Semi-mechanistic modelling of the tumour growth inhibitory effects of LY2157299, a new type I receptor TGF-β kinase antagonist, in mice

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Abstract

Human xenografts Calu6 (non-small cell lung cancer) and MX1 (breast cancer) were implanted subcutaneously in nude mice and LY2157299, a new type I receptor TGF-β kinase antagonist, was administered orally. Plasma levels of LY2157299, percentage of phosphorylated Smad2,3 (pSmad) in tumour, and tumour size were used to establish a semi-mechanistic pharmacokinetic/pharmacodynamic model.

An indirect response model was used to relate plasma concentrations with pSmad. The model predicts complete inhibition of pSmad and rapid turnover rates [t1/2 (min) = 18.6 (Calu6) and 32.0 (MX1)]. Tumour growth inhibition was linked to pSmad using two signal transduction compartments characterised by a mean signal propagation time with estimated values of 6.17 and 28.7 days for Calu6 and MX1, respectively.

The model provides a tool to generate experimental hypothesis to gain insights into the mechanisms of signal transduction associated to the TGF-β membrane receptor type I.

Introduction

Since its discovery almost two decades ago1 the transforming growth factor-β (TGF-β) has received considerable attention, being identified as a key element regulating tumour cell growth.2, 3 The complex and multifunctional activities of TGF-β endow it with both tumour suppressor and tumour promoting activities, depending on the stage of carcinogenesis and the response of the tumour cell.4, 5

Briefly, the TGF-β exerts its regulatory functions binding to the TGF-β type II receptor located in the cell membrane, the constitutively active TGF-β type II receptor recruits a TGF-β type I receptor, to form a heterotetrameric receptor complex, where TGF-β type II receptor phosphorylates the TGF-β type I receptor in the juxtamembrane region, or ‘GS domain’ which is rich in serine and threonine residues.6 Activated TGF-β type I receptor propagates the signal downstream directly by phosphorylating Smad2 and Smad3, which in turn form complexes with Smad4. This combined complex translocates to the nucleus where it regulates numerous gene transcriptions in combination with transcription factors.7

Recently, the in vitro and in vivo pharmacodynamic (PD) characterisation of new TGF-β receptor I kinase inhibitors has been published8, 9 as well as a review of the current drug development programmes for TGF-β receptors antagonists.10 However, to our knowledge in vivo characterisation of novel drugs inhibiting TGF-β receptors based on a pharmacokinetic/pharmacodynamic (PK/PD) approach has not been published yet.

The objective of the present study was to develop a semi-mechanistic PK/PD model for LY2157299, a new and potent specific TGF-β type I receptor antagonist of the family of pyrazoles.11, 12 The model was formulated with the aim of integrating the pharmacokinetics (PK) properties of LY2157299, the percentage of phosphorylated Smad2 and Smad3 (pSmad) considered as a biomarker, and the inhibition of tumour growth.

Section snippets

Materials and methods

106 Calu6 human anaplastic carcinoma lung cells (Calu6) or 106 MX1 human carcinoma breast cells (MX1) were implanted subcutaneously into Charles River nude mice (weight ∼25 mg). Experiments started 7–10 days after tumour implantation. Two different experiments were carried out, the PK/PD experiments which provided the pharmacokinetic and biomarker data, and the tumour growth experiments which provided the tumour size data. The experiments were adhered to the Principles of Laboratory Animal Care

Pharmacokinetic model

Disposition of LY2157299 in plasma was best described with a two compartment model. Absorption process could not be adequately characterised due to the lack of plasma samples for the first 30 min after drug administration. The value of Ka, the first-order rate absorption constant used (8 h−1) was selected as a result of a sensitive analysis, where Ka was fixed to different values (ranging from 0.5 to 15 h−1), selecting the one providing the lowest value in OBJ. Dose and time did not show

Discussion

Pharmacokinetics of LY2157299 was described with standard PK models, and neither time nor dose dependencies were detected. LY2157299 was rapidly eliminated from the plasma and no accumulation was present.

The time course of pSmad was described with an indirect response model,17, 22 where the rate of phosphorylation was inhibited by the drug resembling a mechanism that is supported by experimental findings.10, 23 It is recognised, however, that the model for biomarker represents an

Conflict of interest statement

The authors are employees of Eli Lilly and Company (Dinesh P. de Alwis, Celine Pitou, Jonathan Yingling, Michael Lahn and Sophie Glatt) or have received financial research support from this company (Lorea Bueno and Iñaki F. Trocóniz).

Acknowledgement

This work was supported by Eli Lilly and Company, Windlesham, Surrey, UK

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