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The Tightrope Walker and the Fortune Teller: Key Players in Lead Optimization

By Amanda Woodrooffe, PhD & Kenneth R. Brouwer, PhD, RPh / Nov 19, 2019

Lead optimization is the iterative process of refining the structural properties of a new drug molecule to balance the desired pharmacological effects with appropriate drug-like properties with respect to the disposition and safety of the molecule.

Achieving this balance is critical in identifying a molecule with the highest likelihood of clinical success – a key need for drug developers given the ongoing challenge of attrition during clinical development.

Use of relevant, predictive models for characterizing new molecules is therefore an essential part of finalizing and finessing the selection of candidate molecules for preclinical and clinical development.

Case Study: Efficacy and Safety of the GPR40 agonist TAK 875

Free fatty acids (FFAs) not only act as a primary energy source in the body but are also cell signalling mediators. Free fatty acid receptor 1 (FFAR1; also known as GPR40) is a G protein-coupled receptor (GPCR), predominantly expressed in human and rodent pancreatic β cells. GPR40 mediates enhancement of glucose-stimulated insulin secretion (GSIS) through activation by physiological concentrations of long- and medium-chain FFAs1

A potent and selective GPR40 agonist is theorized to be a safe and effective antidiabetic drug, with little or no risk of hypoglycaemia,2

Fasiglifam (TAK-875), a GPR40 agonist developed to treat type 2 diabetes, was withdrawn from Phase III clinical trials due to drug-induced liver injury (DILI).

Mechanistic investigations revealed that covalent binding to hepatocytes was due to formation of a reactive acyl glucuronide (AG) of TAK-875 and concomitant, potent inhibition of hepatic drug transporters was likely to contribute to liver accumulation of this reactive metabolite. Evidence also suggested that TAK-875 inhibited mitochondrial respiration.3,4 All these mechanisms contribute to drug induced liver injury (DILI). In particular, cholestasis accounts for approximately 30% of all DILI cases.

Using human primary cell-based models, BioIVT generated data to demonstrate the published efficacy of TAK-875 to potentiate GSIS and to confirm the drug-induced liver injury that resulted in the termination of TAK-875 as a treatment for type 2 diabetes.

Efficacy of TAK-875

TAK-875 was investigated in BioIVT’s islet ORGANDOT™ model and was shown to effectively potentiate glucose-stimulated insulin secretion (Figure 1).

Lead Op_Blog Fig 1Figure 1. Effect of the GPR40 receptor agonist TAK-875 on isletORGANDOT™ GSIS responses.

GSIS assays were performed with 6.7mM (black bars) and 16.7mM (grey bars) glucose and various concentrations of TAK-875. Assays were performed with islet ORGANDOT™ cultures from two donors. The data are presented as mean±SEM (n=4 replicates). The student’s t-test was used to compare secretion of insulin in the presence of TAK-875 with that obtained in the absence of the agonist: *=p<0.05; ** = p<0.01, *** = p<0.001.


TAK-875 was evaluated in BioIVT’s C-DILI™ assay which uses Transporter Certified™ hepatocytes in sandwich culture to evaluate the potential for a compound to cause DILI by altering the hepatobiliary disposition of bile acids. TAK-875 was identified as having development-limiting drug safety concerns (Figure 2), with decreased ATP content and increased LDH leakage from human hepatocytes similar to the positive control troglitazone.

Lead Op_Blog Fig 2

Figure 2. Effect of the GPR40 receptor agonist TAK-875 in the C-DILI assay, ATP and LDH readouts.

C-DILI assays were performed with various concentrations of TAK-875 and a standard panel of control drugs (all concentrations are in µM). ATP and LDH data are presented as mean±SEM (n=3 replicates). Two-way ANOVA followed by Dunnett’s multiple comparisons test was used to evaluate differences compared to the DMSO control. (* p ≤0.001)

The effective concentrations of TAK-875 (1-10uM) in our isletORGANDOT GSIS assay did not demonstrate adverse effects in our C-DILI assay (100uM).

Data published by Li et al.4 demonstrate that TAK-875 inhibited hepatobiliary transporters, notably efflux transporter MRP2/Mrp2 and uptake transporters Ntcp and OATP/Oatp, which affect bile acid and bilirubin homeostasis, resulting in hyperbilirubinemia and cholestatic hepatotoxicity. In addition, their pharmacokinetic data for TAK-875 in rat suggest the potential for TAK-875 to accumulate 3-fold in the liver. The steady state plasma concentration of TAK-875 is around 10uM in man.5 Following oral administration, the portal vein concentrations of a drug can be significantly higher than systemic concentrations. Matsuda, et al. observed portal vein concentrations that were 10 to 100-fold higher than systemic concentrations in portal vein cannulated rats.6 Higher portal vein concentrations following oral administration should be taken into account in the design of in vitro experiments. The combination of increased portal vein concentrations along with significant hepatic accumulation could result in high intracellular concentrations of drugs that reach values where evidence of toxicity is observed in the in vitro C-DILI™ assay.

The data presented here demonstrate how human primary cell based, organotypic models can be effectively used to characterize pharmacology at native targets and identify potential safety concerns during lead optimization and candidate selection. This in turn can lead to planned investigations to further mitigate safety risks during ongoing drug development.

Predicting effects and finding the correct balance are essential elements of successful drug discovery. Using human in vitro models to assess both the efficacy and potential side effect liability of novel therapeutics during lead optimization and candidate selection enables a balance of the desired pharmacological effects with acceptable disposition and safety of the molecule.

For more information on ORGANDOT models and C-DILI assay kits and services, please contact us at



  1.  A Novel Antidiabetic Drug, Fasiglifam/TAK-875, Acts as an Ago-Allosteric Modulator of FFAR1. Yabuki C, et al. (2013). PLoS ONE 8(10): e76280.
  2. Discovery of TAK-875: A Potent, Selective, and Orally Bioavailable GPR40 Agonist. Negoro N, et al., (2010). ACS Med. Chem. Lett., 2010, 1 (6), pp 290–294.
  3. Fasiglifam (TAK-875): Mechanistic Investigation and Retrospective Identification of Hazards for Drug Induced Liver Injury. (2018). Otieno MA, et al., Toxicol Sci. 163(2):374-384.
  4. Fasiglifam (TAK-875) Inhibits Hepatobiliary Transporters: A Possible Factor Contributing to Fasiglifam-Induced Liver Injury. (2015). Li X, et al., Drug Metabolism and Disposition. 43 (11) 1751-1759.
  5. A multiple-ascending-dose study to evaluate safety, pharmacokinetics, and pharmacodynamics of a novel GPR40 agonist, TAK-875, in subjects with type 2 diabetes. (2012). Leifke E. et al., Clin Pharmacol Ther 92:29–39
  6. Assessment of Intestinal Availability of Various Drugs in the Oral Absorption Process Using Portal Vein-Cannulated Rats. (2012). Matsuda Y. et al., DMD 40:2231-2238.

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