone. Both drugs are used in cancer patients to reduce chemotherapy and radiotherapy-induced nausea and vomiting, with dexamethasone also used to maintain weight in some circumstances [
52,
53]. The anti-emetic effect of dexamethasone, a synthetic glucocorticoid, is not well understood, although several mechanisms have been put forward, such as anti-inflammatory effects, normalisation of the hypothalamic–pituitary–adrenal axis, and effects on serotonin [
54]. Indeed, we found that also in mice dexamethasone showed a dose-dependent effect on chemotherapy-induced weight loss, with the optimum dosage at 0.2 mg/kg, which is within the dose range that is used in patients in this context [
55]. Ondansetron is a serotonin 5-HT
3 receptor antagonist used for the treatment of chemotherapy-induced nausea [
56]. Chemotherapeutics induce the release of serotonin in the small intestine, which binds 5-HT
3 receptors and induces emesis. Ondansetron outcompetes serotonin, preventing receptor binding and therefore acting as an effective anti-emetic [
57]. It is used to prevent nausea and vomiting after chemotherapy or radiotherapy in humans, but also in company animals such as dogs and cats [
58]. Two 5-HT
3 subunits have been identified in mice, namely A and B subunits while in humans there are five, A-E [
59]. The primary binding of ondansetron to 5-HT
3 is via the subunit A receptor [
60] and so this provided some rational for investigation in our supportive care studies. However, our results show that ondansetron alone is ineffective in reducing cisplatin-induced weight loss or improving clinical condition in mice. This is perhaps not completely unexpected, as ondansetron primarily regulates the vomit reflex along with nausea [
55]. Since rodents are not able to vomit, it might be expected that only an improved appetite, not decreased vomiting would result in reduced weight loss. However, surprisingly, we found that ondansetron abolished the beneficial effect of dexamethasone on preventing chemotherapy-induced weight loss in mice. This is striking since in the clinical setting it is common practice to combine ondansetron with dexamethasone as this combination is better at emetic control than ondansetron alone [
21]. Our data suggest that this combination should not be used in mice for this indication.