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

The tyrosine kinase (TK) inhibitor (TKI) imatinib provides a highly effective treatment for chronic myeloid leukemia (CML) targeting at the causative oncogenic TK BCR-ABL1. However, imatinib exerts off-target effects by inhibiting other TKs that are involved, e.g., in bone metabolism. Clinically, CML patients on imatinib exhibit altered bone metabolism as a side effect, which translates into linear growth failure in pediatric patients. As TKI treatment might be necessary for the whole life, long-term side effects exerted on bone and other developing organs in children are of major concern and not yet studied systematically. Here, we describe a new juvenile rat model to face this challenge. The established model mimics perfectly long-term side effects of TKI exposure on the growing bone in a developmental stage-dependent fashion. Thus, longitudinal growth impairment observed clinically in children could be unequivocally modeled and confirmed. In a “bench-to-bedside” manner, we also demonstrate that this juvenile animal model predicts side effects of newer treatment strategies by second generation TKIs or modified treatment schedules (continuous vs. intermittent treatment) to minimize side effects. We conclude that the results generated by this juvenile animal model can be directly used in the clinic to optimize treatment algorithms in pediatric patients

Effect of sclerostin inactivation in a mouse model of severe dominant osteogenesis imperfecta

Abstract Osteogenesis imperfecta (OI) is a rare bone disease that is associated with fractures and low bone mass. Sclerostin inhibition is being evaluated as a potential approach to increase bone mass in OI. We had previously found that in Col1a1 Jrt/+ mice, a model of severe OI, treatment with an anti-sclerostin antibody had a minor effect on the skeletal phenotype. In the present study, we assessed the effect of genetic sclerostin inactivation in the Col1a1 Jrt/+ mouse. We crossed Col1a1 Jrt/+ mice with Sost knockout mice to generate Sost-deficient Col1a1 Jrt/+ mice and assessed differences between Col1a1 Jrt/+ mice with homozygous Sost deficiency and Col1a1 Jrt/+ mice with heterozygous Sost deficiency. We found that Col1a1 Jrt/+ mice with homozygous Sost deficiency had higher body mass, femur length, trabecular bone volume, cortical thickness and periosteal diameter as well as increased biomechanical parameters of bone strength. Differences between genotypes were larger at the age of 14 weeks than at 8 weeks of age. Transcriptome analysis of RNA extracted from the tibial diaphysis revealed only 5 differentially regulated genes. Thus, genetic inactivation of Sost increased bone mass and strength in the Col1a1 Jrt/+ mouse. It appears from these observations that the degree of Sost suppression that is required for eliciting a beneficial response can vary with the genetic cause of OI

Impact of Long-Term Exposure to the Tyrosine Kinase Inhibitor Imatinib on the Skeleton of Growing Rats

<div><p>The tyrosine kinase (TK) inhibitor imatinib provides a highly effective therapy for chronic myeloid leukemia (CML) via inhibition of the oncogenic TK BCR-ABL1. However, off-target TKs like platelet-derived growth factor receptors (PDGF-R) and colony-stimulating factor-1 receptor (c-fms), involved in bone remodeling, are also inhibited. Thus, pediatric patients with CML on imatinib exhibit altered bone metabolism, leading to linear growth failure. As TKI treatment might be necessary for a lifetime, long-term effects exerted on bone in children are of major concern. Therefore, we studied the skeletal long-term effects of continuous and intermittent imatinib exposure in a juvenile rat model.</p><p>Four-weeks-old male Wistar rats were chronically exposed to imatinib via drinking water over a period of 10 weeks. Animals were exposed to a standard and high imatinib dosage continuously and to the high imatinib dose intermittently. Bone mass and strength were assessed using pQCT, micro-computed tomography (μCT), and biomechanical testing at the prepubertal, pubertal, and postpubertal age. Bone length and vertebral height as well as biochemical markers of bone turnover were analyzed.</p><p>Femoral and tibial bone length were dose-dependently reduced by up to 24% (p<0.0001), femoral and tibial trabecular bone mass density (BMD) were reduced by up to 25% (p<0.01), and femoral breaking strength was lowered by up to 20% (p<0.05). Intermittent exposure mitigated these skeletal effects. Long-term exposure resulted in reduced vertebral height by 15% and lower trabecular BMD by 5%. Skeletal changes were associated with suppressed serum osteocalcin (p<0.01) and non-significantly elevated serum CTX-I and PINP levels.</p><p>In conclusion, imatinib mainly impaired longitudinal growth of long bones rather than the vertebrae of growing rats. Interestingly, intermittent imatinib exposure has less skeletal side effects, which may be beneficial in pediatric patients taking imatinib.</p></div

Femoral BV/TV, Tb.Th, Tb.N, and Tb.C during long-term imatinib exposure.

<p>Three dimensional trabecular microarchitecture of the right femora was assessed by μCT. Trabecular region of interest was defined manually located within the secondary spongiosa in prepubertal femora 196 slices, pubertal femora 226 slices, and in postpubertal femora 256 slices. For trabecular bone regions, bone volume/ total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular connectivity (Tb.C) were calculated. Data represents mean ± 95% CI. Prepubertal = age of rats: 6 weeks, duration of exposure: 2 weeks (n = 4 each group); Pubertal = age of rats: 8 weeks, duration of exposure: 4 weeks (n = 3 each group); Postpubertal = age of rats: 14 weeks, duration of exposure: 10 weeks (n = 3 each group). Statistical analysis at defined time points: * p<0.05 versus age-related controls; ** p<0.01 versus age-related controls; *** p<0.001 versus age-related controls.</p

Fluid intake of the rats during long-term imatinib exposure.

<p>Fluid intake was measured 3 times weekly (Monday, Wednesday, Friday). Data represents mean ± 95% CI.</p

Bone length, total BMD, trabecular BMD, and cortical BMD of the femora (A), tibiae (B), and vertebra L2 (C) during long-term imatinib exposure assessed by pQCT.

<p>BMD = bone mineral density; Prep = prepubertal (age of rats: 6 weeks, duration of exposure: 2 weeks; n = 10 each group); Pub = pubertal (age of rats: 8 weeks, duration of exposure: 4 weeks; n = 8 each group); Postpub = postpubertal (age of rats: 14 weeks, duration of exposure: 10 weeks; n = 8 each group); Data represents mean ± 95% CI. Statistical analysis at defined time points: * p<0.05 versus age-related controls; ** p<0.01 versus age-related controls; *** p<0.001 versus age-related controls.</p