**6. Non bone-related side effects of imatinib treatment**

#### **6.1. Growth hormone**

Main length growth regulating factors at childhood and adolescence are GH and "insulin-like growth factor 1" (IGF-1), thyroid hormone (T3, T4), glucocorticoids, and sex hormones during puberty [79].

GH is secreted by pituitary somatotrophins in a pulsatile manner and acts on peripheral tissues, either directly or indirectly, through the stimulation of IGF-1 synthesis and secretion [80–82]. As reported, the increase in body height during childhood is initiated by promoting chondrocyte proliferation and endochondral ossification in the growth plate or induction of osteoblastogenesis, leading to linear bone growth [80].

Owing to growth, children and/or adolescents going through puberty are particularly vulnerable to a possible GH deficiency (GHD) under long-term imatinib treatment [5, 41]. Mimicking those findings in children on imatinib treatment, the juvenile animal model disclosed significantly lowered serum levels of IGF-1 binding protein 3 (IGF-BP3)—a stable and more accurately measurable degradation product of IGF-1—at all concentrations applied and at all ages investigated [37, 83, 84]. Data of clinical studies in pediatric CML patients under TKI therapy revealed IGF-1 and IGFBP-3 levels almost exclusively in the very low or deep pathological range when compared to age-matched controls, independent of treatment duration [83, 84].

#### **6.2. Vitamin D and bone**

**Figure 5.** Biphasic response (time period 1 [pink background], period 2 [brown background]) of bone remodeling to imatinib treatment in pediatric patients with CML. Data depicted from CML-PAED II study [78]. One hundred and nineteen patients (70 male/49 female, median age 12 years, range 1–18 years) received 260–340 mg imatinib/m<sup>2</sup>

within 1 week after diagnosis of CML (0). Up to 30 patients (range 20–30) out of this cohort could successfully be monitored repeatedly over a median period of 3 years for all parameters planned to be analyzed by collecting blood and urine for 3- months under appropriate circumstances. Assays were performed in a central laboratory as described

previously [77]. Age normalized reference values were used as standard deviation scores (SDS).

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

daily

Within the bone remodeling cycle, vitamin D plays a crucial role by influencing the overall mineralization and bone turnover of the skeleton. The main effects of the active vitamin D metabolite 1.25(OH)<sup>2</sup> D3 comprises of stimulating the absorption of calcium/phosphorus from the gut to create optimal circumstances for bone mineralization, as well as stimulation of the osteoblast-mediated mineralization and osteoclast differentiation [85]. The consequences of vitamin D deficiency are secondary hyperparathyroidism and bone loss, leading to osteoporosis and fractures, mineralization defects, which may lead to osteomalacia in the long-term, and muscle weakness, causing falls and fractures [86].

Hypophosphatemia, associated with low serum levels of 25-(OH)D<sup>3</sup> , 1.25(OH)2 D3 , calcium, and secondary hyperparathyroidism are known side effects in adult patients with CML under imatinib treatment [3]. An explanation for these findings is that imatinib directly stimulates bone formation while restraining resorption, resulting in a net flux of calcium from extracellular fluid into bone, a decreased serum calcium level, and a compensatory rise in the level of parathyroid hormone, which causes phosphaturia and modest hypophosphatemia [50]. Pediatric patients with CML also exhibit moderate secondary hyperparathyroidism in conjunction with pathologically low 25-(OH)D<sup>3</sup> and 1.25(OH)<sup>2</sup> D3 levels but normal serum calcium and phosphate levels under imatinib therapy [77, 78]. Thereby these effects were independent of the duration of imatinib therapy, which underlined once again that regulation and compensatory mechanisms on the growing skeleton are different from those in the adult skeleton.

How imatinib interferes with vitamin D synthesis and metabolism is poorly understood yet. So far only one study investigated *in vitro* the effect of imatinib on keratinocytes yet and revealed a competitive inhibition of CYP27B1, a vitamin D hydroxylating enzyme, by imatinib [87].

#### **6.3. Fertility**

TKs like c-kit and PDGF-R, which are inhibited "off-target" by imatinib, are involved not only in the bone remodeling process but also in the regulation of spermatogenesis [88], raising the question of testicular toxicities by imatinib treatment. Up to now, the influence of TKIs on the male reproductive endocrine system in pediatric patients with CML is still controversially discussed [89].

The first study in neonatal rats revealed that imatinib interferes with postnatal testicular development [90]. Investigations in the juvenile animal model starting at an older age (4 weeks) depicted unchanged testis weight but reduced testosterone levels under longterm imatinib exposure until young adulthood. Inhibin B, a protein that is predominantly produced in the testis controlling follicle stimulating hormone (FSH) [91], did not significantly differ from controls, at all doses, and by all application schemes tested [92]. A clinical study conducted in a small cohort of boys (age: 7.8–18.9 years) with CML receiving TKI treatment revealed testosterone and inhibin B levels within normal age-related reference ranges [83, 84, 92]. Therefore, severe testicular toxicity by imatinib seems to be unlikely.

an age-dependent increase in oxidative stress [100]. This suggests that cardiac monitoring of

**Figure 6.** Number of proliferating testicular epithelium cells (Marker Ki67) in Wistar rats under imatinib exposure [93].

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

http://dx.doi.org/10.5772/intechopen.70006

325

As cardiac side effects were also observed with the use of dasatinib, in the experiment conducted with the juvenile animal model, this 2nd-generation TKI was tested for safety, efficacy, and dose response. Surprisingly, animals died spontaneously in a dose- and exposure timedependent manner (**Figure 7**). Data of the surviving animals that were sacrificed according to the experimental set-up schedule (**Figure 3**) disclosed—dependent on the cumulative dose administered—increased relative heart weights, impaired heart ejection fraction as assessed by echocardiography, and elevated brain natriuretic peptide (BNP) serum levels, an indicator of cardiac dysfunction [101]. Data of this unexpected high toxicity can be explained by the serum elimination half-life time of dasatinib which is rather short and in the range of 2–3 h in rodents [102]. As known from clinical data on treatment of CML by dasatinib, it is not mandatory to achieve steady state drug blood levels as the intracellular concentration of dasatinib is responsible for efficacy, which is sufficiently achieved by once daily drug administration. Initial trials in humans based on drug administration twice daily were characterized by high toxicity requiring treatment interruption or reduction to once daily dosing [103]. Thus, the juvenile rat model also mimics this situation as a continuous intake of small doses of dasatinib

Initially, inhibition of the c-abl kinase was assumed to be the reason for cardiac toxicity by TKI [94]. But an extensive *in vitro* study of 18 TKIs on myocytes showed that their relative ability to inhibit ABL1 or ABL2 did not correlate with myocyte damage, revealing that inhibition

older patients receiving imatinib therapy may be especially warranted.

via the drinking water evidently is associated with higher toxicity.

However, a closer look on spermatogenesis in the juvenile animal model revealed that the spermatogenic cell counts were significantly decreased by high dose imatinib exposure (**Figure 6**). Additionally, during spermatogenesis cell cycle, the stage of the dominant cell proportion was shifted to more immature stages. Low dose and intermittent imatinib exposure attenuated these findings. Interestingly, spermatogenic cell proliferation was significantly lowered at all imatinib doses applied [93]. Thus, a delayed negative effect of long-term imatinib exposure on spermatogenesis cannot be excluded.

#### **6.4. Cardiac side effects of TKI treatment**

In the literature as well as indicated by the manufacturers in the specialist information, cardiotoxic and vascular side effects of imatinib and the next-generation TKIs are of special concern [94–99]. However, this primarily may play a role in older adult patients with CML (age > 65 years) under TKI treatment.

The juvenile animal model under discussion disclosed an increase in the relative heart weight ratio (= ratio of the heart weight to total body weight at sacrifice) under imatinib exposure. Another study found that imatinib treatment led to mitochondrial-dependent myocyte loss and cardiac dysfunction, occurring more severely in older mice, in part due to Studying Side Effects of Tyrosine Kinase Inhibitors in a Juvenile Rat Model with Focus on Skeletal... http://dx.doi.org/10.5772/intechopen.70006 325

regulation and compensatory mechanisms on the growing skeleton are different from those

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

How imatinib interferes with vitamin D synthesis and metabolism is poorly understood yet. So far only one study investigated *in vitro* the effect of imatinib on keratinocytes yet and revealed a competitive inhibition of CYP27B1, a vitamin D hydroxylating enzyme, by ima-

TKs like c-kit and PDGF-R, which are inhibited "off-target" by imatinib, are involved not only in the bone remodeling process but also in the regulation of spermatogenesis [88], raising the question of testicular toxicities by imatinib treatment. Up to now, the influence of TKIs on the male reproductive endocrine system in pediatric patients with CML is still controversially

The first study in neonatal rats revealed that imatinib interferes with postnatal testicular development [90]. Investigations in the juvenile animal model starting at an older age (4 weeks) depicted unchanged testis weight but reduced testosterone levels under longterm imatinib exposure until young adulthood. Inhibin B, a protein that is predominantly produced in the testis controlling follicle stimulating hormone (FSH) [91], did not significantly differ from controls, at all doses, and by all application schemes tested [92]. A clinical study conducted in a small cohort of boys (age: 7.8–18.9 years) with CML receiving TKI treatment revealed testosterone and inhibin B levels within normal age-related reference ranges [83, 84, 92]. Therefore, severe testicular toxicity by imatinib seems to be

However, a closer look on spermatogenesis in the juvenile animal model revealed that the spermatogenic cell counts were significantly decreased by high dose imatinib exposure (**Figure 6**). Additionally, during spermatogenesis cell cycle, the stage of the dominant cell proportion was shifted to more immature stages. Low dose and intermittent imatinib exposure attenuated these findings. Interestingly, spermatogenic cell proliferation was significantly lowered at all imatinib doses applied [93]. Thus, a delayed negative effect of long-term ima-

In the literature as well as indicated by the manufacturers in the specialist information, cardiotoxic and vascular side effects of imatinib and the next-generation TKIs are of special concern [94–99]. However, this primarily may play a role in older adult patients with CML (age

The juvenile animal model under discussion disclosed an increase in the relative heart weight ratio (= ratio of the heart weight to total body weight at sacrifice) under imatinib exposure. Another study found that imatinib treatment led to mitochondrial-dependent myocyte loss and cardiac dysfunction, occurring more severely in older mice, in part due to

tinib exposure on spermatogenesis cannot be excluded.

**6.4. Cardiac side effects of TKI treatment**

> 65 years) under TKI treatment.

in the adult skeleton.

tinib [87].

**6.3. Fertility**

discussed [89].

unlikely.

**Figure 6.** Number of proliferating testicular epithelium cells (Marker Ki67) in Wistar rats under imatinib exposure [93].

an age-dependent increase in oxidative stress [100]. This suggests that cardiac monitoring of older patients receiving imatinib therapy may be especially warranted.

As cardiac side effects were also observed with the use of dasatinib, in the experiment conducted with the juvenile animal model, this 2nd-generation TKI was tested for safety, efficacy, and dose response. Surprisingly, animals died spontaneously in a dose- and exposure timedependent manner (**Figure 7**). Data of the surviving animals that were sacrificed according to the experimental set-up schedule (**Figure 3**) disclosed—dependent on the cumulative dose administered—increased relative heart weights, impaired heart ejection fraction as assessed by echocardiography, and elevated brain natriuretic peptide (BNP) serum levels, an indicator of cardiac dysfunction [101]. Data of this unexpected high toxicity can be explained by the serum elimination half-life time of dasatinib which is rather short and in the range of 2–3 h in rodents [102]. As known from clinical data on treatment of CML by dasatinib, it is not mandatory to achieve steady state drug blood levels as the intracellular concentration of dasatinib is responsible for efficacy, which is sufficiently achieved by once daily drug administration. Initial trials in humans based on drug administration twice daily were characterized by high toxicity requiring treatment interruption or reduction to once daily dosing [103]. Thus, the juvenile rat model also mimics this situation as a continuous intake of small doses of dasatinib via the drinking water evidently is associated with higher toxicity.

Initially, inhibition of the c-abl kinase was assumed to be the reason for cardiac toxicity by TKI [94]. But an extensive *in vitro* study of 18 TKIs on myocytes showed that their relative ability to inhibit ABL1 or ABL2 did not correlate with myocyte damage, revealing that inhibition

Instead of the typical long, smooth proliferating chondrocyte columns at the epiphyseal line, a thin, disorganized layer of proliferative cells was detected after imatinib treatment resulting in a decreased thickness of PZ and increased the thickness of the HZ. This is in line with an *in vitro* study revealing an inhibitory effect of imatinib on chondrocyte proliferation [107]. Nurmio et al. also observed that imatinib treatment led to a bone resorption arrest and

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

http://dx.doi.org/10.5772/intechopen.70006

327

However, combining our data [68] with data from Nurmio et al. [106] and Vandyke et al. [107], it can be hypothesized that imatinib exposure alters metabolism and remodeling of the growing bone in a temporal-spatial stepwise fashion (**Figure 8B**). In the first instance, migration, proliferation, and activity of chondrocytes will be impaired by imatinib leading to a disturbed organization of the growth plate impairing longitudinal bone growth [106]. Altered growth hormone secretion under imatinib treatment as shown before in the growing organism [39, 41, 83] may aggravate this growth impairment. Thereafter, ongoing drug exposure causes a spatial activity shifting of bone remodeling: initially, the formation will be elevated and shifted to the area of the osteochondral junction, whereas the activity of bone resorption remains unchanged but will be spatially shifted to the distal area of the trabecular bone [106]. Finally, under long-term imatinib treatment, osteoblastogenesis and osteoclastogenesis will be impaired [104, 108], hampering bone remodeling during growth.

As an interesting approach, our juvenile animal model demonstrated that intermittent imatinib exposure will ameliorate growth impairment in rats. The inhibitory effect is not irreversible and we assume that during the days "OFF" imatinib exposure catch-up growth occurred. Therefore, drug administration following a schedule with "days on drug" and "days off drug" might reduce some skeletal side effects in pediatric patients. A single trial in older adults has already proven that intermittent TKI treatment is sufficient to control CML once remission

**Figure 8.** Schematic overview of physiologic bone growth (A) and under imatinib exposure (B). On the left side, longitudinal section of the epiphyseal line of a rodent proximal tibial metaphysis is depicted [109]. The epiphyseal plate separates the epiphysis from the metaphysis and is important for endochondral bone formation. The growth plate is divided into reserve zone (RZ), proliferative zone (PZ), and the hypertrophic zone (HZ). The transition of HZ to the primary spongiosa (PS)—the initial trabecular network formed after the vascular invasion and matrix calcification—is the osteochondral junction (OJ). Under physiological conditions, longitudinal growth occurs by endochondral ossification. In this process, new cartilage is formed at one side of the epiphyseal growth plate and is gradually replaced by bone. Chondrocytes of the growth plate are initially in a resting state in the RZ. They differentiate through proliferative and hypertrophic stages (PZ, HZ) as the growth plate moves past. This programmed differentiation pathway ends in cell death in the HZ and the replacement of cartilage by bone by osteoblasts in the OJ resulting in the PS. (For detailed

increased bone formation at the OJ [106].

review, see Ref. [110]).

**Figure 7.** Survival rate of juvenile Wistar rats under chronic dasatinib exposure [101].

of other kinases like MEK1 and MEK2 could be responsible for the cardiotoxicity. However, it was reported that all TKIs induce myocyte damage correlating with their kinase inhibitor selectivity [97]. So, we conclude that it might be prudent to carefully monitor cardiac function in still growing individuals with CML if treated with TKI continuously over long periods.

### **7. Hypothesized model of osseous damage and clinical relevance**

#### **7.1. Model of action of imatinib on bone remodeling**

Despite the knowledge accumulated so far, the detailed mechanism how imatinib impairs bone remodeling and growth remains yet speculative. In *in vitro* studies, it was shown that imatinib impairs osteoblastogenesis as well as osteoclastogenesis revealing its effect on bone remodeling [49, 50, 104, 105]. However, long bone growth is not only based on the balanced action of bone formation and bone resorption but also depends on the endochondral bone formation at the epiphyseal line of the long bones. Here, the column structure of the epiphyseal line, achieved and maintained by chondrocytes, is of main importance. In general, the epiphyseal line or growth plate is divided into different zones: reserve zone (RZ), proliferative zone (PZ), and the hypertrophic zone (HZ) followed by the primary spongiosa (PS)—the initial trabecular bone. The transition zone between HZ and PS is the osteochondral junction (OJ) (**Figure 8A**).

During growth, new cartilage is formed at one side of the epiphyseal growth plate and is gradually replaced by bone. The work by Nurmio et al. disclosed a disorganization of the epiphyseal line by imatinib treatment of neonatal rats (1–15 days old) (**Figure 9**) [106]. Instead of the typical long, smooth proliferating chondrocyte columns at the epiphyseal line, a thin, disorganized layer of proliferative cells was detected after imatinib treatment resulting in a decreased thickness of PZ and increased the thickness of the HZ. This is in line with an *in vitro* study revealing an inhibitory effect of imatinib on chondrocyte proliferation [107]. Nurmio et al. also observed that imatinib treatment led to a bone resorption arrest and increased bone formation at the OJ [106].

However, combining our data [68] with data from Nurmio et al. [106] and Vandyke et al. [107], it can be hypothesized that imatinib exposure alters metabolism and remodeling of the growing bone in a temporal-spatial stepwise fashion (**Figure 8B**). In the first instance, migration, proliferation, and activity of chondrocytes will be impaired by imatinib leading to a disturbed organization of the growth plate impairing longitudinal bone growth [106]. Altered growth hormone secretion under imatinib treatment as shown before in the growing organism [39, 41, 83] may aggravate this growth impairment. Thereafter, ongoing drug exposure causes a spatial activity shifting of bone remodeling: initially, the formation will be elevated and shifted to the area of the osteochondral junction, whereas the activity of bone resorption remains unchanged but will be spatially shifted to the distal area of the trabecular bone [106]. Finally, under long-term imatinib treatment, osteoblastogenesis and osteoclastogenesis will be impaired [104, 108], hampering bone remodeling during growth.

As an interesting approach, our juvenile animal model demonstrated that intermittent imatinib exposure will ameliorate growth impairment in rats. The inhibitory effect is not irreversible and we assume that during the days "OFF" imatinib exposure catch-up growth occurred. Therefore, drug administration following a schedule with "days on drug" and "days off drug" might reduce some skeletal side effects in pediatric patients. A single trial in older adults has already proven that intermittent TKI treatment is sufficient to control CML once remission

of other kinases like MEK1 and MEK2 could be responsible for the cardiotoxicity. However, it was reported that all TKIs induce myocyte damage correlating with their kinase inhibitor selectivity [97]. So, we conclude that it might be prudent to carefully monitor cardiac function in still growing individuals with CML if treated with TKI continuously over long periods.

Despite the knowledge accumulated so far, the detailed mechanism how imatinib impairs bone remodeling and growth remains yet speculative. In *in vitro* studies, it was shown that imatinib impairs osteoblastogenesis as well as osteoclastogenesis revealing its effect on bone remodeling [49, 50, 104, 105]. However, long bone growth is not only based on the balanced action of bone formation and bone resorption but also depends on the endochondral bone formation at the epiphyseal line of the long bones. Here, the column structure of the epiphyseal line, achieved and maintained by chondrocytes, is of main importance. In general, the epiphyseal line or growth plate is divided into different zones: reserve zone (RZ), proliferative zone (PZ), and the hypertrophic zone (HZ) followed by the primary spongiosa (PS)—the initial trabecular bone. The transition zone between HZ and PS is the osteochondral junction (OJ) (**Figure 8A**).

During growth, new cartilage is formed at one side of the epiphyseal growth plate and is gradually replaced by bone. The work by Nurmio et al. disclosed a disorganization of the epiphyseal line by imatinib treatment of neonatal rats (1–15 days old) (**Figure 9**) [106].

**7. Hypothesized model of osseous damage and clinical relevance**

**7.1. Model of action of imatinib on bone remodeling**

**Figure 7.** Survival rate of juvenile Wistar rats under chronic dasatinib exposure [101].

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

**Figure 8.** Schematic overview of physiologic bone growth (A) and under imatinib exposure (B). On the left side, longitudinal section of the epiphyseal line of a rodent proximal tibial metaphysis is depicted [109]. The epiphyseal plate separates the epiphysis from the metaphysis and is important for endochondral bone formation. The growth plate is divided into reserve zone (RZ), proliferative zone (PZ), and the hypertrophic zone (HZ). The transition of HZ to the primary spongiosa (PS)—the initial trabecular network formed after the vascular invasion and matrix calcification—is the osteochondral junction (OJ). Under physiological conditions, longitudinal growth occurs by endochondral ossification. In this process, new cartilage is formed at one side of the epiphyseal growth plate and is gradually replaced by bone. Chondrocytes of the growth plate are initially in a resting state in the RZ. They differentiate through proliferative and hypertrophic stages (PZ, HZ) as the growth plate moves past. This programmed differentiation pathway ends in cell death in the HZ and the replacement of cartilage by bone by osteoblasts in the OJ resulting in the PS. (For detailed review, see Ref. [110]).

Our hypothesis of spatiotemporal shifting of skeletal formation and resorption under imatinib is supported by clinical observations of a biphasic reaction of corresponding osseous metabolism serum markers in adult and pediatric patients with CML. In adult patients, an increase in bone formation occurred accompanied by elevated bone formation markers in the serum within the first months of therapy [74]. Pediatric CML patients display a biphasic response of bone formation and bone resorption by increasing levels within the first 3 months of imatinib treatment followed by a significant decline until 12 months of treatment (**Figure 5**) [77, 78]. Furthermore, we could show that long-term imatinib exposure may result in reduced bone strength possibly posing an elevated fracture risk in pediatric patients. Since 2001, adult CML patients are treated with imatinib but until now, no elevated fracture rates have been described in these patients [113]. As pediatric CML patients are treated with imatinib only since the beginning of this millennium, there is still no long-term experience. Our animal model also revealed that intermittent imatinib treatment mitigated skeletal effects on the growing bone, thus pointing toward a possibility to improve the risk-benefit ratio of long-term TKI exposure in pediatric patients. First clinical data in adults look promising but further studies must be carried out to determine whether the intermittent exposure is also sufficiently effective for the control of CML [111, 112]. Regarding pediatric patients, the results from the juvenile animal model and the clinical experience from adult patients with CML should be combined. This

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

http://dx.doi.org/10.5772/intechopen.70006

329

approach can be expected to harbor great potential in translational research.

mice are used mostly in orthotopic animal models [120–123].

The aim of the animal model described in this chapter was to evaluate side effects on bone remodeling rather than gaining further insight into the biology of CML (e.g., to study elementary mechanisms of CML disease progression) or on a more efficient antileukemic treatment exerted by new drugs (e.g., exploring why resistance develops under TKI therapy) [114, 115]. For these essential questions, the reader is kindly referred to the detailed body of literature on establishing and maintaining acute lymphatic or myeloid leukemic cells in xenograft models, transgenic models, and syngeneic models using a broad range of species [116–119], whereas

Our research described, focused on the question how bone metabolism is affected by TKI treatment as an off-targeted side effect and therewith induced structural and mechanical osseous changes in healthy not-outgrown animals [124]. Bone remodeling has been studied in many species and resulted in the current available knowledge [125–131]. Evidently, the financial burden of animal maintenance and drug doses to be administered when sequelae of chronic exposure are investigated are much lower using small animals like mice and rats. Especially in these species, the time periods concerning defined stages of development are shorter, thus requiring drug exposure only for 2–3 months in order to mimic one to two

Most importantly, any intervention on the bone during chronic TKI exposure of the animals was minimized. Bone growth and repair is governed by regulatory mechanisms other than that of the outgrown organism. Therefore, the model described here differs principally from

**8. Other animal models**

decades in humans [132].

**Figure 9.** Disorganization of the femoral epiphyseal line by long-term imatinib exposure. 2 μm sections of decalcified femora were stained with hematoxylin-eosin (magnification 100 ×). Controls show the typical "column" structure of the epiphyseal line and its physiological narrowing with increasing age. However, under imatinib exposure, the cellular architecture is more disorganized in a dose- and time-dependent manner.

has been achieved [111, 112]. However, the length and frequency of intervals to allow catchup growth in children on TKI treatment still have to be defined and at least in our rat model, this approach did not recover the biomechanical strength of the long bones.

#### **7.2. Clinical relevance**

The established juvenile rat model mimics to a gross extent side effects of long-term TKI exposure on the growing bone in a developmental stage-dependent fashion. Impairment of longitudinal growth, as observed in children under imatinib treatment, could be unequivocally modeled and confirmed.

Our hypothesis of spatiotemporal shifting of skeletal formation and resorption under imatinib is supported by clinical observations of a biphasic reaction of corresponding osseous metabolism serum markers in adult and pediatric patients with CML. In adult patients, an increase in bone formation occurred accompanied by elevated bone formation markers in the serum within the first months of therapy [74]. Pediatric CML patients display a biphasic response of bone formation and bone resorption by increasing levels within the first 3 months of imatinib treatment followed by a significant decline until 12 months of treatment (**Figure 5**) [77, 78].

Furthermore, we could show that long-term imatinib exposure may result in reduced bone strength possibly posing an elevated fracture risk in pediatric patients. Since 2001, adult CML patients are treated with imatinib but until now, no elevated fracture rates have been described in these patients [113]. As pediatric CML patients are treated with imatinib only since the beginning of this millennium, there is still no long-term experience. Our animal model also revealed that intermittent imatinib treatment mitigated skeletal effects on the growing bone, thus pointing toward a possibility to improve the risk-benefit ratio of long-term TKI exposure in pediatric patients. First clinical data in adults look promising but further studies must be carried out to determine whether the intermittent exposure is also sufficiently effective for the control of CML [111, 112]. Regarding pediatric patients, the results from the juvenile animal model and the clinical experience from adult patients with CML should be combined. This approach can be expected to harbor great potential in translational research.
