**3. Imatinib as front-line treatment for pediatric CML**

As imatinib has proven very effective in adult CML, its accelerated clinical approval was given in the year 2001 for adults with CML and without age restriction in 2003. In several studies, imatinib showed similar antileukemic efficacy in children compared to adults (**Figure 1**) [26, 27]. Typically, standard dose of imatinib (300 mg/m<sup>2</sup> ) achieved a complete hematologic remission in 95% of the pediatric patients after 3 months, a complete cytogenetic remission in 80% after 12 months, and a major molecular remission (MR3.0 = 0.1 % ratio copy number of gene transcripts BCR-ABL1/control gene) in 60% after 18 months of treatment [16, 28].

Although imatinib acts relatively specifically against the dysregulated BCR-ABL1 TK, it is known that imatinib exerts off-target effects at therapeutic blood levels on other TKs (**Table 1**). The reason for this is the affinity of imatinib to the ATP-binding pocket of the kinase domain. This domain is a characteristic feature of many members of the kinome, including TKs (review of the human kinome: [29, 30], review structure of the TK: [8, 9]).

Nevertheless, imatinib treatment is generally well tolerated, showing mostly mild side effects. Neutropenia, thrombocytopenia, and anemia occur in up to 45, 20, and 10% of patients, respectively, who are in the chronic phase of CML and receive standard dose imatinib [2]. Nonhematologic adverse effects include nausea, skin rashes, peripheral edemas, muscle cramps, and elevated liver transaminase levels [2].

Underlying dynamic processes of the growing skeleton are subject to strict regulation/communication of bone formation and resorption and can be easily influenced by interfering factors. At present, it is not possible to study simultaneously these complex bone remodeling processes such as the interaction of bone forming osteoblasts and bone resorbing osteoclasts by culturing systems *in vitro*. Therefore, it is only possible to study bone breakdown, bone structure, changes in the mineral content, and the overall structure of the bone *in vivo*

**Figure 2.** Growth failure in pediatric CML patients during imatinib treatment [4]. SDS: Standard deviation score. One hundred and two patients (54 male/48 female; median age 12 years, range: 1–18 years) at diagnosis of CML receiving imatinib as upfront treatment were enrolled retrospectively in the trial CML-PAED II during the period 02/2006 to 06/2014. Height standard deviation scores (SDS) were derived from WHO-AnthroPlus, version 1.04 software, a global growth-monitoring tool providing normal range values for the age cohorts from birth till 19 years. Eighty-one out of 102 patients fulfilled the criteria for continuous assessment of growth scheduled at 3- months intervals during imatinib exposure. Twenty-one patients were analyzed at intervals ≠ 3 months. Calculation: Δ SDS = SDSTKI therapy–SDSDiagnosis; data

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In addition, TKI treatment for CML is not curative in most patients. Although first results from stopping TKI trials in adult patients after achieving sustainable deep molecular remission look promising most patients probably require a lifelong TKI treatment. This poses an increased risk to pediatric CML patients exposed to TKI treatment for decades as the long-term side effects on bone or other organs in a still growing organism presently are totally unknown.

All regulatory authorities (Food and Drug Administration (FDA), Health Canada, European Medical Agency (EMA)) require animal tests to be conducted before humans are exposed to a new molecular entity. In drug developmental process, every potential new therapeutic agent

in appropriate juvenile animal models.

are shown as Whiskers box plot (median ± 5th/95th percentile).

**4. Juvenile animal model for chronic TKI exposure**

Studies with imatinib in adult patients also showed disturbed bone metabolism as a specific side effect [3] comprising altered calcium metabolism as well as increased trabecular mineralization and increased bone density in stamp biopsies [32]. In children, imatinib therapy has been associated with severe longitudinal growth retardation [4, 5, 33–41] (**Figure 2**), but the detailed mechanism how imatinib interferes with bone metabolism and the final consequences are not fully understood.

Regardless the type of kinase, imatinib binds to all structurally accessible ATP-binding pockets. Accordingly, other membrane-bound and cytosolic TKs, e.g., c-abl, PDGF-R α/β, c-KIT, and c-FMS [31, 42–44], are inhibited which play a major role in bone remodeling.


**Table 1.** Inhibitory effect of imatinib on selected TKs [31].

Studying Side Effects of Tyrosine Kinase Inhibitors in a Juvenile Rat Model with Focus on Skeletal... http://dx.doi.org/10.5772/intechopen.70006 315

However, some patients develop resistance to imatinib resulting in loss of treatment response or even leukemic relapse. Among other underlying mechanisms, BCR-ABL1 kinase domain mutations can cause varying degrees of drug insensitivity [24]. In order to counter these mechanisms, next generation TKIs have been developed like dasatinib (SPRYCEL®, Bristol-Myers-Squibb), nilotinib (Tasigna®, Novartis), bosutinib (BOSULIF®, Pfizer), and ponatinib (ICLUSIG®, Ariad Pharmaceuticals) with different affinities to the ATP-binding pocket of the BCR-ABL1 TK [25].

As imatinib has proven very effective in adult CML, its accelerated clinical approval was given in the year 2001 for adults with CML and without age restriction in 2003. In several studies, imatinib showed similar antileukemic efficacy in children compared to adults (**Figure 1**) [26, 27].

in 95% of the pediatric patients after 3 months, a complete cytogenetic remission in 80% after 12 months, and a major molecular remission (MR3.0 = 0.1 % ratio copy number of gene transcripts

Although imatinib acts relatively specifically against the dysregulated BCR-ABL1 TK, it is known that imatinib exerts off-target effects at therapeutic blood levels on other TKs (**Table 1**). The reason for this is the affinity of imatinib to the ATP-binding pocket of the kinase domain. This domain is a characteristic feature of many members of the kinome, including TKs (review

Nevertheless, imatinib treatment is generally well tolerated, showing mostly mild side effects. Neutropenia, thrombocytopenia, and anemia occur in up to 45, 20, and 10% of patients, respectively, who are in the chronic phase of CML and receive standard dose imatinib [2]. Nonhematologic adverse effects include nausea, skin rashes, peripheral edemas, muscle

Studies with imatinib in adult patients also showed disturbed bone metabolism as a specific side effect [3] comprising altered calcium metabolism as well as increased trabecular mineralization and increased bone density in stamp biopsies [32]. In children, imatinib therapy has been associated with severe longitudinal growth retardation [4, 5, 33–41] (**Figure 2**), but the detailed mechanism how imatinib interferes with bone metabolism and the final conse-

Regardless the type of kinase, imatinib binds to all structurally accessible ATP-binding pockets. Accordingly, other membrane-bound and cytosolic TKs, e.g., c-abl, PDGF-R α/β, c-KIT, and c-FMS [31, 42–44], are inhibited which play a major role in bone remodeling.

**Tyrosine kinase BCR-ABL1 c-abl c-Kit PDGF-Rα PDGF-Rβ c-FMS IC50(µM)** 0.25 0.19 0.15 0.10 0.39 1.42

) achieved a complete hematologic remission

**3. Imatinib as front-line treatment for pediatric CML**

314 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

BCR-ABL1/control gene) in 60% after 18 months of treatment [16, 28].

of the human kinome: [29, 30], review structure of the TK: [8, 9]).

Typically, standard dose of imatinib (300 mg/m<sup>2</sup>

cramps, and elevated liver transaminase levels [2].

quences are not fully understood.

**Table 1.** Inhibitory effect of imatinib on selected TKs [31].

IC, inhibitory concentration.

**Figure 2.** Growth failure in pediatric CML patients during imatinib treatment [4]. SDS: Standard deviation score. One hundred and two patients (54 male/48 female; median age 12 years, range: 1–18 years) at diagnosis of CML receiving imatinib as upfront treatment were enrolled retrospectively in the trial CML-PAED II during the period 02/2006 to 06/2014. Height standard deviation scores (SDS) were derived from WHO-AnthroPlus, version 1.04 software, a global growth-monitoring tool providing normal range values for the age cohorts from birth till 19 years. Eighty-one out of 102 patients fulfilled the criteria for continuous assessment of growth scheduled at 3- months intervals during imatinib exposure. Twenty-one patients were analyzed at intervals ≠ 3 months. Calculation: Δ SDS = SDSTKI therapy–SDSDiagnosis; data are shown as Whiskers box plot (median ± 5th/95th percentile).

Underlying dynamic processes of the growing skeleton are subject to strict regulation/communication of bone formation and resorption and can be easily influenced by interfering factors. At present, it is not possible to study simultaneously these complex bone remodeling processes such as the interaction of bone forming osteoblasts and bone resorbing osteoclasts by culturing systems *in vitro*. Therefore, it is only possible to study bone breakdown, bone structure, changes in the mineral content, and the overall structure of the bone *in vivo* in appropriate juvenile animal models.

In addition, TKI treatment for CML is not curative in most patients. Although first results from stopping TKI trials in adult patients after achieving sustainable deep molecular remission look promising most patients probably require a lifelong TKI treatment. This poses an increased risk to pediatric CML patients exposed to TKI treatment for decades as the long-term side effects on bone or other organs in a still growing organism presently are totally unknown.

#### **4. Juvenile animal model for chronic TKI exposure**

All regulatory authorities (Food and Drug Administration (FDA), Health Canada, European Medical Agency (EMA)) require animal tests to be conducted before humans are exposed to a new molecular entity. In drug developmental process, every potential new therapeutic agent has to pass clinical phase I-III studies in humans to verify safety, dosage, efficacy, side effects, and monitoring adverse reactions. All these studies are done in adult volunteers or adult patients if the disease under study occurs not exclusively at pediatric age [45]. Thus, in order to gain insight into side effects occurring specifically in the still growing organism during the preclinical research phase, *in vivo* studies in young growing animals are of main importance.

long inbreeding of this strain, whereas outbred strains have a diverse genetic background. Due to this genetic characteristic, animals of inbred strains react nearly identical to a specific intervention, like medical treatment, surgery, etc., wherefore the influence of this intervention on a particular parameter can be identified more precisely. However, outbred strains reflect the natural situation more accurately as every individual is genetically different from the other. Every animal of an outbred strain will react slightly different to a specific intervention, which discloses all possible effects of this intervention on the metabolism and mimics more the situation in the clinic. Therefore, depending on the experimental question and if you need a genetic diversity in your test population, inbred or outbred strains are used. Our study focused on side effects of long-term TKI treatment on bone remodeling and to mimic

Studying Side Effects of Tyrosine Kinase Inhibitors in a Juvenile Rat Model with Focus on Skeletal...

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317

We exclusively studied Wistar rats of male gender, as males tend to be more sensitive to bone influencing agents than female animals due to more rapid weight development and gender-

Prepubertal young Wistar rats triple their body weight, regardless of sex, from about 60 to 180 g in 14 days from the 3rd to 5th week of life due to the increasing growth hormone (GH) pulse amplitudes. The duration of GH pulses is significantly longer in males versus females, a pattern that continues throughout adulthood. Between 5th and 7th week of life, GH pulse amplitudes are similarly increased in both sexes [53, 54]. The rapid skeletal growth associated with this is particularly strongly influenced by interfering factors. In postpuberty, the growth slows down, especially in female rats, who weigh 200 g in the 8th week of life and 220 g in the 10th week of life. Contrary, male animals reach a body weight of 300 g postpubertally in the 8th week of life and 390 g in the 10th week of life. These differences in growth dynamics should also make postpubertal bone alterations due to TKI exposure more prominent in male animals.

Nevertheless, additional factors especially endocrine changes in hormones, such as testosterone, 17ß-estradiol, and corticosterone, inducing and associated with the onset of puberty and puberty itself may be more important than GH to decide about the sex when setting up an animal model. It is commonly considered that puberty lasts until the 8th week of age [54]. However, onset of puberty in the rat (as measured by the age at vaginal opening and the onset of estrous cyclicity) occurs between 4th and 5th week in females, whereas in males (as measured by preputial separation which is an androgen-dependent event) occurs around 7th week of life depending on the strain used [55]. The onset of puberty in male Wistar rats based on the increase in plasma testosterone levels starts at 46–50 days of age and progressively increases until 76 days of age [56–58]. However, related to the increased production of estrogen and its positive influence on bone formation, trabecular bone density increases significantly both in women and in female rats with the onset of puberty [59, 60]. Because of this hormonal influence, effects on the bones,

For chronic drug exposure, we choose administration via the drinking water. Drug application via subcutaneous (s.c.) or intraperitoneal injection (i.p.) or oral gavage is the most

which are only mild, would be more difficult to detect in the female organism.

the human situation, we choose juvenile rats of the outbred strain "Wistar."

**4.3. Male versus female**

**4.4. Drug administration**

specific hormones.

However, almost in all instances, primarily adult animal models are used in preclinical research (for reviews about the ongoing debate about animal models in clinical research see Refs. [46–48]). Adult animals were also used to study the influence of imatinib on the skeletal system [49, 50]. But as the growth process of the juvenile bone differs significantly from a mature bone, results described so far in adult patients/animals cannot readily be transferred to pediatric cohorts. Furthermore, Juvenile animal models cannot easily be selected as they are not established to match every single "research question" or disease on a routine basis.

Therefore, we describe here our established juvenile animal model to study side effects of a **chronic exposure** of imatinib primarily on the **growing bone** and to a lesser extent on other organs.

When establishing a juvenile animal model, several issues should be considered like the rodent species itself (mouse vs. rat), the strain (inbred vs. outbred), the overall speed of development (age when puberty starts), and convenient ways of drug administration in the situation of long-term exposure (intraperitoneal vs. subcutaneous vs. oral gavages vs. microosmotic pumps). Overall, the developmental stages must be comparable to human life.

#### **4.1. Mice versus rat**

Most of the animals used in biomedical research are mice and rats because of their availability, ease of handling, and fast reproduction rate. Mice are an excellent model for human diseases because genetically they share 98% homology with human genome as well as a similar organization of their DNA and gene expression. However, the genome of a rat is smaller than its human equivalent but larger than that of a mouse.

Compared to mice, rats offer many advantages as, for example, their physiology is easier to monitor and is more like the corresponding human condition. But the most important advantage of the rat is its bigger size, not just because of the added ease to perform surgical procedures, but because of larger substructures (e.g., bone growth line, metaphysis) in organs thus influencing (i) which ratio of the organ is prone to an experimental lesion and (ii) the distance effects drugs exert to a specific anatomical area [51].

As we questioned about side effects of a chronic imatinib exposure on the growing organism, we were interested in the side effects on the long bones, which are much bigger in rats as compared to mice. As an additional benefit, we could also monitor side effects on other growing and developing organs like heart and testis as rats are a preferred model in cardiac and reproduction questions [52].

#### **4.2. Inbred versus outbred strain**

In general, the difference between outbred and inbred strains lies in their genetic background. Inbred strains are characterized by almost 99% homogeneity of the genome resulting from a long inbreeding of this strain, whereas outbred strains have a diverse genetic background. Due to this genetic characteristic, animals of inbred strains react nearly identical to a specific intervention, like medical treatment, surgery, etc., wherefore the influence of this intervention on a particular parameter can be identified more precisely. However, outbred strains reflect the natural situation more accurately as every individual is genetically different from the other. Every animal of an outbred strain will react slightly different to a specific intervention, which discloses all possible effects of this intervention on the metabolism and mimics more the situation in the clinic. Therefore, depending on the experimental question and if you need a genetic diversity in your test population, inbred or outbred strains are used. Our study focused on side effects of long-term TKI treatment on bone remodeling and to mimic the human situation, we choose juvenile rats of the outbred strain "Wistar."

#### **4.3. Male versus female**

has to pass clinical phase I-III studies in humans to verify safety, dosage, efficacy, side effects, and monitoring adverse reactions. All these studies are done in adult volunteers or adult patients if the disease under study occurs not exclusively at pediatric age [45]. Thus, in order to gain insight into side effects occurring specifically in the still growing organism during the preclinical research phase, *in vivo* studies in young growing animals are of main importance. However, almost in all instances, primarily adult animal models are used in preclinical research (for reviews about the ongoing debate about animal models in clinical research see Refs. [46–48]). Adult animals were also used to study the influence of imatinib on the skeletal system [49, 50]. But as the growth process of the juvenile bone differs significantly from a mature bone, results described so far in adult patients/animals cannot readily be transferred to pediatric cohorts. Furthermore, Juvenile animal models cannot easily be selected as they are not established to match every single "research question" or disease on a routine basis. Therefore, we describe here our established juvenile animal model to study side effects of a **chronic exposure** of imatinib primarily on the **growing bone** and to a lesser extent on other

316 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

When establishing a juvenile animal model, several issues should be considered like the rodent species itself (mouse vs. rat), the strain (inbred vs. outbred), the overall speed of development (age when puberty starts), and convenient ways of drug administration in the situation of long-term exposure (intraperitoneal vs. subcutaneous vs. oral gavages vs. microosmotic pumps). Overall, the developmental stages must be comparable to human life.

Most of the animals used in biomedical research are mice and rats because of their availability, ease of handling, and fast reproduction rate. Mice are an excellent model for human diseases because genetically they share 98% homology with human genome as well as a similar organization of their DNA and gene expression. However, the genome of a rat is smaller than

Compared to mice, rats offer many advantages as, for example, their physiology is easier to monitor and is more like the corresponding human condition. But the most important advantage of the rat is its bigger size, not just because of the added ease to perform surgical procedures, but because of larger substructures (e.g., bone growth line, metaphysis) in organs thus influencing (i) which ratio of the organ is prone to an experimental lesion and (ii) the distance

As we questioned about side effects of a chronic imatinib exposure on the growing organism, we were interested in the side effects on the long bones, which are much bigger in rats as compared to mice. As an additional benefit, we could also monitor side effects on other growing and developing organs like heart and testis as rats are a preferred model in cardiac and

In general, the difference between outbred and inbred strains lies in their genetic background. Inbred strains are characterized by almost 99% homogeneity of the genome resulting from a

organs.

**4.1. Mice versus rat**

its human equivalent but larger than that of a mouse.

effects drugs exert to a specific anatomical area [51].

reproduction questions [52].

**4.2. Inbred versus outbred strain**

We exclusively studied Wistar rats of male gender, as males tend to be more sensitive to bone influencing agents than female animals due to more rapid weight development and genderspecific hormones.

Prepubertal young Wistar rats triple their body weight, regardless of sex, from about 60 to 180 g in 14 days from the 3rd to 5th week of life due to the increasing growth hormone (GH) pulse amplitudes. The duration of GH pulses is significantly longer in males versus females, a pattern that continues throughout adulthood. Between 5th and 7th week of life, GH pulse amplitudes are similarly increased in both sexes [53, 54]. The rapid skeletal growth associated with this is particularly strongly influenced by interfering factors. In postpuberty, the growth slows down, especially in female rats, who weigh 200 g in the 8th week of life and 220 g in the 10th week of life. Contrary, male animals reach a body weight of 300 g postpubertally in the 8th week of life and 390 g in the 10th week of life. These differences in growth dynamics should also make postpubertal bone alterations due to TKI exposure more prominent in male animals.

Nevertheless, additional factors especially endocrine changes in hormones, such as testosterone, 17ß-estradiol, and corticosterone, inducing and associated with the onset of puberty and puberty itself may be more important than GH to decide about the sex when setting up an animal model. It is commonly considered that puberty lasts until the 8th week of age [54]. However, onset of puberty in the rat (as measured by the age at vaginal opening and the onset of estrous cyclicity) occurs between 4th and 5th week in females, whereas in males (as measured by preputial separation which is an androgen-dependent event) occurs around 7th week of life depending on the strain used [55]. The onset of puberty in male Wistar rats based on the increase in plasma testosterone levels starts at 46–50 days of age and progressively increases until 76 days of age [56–58]. However, related to the increased production of estrogen and its positive influence on bone formation, trabecular bone density increases significantly both in women and in female rats with the onset of puberty [59, 60]. Because of this hormonal influence, effects on the bones, which are only mild, would be more difficult to detect in the female organism.

#### **4.4. Drug administration**

For chronic drug exposure, we choose administration via the drinking water. Drug application via subcutaneous (s.c.) or intraperitoneal injection (i.p.) or oral gavage is the most accurate type of body weight-related exposure. However, young animals are prone to risks of injury and subsequent infection in the pharynx and/or esophagus [61, 62]. Micro-osmotic pumps could also be considered for s.c. administration but repeated implantation and removal of the pumps combined with the increased risk of infection should be taken into account [63]. For a detailed review of routes for chronic drug administration, see ref. [64]. However, due to the pharmacodynamics of the TKI, single shot by s.c. or i.p. administration would need at least two TKI applications daily over 10 weeks. Considering animal ethics as well as personal resources over several weeks including shifts on weekends, these numerous manipulations are hardly tolerable and affordable. For these reasons, the chosen intake of the drug via the drinking water was the most adequate and convenient form of chronic TKI exposure. Also, the stability of the TKIs in aqueous solution at room temperature facilitated this approach. Hence, the drug intake is dependent on the daily drinking volume considering age and associated body weight. Other possible interfering factors are loss of liquid when changing the water bottles or leaking water bottles and changes in the drinking behavior due to changes in the environment like fluctuation in the room temperature or humidity, or social conflicts between the animals. To counteract this, the care of the experimental animals, the measuring of the drinking volume, and the determination of weight gain were always carried out on a fixed schedule and by identical staff members including weekends.

Considering the rapid maturation of the rats and the objective of examining the development (infancy, puberty, and young adulthood), we selected an exposure period of 10 weeks starting

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Summing key issues in the juvenile animal model described, we chronically exposed healthy 4-week-old male Wistar rats to varying concentrations (low dose vs. high dose) of imatinib via drinking water over a period of 10 weeks while growing. We applied different treatment schedules to mimic possible new treatment strategies (continuous vs. intermittent). During the entire exposure time, the developmental stages from the end of weaning until young adolescence were covered (**Figure 3**). During ongoing imatinib exposure, a defined number of animals from each cohort were humanely sacrificed at prepubertal stage (age 6 weeks; after 2 weeks of exposure), at pubertal stage (age 8 weeks; after 4 weeks of exposure), and at postpubertal stage (age 14 weeks; after 10 weeks of

**5. Side effects of chronic imatinib treatment on growing bone**

**Figure 3.** Experimental design of the juvenile animal model.

At defined time points of analysis (**Figure 3**), blood serum was collected to measure TKI concentration by high-performance liquid chromatography (HPLC), biochemical markers of bone turnover, and hormone levels by ELISA technique. Long bones (tibia and femur) and lumbar vertebrae L1–L4 were isolated to determine bone length, vertebral height, bone mass,

at 4 weeks of age.

exposure) [68].

Age-dependent drinking behavior of mammals varies. According to body weight, higher volumes are ingested by younger animals [65, 66]. Furthermore, rodents show a circadian rhythm of their food and drinking water intake. About 80% of the maximum daily intake of liquids occur at night [64]. This allows the conclusion that by administration via the drinking water, a peak level was achieved during the night, comparable to the single administration in human patients during the daytime.

#### **4.5. Developmental stages**

Due to the well-documented developmental stages of the rat, it is possible to carry out a comparison with human developmental stages in order to interpret the generated data in an orientated manner (**Table 2**).


**Table 2.** Developmental stages of rat and human [67].

Considering the rapid maturation of the rats and the objective of examining the development (infancy, puberty, and young adulthood), we selected an exposure period of 10 weeks starting at 4 weeks of age.

Summing key issues in the juvenile animal model described, we chronically exposed healthy 4-week-old male Wistar rats to varying concentrations (low dose vs. high dose) of imatinib via drinking water over a period of 10 weeks while growing. We applied different treatment schedules to mimic possible new treatment strategies (continuous vs. intermittent). During the entire exposure time, the developmental stages from the end of weaning until young adolescence were covered (**Figure 3**). During ongoing imatinib exposure, a defined number of animals from each cohort were humanely sacrificed at prepubertal stage (age 6 weeks; after 2 weeks of exposure), at pubertal stage (age 8 weeks; after 4 weeks of exposure), and at postpubertal stage (age 14 weeks; after 10 weeks of exposure) [68].

**Figure 3.** Experimental design of the juvenile animal model.

accurate type of body weight-related exposure. However, young animals are prone to risks of injury and subsequent infection in the pharynx and/or esophagus [61, 62]. Micro-osmotic pumps could also be considered for s.c. administration but repeated implantation and removal of the pumps combined with the increased risk of infection should be taken into account [63]. For a detailed review of routes for chronic drug administration, see ref. [64]. However, due to the pharmacodynamics of the TKI, single shot by s.c. or i.p. administration would need at least two TKI applications daily over 10 weeks. Considering animal ethics as well as personal resources over several weeks including shifts on weekends, these numerous manipulations are hardly tolerable and affordable. For these reasons, the chosen intake of the drug via the drinking water was the most adequate and convenient form of chronic TKI exposure. Also, the stability of the TKIs in aqueous solution at room temperature facilitated this approach. Hence, the drug intake is dependent on the daily drinking volume considering age and associated body weight. Other possible interfering factors are loss of liquid when changing the water bottles or leaking water bottles and changes in the drinking behavior due to changes in the environment like fluctuation in the room temperature or humidity, or social conflicts between the animals. To counteract this, the care of the experimental animals, the measuring of the drinking volume, and the determination of weight gain were always carried out on a fixed schedule and by identical staff members

318 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

Age-dependent drinking behavior of mammals varies. According to body weight, higher volumes are ingested by younger animals [65, 66]. Furthermore, rodents show a circadian rhythm of their food and drinking water intake. About 80% of the maximum daily intake of liquids occur at night [64]. This allows the conclusion that by administration via the drinking water, a peak level was achieved during the night, comparable to the single administration in

Due to the well-documented developmental stages of the rat, it is possible to carry out a comparison with human developmental stages in order to interpret the generated data in an

**Developmental stages Rat Human** Weaning 3 weeks 6 months Puberty 7 weeks 12–14 years Adolescent 8–11 weeks 15–20 years Adult >12 weeks >20 years Death 2–3 years 70–80 years

including weekends.

human patients during the daytime.

**Table 2.** Developmental stages of rat and human [67].

**4.5. Developmental stages**

orientated manner (**Table 2**).
