TGF-β plays a key role in several disease states, including cancer, cardiovascular disease, and fibrosis, which has made the TGF-β signaling pathway a logical and attractive target of pharmaceutical development. However, TGF-β signaling has posed a challenge with respect to understanding safe therapeutic doses that do not confer toxicities associated with prolonged inhibition of this critical pathway [16
]. We characterized the toxicity profile of LY2157299 in nonclinical species for up to 6 months of daily dosing. LY2157299 was well tolerated in the rat and the dog for one month of daily dosing and in the rats for 3 months of intermittent dosing. Long-term daily dosing of =3 months caused alterations in the gastrointestinal, immune, musculoskeletal, renal and cardiovascular systems of animals, which is anticipated based on the integral role for TGF-β signaling for maintaining homeostasis.
Other pharmaceutical companies have published similar valvular toxicities with inhibitors from their development efforts, indicating that the lesions described are likely an on target effect [12
]. Many explanations have been considered to understand mechanism of this phenomenon; no definitive explanation has been provided at this time [12
]. TGF-β is a required mediator for valve structural and functional homeostasis [20
]. In response to injury, valvularmyofibroblasts are activated by TGF-β and results in increases in a-SMA expression [21
]. This paradoxical inhibition of TGF-β signaling with LY2157299 -treated animals resulting in increased SMA in their heart valves is not understood and awaits further clarification.
We have characterized cardiac effects in nonclinical species that extend beyond the previously reported valvulopathy and, in contrast to the valvulopathy, constituted a pathologic lesion defined as dose limiting within the context of a given study. In both species, the aorta and arteries of the heart are major target organs for toxicity. The vascular lesions in the rat are characterized by inflammation and hemorrhage with associated disruption of the mural organization, sometimes resulting in vessel rupture and hemorrhage into the thoracic cavity. In contrast to the rat, the dog showed changes in the base of the ascending aorta, characterized by degeneration and disorganization of the mural elastic lamellae and often increased prominence of mucopolysaccharide-rich ground substance without accompanying inflammation. The reason for this apparent species difference is unknown.
Our findings support that TGF-β plays a pleiotropic role in cell growth and on the extracellular matrix. It is important in both production of matrix proteins and the degradation of the matrix [22
]. Two well-studied genetic diseases Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS) affect the skeletal, ocular, and cardiovascular systems [23
]. Clinically, these syndromes present with overlapping symptoms including aortic aneurysms, dissections and mitral valve prolapse [24
]. MFS is caused by mutations in the gene encoding fibrillin-1 that interacts with latent TGF-β binding protein to upregulate TGF-β bioavailability and activity [25
]. LDS is an autosomal dominant aortic aneurysm syndrome caused by mutations in the promoter of TGF-β R1 and R2 with increased TGF-β activity [23
]. In LDS, the microscopic aortic lesion is characterized by fragmentation of elastic fibers, loss of elastin content, and accumulation of amorphous matrix components in the aortic media, changes very similar to those observed in the 6-month dog study with LY2157299 [23
]. In cardiac tissue from affected patients and mouse models of MFS, TGF-β signaling is increased, potentially due toaltered receptor processing or alternative pathways responsible for upregulation of TGF-β [27
]. Mutations in the TGF-β signaling pathway that should lead to decreased TGF-β signaling resulting in an upregulation of this pathway underscores that in vivo, precise regulation of multiple members is required to maintain homeostasis and that canonical and non-canonical signaling pathways play an important role [28
The findings described in the aorta and cardiac arteries in the toxicology studies in the rat and the dog, based on similarity with genetic syndromes, appear to be more consistent with an upregulation rather than inhibition of the TGF-β pathway. The contributions of the non-canonical TGF-β signaling are not yet understood. Activation of the ERK pathway contributes to progression of aneurysms in Fbn1
mice. RDEA119, a selective MEK inhibitor, reduced aortic root growth independent of increased Smad2
activation, suggesting that non-canonical pathway modulation may contribute to progression to aneursyms in Fbn1
]. As with MFS and LDS, which are genetic mutations resulting in a clinical phenotype, additional genetic differences may also play a role in susceptibility to cardiac injury observed in nonclinical studies with inhibitors of TGF-β signaling. Behmoaraset al. evaluated seven genetically different strains in rats for their differences in composition of the extracellular matrix and noted a differentiation among strains with respect to matrix composition and occurrence of internal elastic lamina rupture [30
The changes to the heart following long-term treatment of LY2157299 at toxicologically relevant doses are of largest concern with respect to clinical safety, inhibition of TGF-β signaling resulted in changes to several other organ systems in both the rat and dog. LY2157299 resulted in changes to the bone similar to previous reports [12
]. Reversibility of bone changes was assessed following the 1-month study in rats revealing a band of increased trabecular density that was separated from the normal physis by a zone of normal endochondral ossification. Following 6 months of treatment in the rat, degeneration in the joints of the sternebrum and stifle was reported. Dominant negative mutant TGF-β receptor II mice are reported to have bifurcated xiphoid process and sternum as well as a progressive osteoarthritis-like disease [31
]. Dogs used in the 6-month study were between 6-8 months of age and are expected to still have active longitudinal growth in their bones; therefore, alterations in the endochondral cartilage similar to what was described in the rat may be anticipated [32
TGF-β also plays a critical role in epithelial biology, acting as a tumor suppressor or promoter [33
]. Alteration in TGF-β signaling is common in many cancers [34
]. Disruption of TGF-β signaling in either epithelial or stromal cells increases inflammatory responses that promote tumor initiation, progression, and metastasis [35
]. TGFβ1-/- Rag2 -/-
mice develop inflammation-associated adenomas and carcinomas through an inability to maintain epithelial tissue organization [36
]. Mice genetically deficient in TGF-β R2 signaling have increased susceptibility to cancer [37
]. Similarly, inactivation of downstream proteins Smad3
results in carcinomas of the intestine [38
]. The observations of hyperplasia, adenoma, and carcinoma of the intestine in rats treated with LY2157299 for 6 months are consistent with the well-characterized biology.
The use of intermittent dosing is common practice in oncology: clinicians will reduce the dose or permit a drug holiday to allow the patient to recover from toxicities prior to receiving another dose. Studies have shown that anticancer agents were more tolerable and efficacious when administered in intermittent dosing schedules [39
]. In contrast to a dosing schedule based on clinical findings, clinical dosing with LY2157299 proactively applied an indirect model to support a clinical dosing regimen that would support a desired pharmacologic effect and avoid unwanted toxicity. Buenoet al. have been able to relate plasma concentrations to pSmad inhibition [41
]. Tumor growth inhibition was then linked to pSmad inhibition, providing a tool to titrate the dose required for efficacy. Furthermore, the data support the concept of intermittent dosing in maintaining a similar tumor response. The ability to maintain plasma concentrations required for efficacy lower than those observed to cause adverse toxicities in nonclinical species is important, especially when considering drugs targeting signaling pathways such as TGF-β that have been demonstrated to be critical in maintaining normal function. We demonstrate continuous administration of LY2157299 at high doses or durations =3 months in nonclinical species results in adverse toxicities to numerous organ systems including cardiovascular, gastrointestinal, immune, bone/cartilage, reproductive, and renal. The cardiovascular system appears to be most sensitive to abrogation of TGF-β signaling with the small molecule inhibitor LY2157299. Our data have defined a NOEL for cardiovascular lesions of 150 mg/kg and 20 mg/kg, in rat and dog, respectively, after one month of daily dosing and 50 mg/kg for 3 months on a 2-week-on/2-week-off dosing schedule in the rat. An intermittent dosing paradigm demonstrated that the severity and incidence of cardiac lesions could be lessened. The data from the 3-month intermittent dosing study in rats along with the absence of cardiovascular lesions in the 1-month rat and dog study established a nonclinical data package that supports a clinical dosing schedule of 2 weeks on/2 weeks off LY2157299. Successful development of such inhibitors will need to have a thorough characterization of nonclinical toxicity and an understanding of schedule and plasma concentrations needed to demonstrate anti-tumor effect and determine if a sufficient therapeutic window exists to test the clinical efficacy of molecules involved in inhibiting key pathways.