Src Inhibitors Activity In Vivo In Bone Loss Models

The in vivo activity of the pyrrolopyrimidine CGP77675 was previously reported in bone loss models (19). Here we present new data with other compounds in several bone loss models: young growing and skeletally mature female ovariectomized (OVX) rats and young female and male retinoid-treated intact or thyroparathyroidectomized (TPTX) rats. Table 4 gives details on treatment regimen and methods of measurement and summarizes activities of several Src inhibitors in these models. The highest activity was displayed by two compounds: the pyrrolopyrimidine CGP76030 and the olomoucine NVP-AAK980. Two other compounds—CGP81699 and CGP79833, also a pyrrolopyrimidine and an olomoucine—showed less activity and more side effects (see Heading 6). Various activities and side-effect profiles within the same chemical classes suggested that the effects were not linked to the core compound structure, but rather to different biological selectivity profiles of each compound.

The most extensively studied compound was the pyrrolopyrimidine CGP76030, which showed activity in all models, even at doses as low as 10 mg/kg by mouth once or twice daily (Table 4). The compound fully prevented hypercalcemia and bone loss in retinoid-treated male TPTX and intact

Table 4

In Vivo Activity of Several Src Inhibitors in Models of Bone Loss





Measurements: Results/bone:






8- and 40 to 44-wk-old female OVX Sprague-Dawley or Wistar rats0

6- or 12-wk-old retinoid-treated, TPTX, and intact Wistar rats6 10 to 100 mg/kg, p.o., administered once or twice daily, 6-12 wka

10 to 100 mg/kg, p.o., once daily for 4 db Chemical analyses0, DEXA0b, pQCT0, serum calcium6 At 10 mg/kg, twice daily, significant prevention of trabecular bone loss in 8- and 40-wk-old OVX rats (chemical analyses) Nonsignificant protection at some sites by in 40 to 44-wk-old

OVX rats (pQCT) No effects by DEXA in OVX model Dose-dependent protection in retinoid model: Calcium protection 29% at 10 mg/kg, significant 53% at

50 mg/kg, and full at 100 mg/kg, once daily Full, significant bone loss protection at 100 mg/kg (DEXA)

44-wk-old female OVX Sprague-Dawley rats

3, 10, and 30 mg/kg p.o., daily, for 12 wk pQCT in proximal tibial metaphysis, DEXA in femur and lumbar vertebral bodies No protection of bone by pQCT or DEXA



Model: Treatment: Measurement: Results/bone:


Model: Treatment: Measurement: Results/bone:

6 to 7-wk-old male Wistar rats, thyroxin- and retinoid-treated 10, 30, and 75 mg/kg, daily, for 4 d Serum calcium, 3 h after the last daily injection Inhibition of serum calcium: none at 10 mg/kg, significant 39% at 30 mg/kg, and full at 100 mg/kg, ED50 of 34 mg/kg

6 to 7-wk-old male Wistar rats, thyroxin- and retinoid-treated 30 and 75 mg/kg, daily, for 4 d Serum calcium, 3 h after the last daily injection Inhibition of serum calcium: 30% at 30 mg/kg, significant 37% at 100 mg/kg a and b indicate the corresponding models, treatments, and measurements. OVX, ovariectomized; TPTX, thyroparathyroidectomized; DEXA, dual energy X-ray absorptiometry; pQCT, peripheral quantitative computed tomography.

Fig. 4. Protective effect of CGP76030 on vertebral trabecular bone. Three-dimensional microcomputed tomographic reconstruction of rat lumbar vertebrae (LV2). (A) The central portion (1.3-mm length) of a complete vertebra of a sham-operated rat is shown. (B) Enlarged reconstruction of the isolated vertebral body from vertebrae shown in A. (C,D) Vertebral bodies of 8-wk-old ovariectomized rats without (C) or with (D) treatment with 50 mg/kg orally CGP76030 twice daily. The compound partially prevented microarchitectural changes caused by ovariectomy. OVX, ovariectomized.

female rats at 100 mg/kg by mouth on day 4, but could not be tested at such high doses in a long-term (12 wk) model of OVX-induced bone loss in skeletally mature rats owing to side effects (see Heading 6). Deaths occurred already at 30 mg/kg/day in 40 to 44-wk-old OVX female rats, but not up to 50 mg/kg/twice daily in young 8-wk-old rats. The reasons for this difference are probably connected to overproportional exposure at higher doses, particularly in old rats, and were facilitated by the long treatment period (12 wk). In OVX rat models, the protective effects of CGP76030 on bone measured by peripheral quantitative computed tomography and dual energy X-ray absorptionmetry were partial and often not significant, which can be attributed to relatively low bone loss by OVX. However, chemical analyses of bone content by measurement of bone calcium and hydroxyproline detected significant prevention of bone loss at 10-20 mg/kg by mouth in both young and old OVX rats. In addition, OVX-induced loss in trabecular bone microarchitecture was partially prevented by CGP76030, as illustrated in Fig. 4.

The olomoucine NVP-AAK980 was tested only in the short-term retinoid-treated rat model. The compound was active at 30 and 100 mg/kg by mouth, with the latter dose showing a full protection of hypercalcemia. Further tests were not done because of an insufficient therapeutic window (see Heading 6).

In conclusion, the pyrrolopyrimidine CGP76030 and the olomoucine NVP-AAK980 were active in preventing bone loss in the OVX and retinoid-treated rat models (from 10 and 30 to 100 mg/kg/by mouth). In the retinoid-treated rat model, they showed a full protection against hypercalcemia at higher doses (both compounds) and bone loss (only CGP76030 tested), an effect that is matched only by bisphosphonates and calcitonin, recognized bone resoprtion inhibitors already used in osteoporosis therapy.

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