Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs ␣ and ␤) that bind triiodothyronine (T 3 , 3,5,3-triiodo-L-thyronine) with high affinity, and its precursor thyroxine (T 4 , 3,5,3,5-tetraiodo-L-thyronine) with lower affinity. T 4 contains a bulky 5 iodine group absent from T 3 . Because T 3 is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5 substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T 4 affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T 4 complexes adopt a conformation that differs from TR-T 3 complexes in solution. Nonetheless, T 4 behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T 3 does not contribute to agonist activity. We determined x-ray crystal structures of the TR␤ LBD in complex with T 3 and T 4 at 2.5-Å and 3.1-Å resolution. Comparison of the structures reveals that TR␤ accommodates T 4 through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5 iodine and complete the coactivator binding surface. While T 3 is the major active TH, our results suggest that T 4 could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5 extension should be considered in TR ligand design. Thyroid hormone (TH)1 plays important regulatory roles in metabolism, homeostasis, and development by binding and altering the transcriptional regulatory properties of two related nuclear receptors (NRs), the thyroid hormone receptors (TRs) ␣ and ␤ (1, 2). Most TH produced in the thyroid gland is secreted in the form of thyroxine (T 4 ; 3,5,3Ј,5Ј-tetraiodo-L-thyronine) (2, 3). The thyroid gland also produces smaller amounts of triiodothyronine (T 3 ; 3,5,3Ј-triiodo-L-thyronine) and reverse T 3 (rT 3 ; 3,3Ј,5Ј-triiodo-L-thyronine), and 80% of T 4 is converted to T 3 and rT 3 in peripheral tissues by two selenium deiodinases, which are tissue-specific (4). Current beliefs are that T 3 is the dominant active form of TH; T 3 binds the TRs with an affinity about 20 -30 times higher than that of T 4 (5-9), and some studies suggest that T 3 is present at higher concentrations in the nucleus than T 4 (10, 11). Nonetheless, the question of whether T 4 is simply a prohormone or an active TH species is not completely resolved. T 4 exerts rapid nongenomic effects at several loci distinct from TRs (12). Moreover, saturating levels of T 4 activate transcription of TH-responsive genes in cell culture (see for example Ref. 5). Whereas it is possible that at least some of this activity is due to T 3 generated from T 4 in the cell, these results suggest that T 4 may act as a TR agonist. Normal concentrations of plasma-free T 4 are about 4 -6-fold higher than th...

1. Intestinal loss, 1 - (Fobs/fH), is the missing fraction of the dose that is unexplained by systemic clearance. Here, we investigated whether intestinal loss in rat is predictive for human, and whether intestinal metabolism explained observed differences between rat and human. 2. For 81 marketed drugs, human and rat intestinal loss values were calculated from the literature and in-house sources. To examine the contribution of intestinal cytochrome P450-mediated metabolism to the high observed intestinal loss in the rat, metabolism was determined in rat and human intestinal microsomes for 15 compounds. 3. Oral bioavailability poorly correlated between rat and human. Twenty-two compounds in the human and 47 compounds in the rat showed an intestinal loss of more than 20%. The intestinal availability for many compounds was higher in human than in rat. Selected compounds, however, were more stable in rat than in human intestinal microsomes. 4. The rat poorly predicts the risk for intestinal loss in human; many compounds in rat had lower bioavailability than anticipated based on the hepatic clearance, but demonstrated little intestinal loss in human. This discrepancy appeared not to be caused by a higher cytochrome P450-mediated intestinal metabolism in the rat.

A well-defined work flow of DMPK activities from early lead identification up to the selection of a candidate drug was developed. This resulted in a cost effective and efficient optimization of chemical series, and facilitated informed decision making throughout project progress.

1. A set of reference compounds for time-dependent inhibition (TDI) of cytochrome P450 with available literature data for k and K was used to predict clinical implications using the GastroPlus software. Comparisons were made to in vivo literature interaction data. 2. The predicted AUC ratios (AUC/AUC) could be compared with the observed ratios from literature for all compounds with detailed information about in vivo administration, pharmacokinetics and in vivo interactions (N = 21). For this dataset, the difference between predicted and observed AUC ratios for interactions with midazolam was within twofold for all compounds except one (telaprevir, for which non-CYP-mediated metabolism likely plays a role after multiple dosing). 3. The sensitivity, specificity and accuracy of the GastroPlus predictions using a binary classification as no-to-weak interaction versus moderate-to-strong interaction for all compounds with available in vivo interaction data, were 80%, 82% and 81%, respectively (N = 31). 4. As a result of our evaluations of the DDI module in GastroPlus, we have implemented an early TDI risk assessment decision tree for our drug discovery projects involving in vitro screening and early GastroPlus predictions. Shifted IC values are determined and k/K estimated (by using a regression line established with in house-shifted IC values and literature k/K ratios), followed by GastroPlus predictions.

The introduction of anti-amyloid monoclonal antibodies against Alzheimer’s disease (AD) is of high importance. However, even though treated patients show very little amyloid pathology, there is only a modest effect on the rate of cognitive decline. Although this effect can possibly increase over time, there is still a need for alternative treatments that will improve cognitive function in patients with AD. Therefore, the purpose of this study was to characterize the triazinetrione ACD856, a novel pan-Trk positive allosteric modulator, in multiple models to address its neuroprotective and potential disease-modifying effects. The pharmacological effect of ACD856 was tested in recombinant cell lines, primary cortical neurons, or animals. We demonstrate that ACD856 enhanced NGF-induced neurite outgrowth, increased the levels of the pre-synaptic protein SNAP25 in PC12 cells, and increased the degree of phosphorylated TrkB in SH-SY5Y cells. In primary cortical neurons, ACD856 led to increased levels of phospho-ERK1/2, showed a neuroprotective effect against amyloid-beta or energy-deprivation-induced neurotoxicity, and increased the levels of brain-derived neurotrophic factor (BDNF). Consequently, administration of ACD856 resulted in a significant increase in BDNF in the brains of 21 months old mice. Furthermore, repeated administration of ACD856 resulted in a sustained anti-depressant effect, which lasted up to seven days, suggesting effects that go beyond merely symptomatic effects. In conclusion, the results confirm ACD856 as a cognitive enhancer, but more importantly, they provide substantial in vitro and in vivo evidence of neuroprotective and long-term effects that contribute to neurotrophic support and increased neuroplasticity. Presumably, the described effects of ACD856 may improve cognition, increase resilience, and promote neurorestorative processes, thereby leading to a healthier brain in patients with AD.