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

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, and strength by using quantitative computed tomography (pQCT), micro-computed tomography (μCT), and biomechanical testing [68].

trabecular bone mineral density (BMD) by imatinib exposure. Analysis of the 3D trabecular structure by μCT emphasizes these findings by demonstration of reduced bone volume density in combination with reduced trabecular number and connectivity [68]. Furthermore, our findings also indicated unchanged cortical BMD and cortical thickness during growth doseand time-independently, whereas the bone strength of the femora was decreased after longterm exposure to high dose imatinib. This could be explained by decreased cross-sectional area, periosteal, and endosteal circumference of the femora, suggesting a blunted radial appositional bone growth [68]. With regard to pediatric patients, BMD measurements or increased fracture rates under long-term imatinib treatment are not published yet. However, intermittent treatment of the high dose mitigated all bony side effects of the long bones, which might

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Concerning lumbar vertebra, 10-week imatinib exposure significantly reduced vertebral height combined with reduced trabecular BMD dose-dependently, whereas total BMD, cortical BMD, cross-sectional area, and cortical thickness were not affected [68, 73]. At the moment, only limited data are available on the effect of imatinib on vertebrae. In adult patients with CML, O´Sullivan et al. observed significantly increased lumbar spine BMD after 24 months of imatinib treatment as assessed by dual energy x-ray absorptiometry (DXA) [74], whereas Vandyke et al. observed unchanged BMD [75]. We predict from our animal model that ima-

tinib also alters vertebral properties, but not to the same extent as in long bones [68].

The bone resorption marker tartrate-resistant acidic phosphatase (TRAP) revealed significantly decreased serum levels under continuous imatinib exposure indicating reduced osteoclast activity at all developmental stages [68]. This is confirmed by *in vitro* studies showing that imatinib impairs osteoclastogenesis leading to diminished numbers of TRAP-positive osteoclasts [49, 76]. However, bone resorption marker C-terminal collagen cross-links (CTX-I) revealed by trend elevated serum levels prepubertally, but normal levels during the ongoing exposure time, indicating nearly unchanged osteoclast activity during growth [68]. This is consistent with data from pediatric patients with CML describing by trend elevated CTX-I

Under imatinib exposure, bone formation marker osteocalcin was decreased but procollagen type I (PINP) levels were by trend elevated, pointing to improved bone formation and mineralization [68]. *In vitro* assays using human isolated mesenchymal stem cells, primary rat osteoblasts, and mouse osteoblast-like cell line MC3T3-E1 revealed all increased mineralization combined with reduced proliferation under therapeutic imatinib concentration [50].

However, bone turnover markers of pediatric patients with CML exhibited a biphasic response during imatinib therapy with increasing levels within the first 3 months of treatment and a

significant decline during long-term treatment (**Figure 5**) [77, 78].

offer a new perspective for pediatric patients.

**5.3. Vertebrae height and quality**

**5.4. Bone turnover markers**

levels prepubertally while on imatinib [77].

#### **5.1. TKI serum concentration**

Imatinib mean serum levels of 1600 and 5600 ng/mL were achieved by continuous drug exposure via the drinking water to either low or high dose, respectively [68]. These serum concentrations match well with therapeutic imatinib levels of pediatric patients ranging from 2000 to 8000 ng/mL on imatinib administered at doses of 260–570 mg/m<sup>2</sup> daily [26], whereas in adult patients, serum levels in the range of ~1000–3400 ng/mL on imatinib doses of 400–600 mg daily were measured [69, 70]. Reflecting the half-life of imatinib in rats reported to be 12.3 h [71], serum levels of animals receiving high dose imatinib intermittently were below the detection limit of the assay (10.0 ng/mL) when serum was collected at the end of a 4-day period without drug exposure.

#### **5.2. Long bone length and bone quality**

During growth, a 10-week exposure to imatinib caused a significant reduction of the long bone length dose-dependently (**Figure 4**) [68]. These findings match with clinical data in children indicating that continuous administration of imatinib—even in high doses—does not result in a complete stop of growth, rather in a decelerated growth rate of the long bones [5, 36, 38–41, 72]. During growth, pQCT analysis of the bones revealed significantly reduced

**Figure 4.** Growth impairment of long bones by imatinib is dependent on the cumulative dose [68]. Prep: Prepubertal; Pub: Pubertal; Postpub: Postpubertal. Compared to controls, high dose imatinib (1000 mg/L daily) causes stronger longitudinal growth impairment than low dose exposure (500 mg/L daily). "On/off" exposure (3 days "on", 4 days "off") to high dose imatinib mitigates this effect. Of note, the cumulative dose resulting from 1000 mg/L administered "on/off" is approximately identical to 500 mg/L daily administered continuously. The resulting reduction in length reflects the cumulative dose administered.

trabecular bone mineral density (BMD) by imatinib exposure. Analysis of the 3D trabecular structure by μCT emphasizes these findings by demonstration of reduced bone volume density in combination with reduced trabecular number and connectivity [68]. Furthermore, our findings also indicated unchanged cortical BMD and cortical thickness during growth doseand time-independently, whereas the bone strength of the femora was decreased after longterm exposure to high dose imatinib. This could be explained by decreased cross-sectional area, periosteal, and endosteal circumference of the femora, suggesting a blunted radial appositional bone growth [68]. With regard to pediatric patients, BMD measurements or increased fracture rates under long-term imatinib treatment are not published yet. However, intermittent treatment of the high dose mitigated all bony side effects of the long bones, which might offer a new perspective for pediatric patients.

#### **5.3. Vertebrae height and quality**

and strength by using quantitative computed tomography (pQCT), micro-computed tomog-

Imatinib mean serum levels of 1600 and 5600 ng/mL were achieved by continuous drug exposure via the drinking water to either low or high dose, respectively [68]. These serum concentrations match well with therapeutic imatinib levels of pediatric patients ranging

whereas in adult patients, serum levels in the range of ~1000–3400 ng/mL on imatinib doses of 400–600 mg daily were measured [69, 70]. Reflecting the half-life of imatinib in rats reported to be 12.3 h [71], serum levels of animals receiving high dose imatinib intermittently were below the detection limit of the assay (10.0 ng/mL) when serum was collected at

During growth, a 10-week exposure to imatinib caused a significant reduction of the long bone length dose-dependently (**Figure 4**) [68]. These findings match with clinical data in children indicating that continuous administration of imatinib—even in high doses—does not result in a complete stop of growth, rather in a decelerated growth rate of the long bones [5, 36, 38–41, 72]. During growth, pQCT analysis of the bones revealed significantly reduced

**Figure 4.** Growth impairment of long bones by imatinib is dependent on the cumulative dose [68]. Prep: Prepubertal; Pub: Pubertal; Postpub: Postpubertal. Compared to controls, high dose imatinib (1000 mg/L daily) causes stronger longitudinal growth impairment than low dose exposure (500 mg/L daily). "On/off" exposure (3 days "on", 4 days "off") to high dose imatinib mitigates this effect. Of note, the cumulative dose resulting from 1000 mg/L administered "on/off" is approximately identical to 500 mg/L daily administered continuously. The resulting reduction in length reflects the

daily [26],

from 2000 to 8000 ng/mL on imatinib administered at doses of 260–570 mg/m<sup>2</sup>

raphy (μCT), and biomechanical testing [68].

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

the end of a 4-day period without drug exposure.

**5.2. Long bone length and bone quality**

cumulative dose administered.

**5.1. TKI serum concentration**

Concerning lumbar vertebra, 10-week imatinib exposure significantly reduced vertebral height combined with reduced trabecular BMD dose-dependently, whereas total BMD, cortical BMD, cross-sectional area, and cortical thickness were not affected [68, 73]. At the moment, only limited data are available on the effect of imatinib on vertebrae. In adult patients with CML, O´Sullivan et al. observed significantly increased lumbar spine BMD after 24 months of imatinib treatment as assessed by dual energy x-ray absorptiometry (DXA) [74], whereas Vandyke et al. observed unchanged BMD [75]. We predict from our animal model that imatinib also alters vertebral properties, but not to the same extent as in long bones [68].

#### **5.4. Bone turnover markers**

The bone resorption marker tartrate-resistant acidic phosphatase (TRAP) revealed significantly decreased serum levels under continuous imatinib exposure indicating reduced osteoclast activity at all developmental stages [68]. This is confirmed by *in vitro* studies showing that imatinib impairs osteoclastogenesis leading to diminished numbers of TRAP-positive osteoclasts [49, 76]. However, bone resorption marker C-terminal collagen cross-links (CTX-I) revealed by trend elevated serum levels prepubertally, but normal levels during the ongoing exposure time, indicating nearly unchanged osteoclast activity during growth [68]. This is consistent with data from pediatric patients with CML describing by trend elevated CTX-I levels prepubertally while on imatinib [77].

Under imatinib exposure, bone formation marker osteocalcin was decreased but procollagen type I (PINP) levels were by trend elevated, pointing to improved bone formation and mineralization [68]. *In vitro* assays using human isolated mesenchymal stem cells, primary rat osteoblasts, and mouse osteoblast-like cell line MC3T3-E1 revealed all increased mineralization combined with reduced proliferation under therapeutic imatinib concentration [50].

However, bone turnover markers of pediatric patients with CML exhibited a biphasic response during imatinib therapy with increasing levels within the first 3 months of treatment and a significant decline during long-term treatment (**Figure 5**) [77, 78].

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

osteoblastogenesis, leading to linear bone growth [80].

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].

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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

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

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

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 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 hyperparathyroid-

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

comprises of stimulating the absorption of calcium/phosphorus from

and 1.25(OH)<sup>2</sup>

, 1.25(OH)2

D3

D3

levels but normal

, calcium,

**6.1. Growth hormone**

duration [83, 84].

**6.2. Vitamin D and bone**

metabolite 1.25(OH)<sup>2</sup>

D3

and muscle weakness, causing falls and fractures [86].

ism in conjunction with pathologically low 25-(OH)D<sup>3</sup>

Hypophosphatemia, associated with low serum levels of 25-(OH)D<sup>3</sup>

**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> daily 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).
