Strategy for Extending Half-life in Drug Design and Its Significance

Preclinical optimization of compounds toward viable drug candidates requires an integrated understanding of properties that impact predictions of the clinically efficacious dose. The importance of optimizing half-life, unbound clearance, and potency and how they impact dose predictions are discussed in this letter. Modest half-life improvements for short half-life compounds can dramatically lower the efficacious dose. The relationship between dose and half-life is nonlinear when unbound clearance is kept constant, whereas the relationship between dose and unbound clearance is linear when half-life is kept constant. Due to this difference, we show that dose is more sensitive to changes in half-life than changes in unbound clearance when half-lives are shorter than 2 h. Through matched molecular pair analyses, we also show that the strategic introduction of halogens is likely to increase half-life and lower projected human dose even though increased lipophilicity does not guarantee extended half-life

A human tissue-based functional assay platform to evaluate the immune function impact of small molecule inhibitors that target the immune system

<div><p>While the immune system is essential for the maintenance of the homeostasis, health and survival of humans, aberrant immune responses can lead to chronic inflammatory and autoimmune disorders. Pharmacological modulation of drug targets in the immune system to ameliorate disease also carry a risk of immunosuppression that could lead to adverse outcomes. Therefore, it is important to understand the ‘immune fingerprint’ of novel therapeutics as they relate to current and, clinically used immunological therapies to better understand their potential therapeutic benefit as well as immunosuppressive ability that might lead to adverse events such as infection risks and cancer. Since the mechanistic investigation of pharmacological modulators in a drug discovery setting is largely compound- and mechanism-centric but not comprehensive in terms of immune system impact, we developed a human tissue based functional assay platform to evaluate the impact of pharmacological modulators on a range of innate and adaptive immune functions. Here, we demonstrate that it is possible to generate a qualitative and quantitative immune system impact of pharmacological modulators, which might help better understand and predict the benefit-risk profiles of these compounds in the treatment of immune disorders.</p></div

The impact of small molecule inhibitors on human T cell function.

<p>The production of IL-2 as well as the proliferation of CD4⁺ and CD8⁺ T cells was evaluated in a human <i>in vitro</i> PBMC assay. The production of IL-2 was evaluated 24h following stimulation with α-CD3 and α-CD28 while the proliferation of CD4⁺ and CD8⁺ T cells was evaluated in the same cultures 72h following α-CD3 and α-CD28 stimulation. Small molecule inhibitors suppress α-CD3 and α-CD28 induced IL-2 production (A), CD4⁺ T cell proliferation (B) and CD8⁺ T cell proliferation (C) in human PBMCs. For each compound, the potency (red circles) is plotted along the top X-axis and the percentage maximal inhibition achieved in the assay (black squares) are plotted along the bottom X-axis. The potencies of the evaluated compounds in these assays are displayed as IC₅₀ ovalues. A maximum of 100% inhibition is possible for small molecule inhibitors in these assays. The reported potency and % max inhibition values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound.</p

Immune function impact profiles of small molecule inhibitors.

<p>Small molecule inhibitors were evaluated in six different functional assays with nine different read-outs. For a given compound on the Y-axis, each symbol on the X-axis represents the protein binding corrected cellular potency of that compound in the corresponding assay. The potencies of the evaluated compounds in these assays are displayed as IC₅₀ values in the X-axis. The reported IC₅₀ values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound in each assay. Immune function impact of a broader set (A) or a subset (B) of inhibitors are presented.</p

Assays to evaluate immune function impact of small molecule inhibitors.

<p>All the assays were established using healthy donor peripheral blood mononuclear cells (PBMCs) or whole blood. The immune functions evaluated, the tissues analyzed, stimulations used as well as the tissue analysis methods are listed for each assay. Small molecule inhibitors that are known to inhibit these functions were used as positive controls (assay controls) in each run of the assay, to ensure consistent technical performance and data robustness.</p

A conceptual framework to develop an ‘immune impact score’ for compounds.

<p>The impact of compounds in immune system-based functional assays, -omics approaches, PK/PD information and chemical informatics (that classify and predict compound-function relationships based on available information) might be integrated with the aim of assigning an ‘immune impact score’ for individual compounds. Further integration of available or modeled efficacy and adverse event data for compounds with the immune impact score might be useful in better understanding and predicting the efficacy, adverse event profile and differentiation of these compounds in a clinical setting. PK/PD–Pharmacokinetics/Pharmacodynamics, AE–adverse events.</p

Impact of small molecule inhibitors on gene expression profiles in the T cell stimulation assay.

<p>(A) A nanostring gene expression panel was used to evaluate mRNA profiles of PBMCs 24 h following treatment with 1μM of the SM inhibitors in the T cell stimulation assay. The gene expression profiles of unstimulated and compound treated stimulated samples are shown. All data were normalized to housekeeping genes and stimulated DMSO control samples. Hierarchical agglomerative clustering of genes with greater than a 2-fold change (p-value<0.05) is shown. (B) Transcript expression of IL-2 under unstimulated, stimulated, and compound treated conditions. The mRNA expression levels of the cytokine genes IFNγ (C), IL4 (D), IL13 (E), IL17F (F) as well as the transcription factors Tbx21 (G), GATA3 (H), RORc (I) and Foxp3 (J) are depicted as examples from the gene expression dataset. The gene expression profiles in (A) is a composite of PBMCs from n = 3 donors, while the individual gene expression profiles in (B-J) are mean±SEM of mRNA expression from n = 3 PBMC donors.</p

Impact of SYK/ZAP-70 inhibitor and prednisolone on gene expression profiles in the T cell stimulation assay.

<p>(A) A nanostring gene expression panel was used to evaluate mRNA profiles of PBMCs 3, 6 and 24 h following treatment with three different concentrations of SYK/ZAP-70 inhibitor and prednisolone. The gene expression profiles of unstimulated and compound treated stimulated samples are shown. All data were normalized to housekeeping genes and stimulated DMSO control samples. Hierarchical agglomerative clustering of genes with greater than a 2-fold change (p-value<0.05) is shown. Transcript expression of IL-2 under unstimulated, stimulated, and stimulated plus SYK/ZAP-70 inhibitor or prednisolone treated conditions at the 3h (B), 6h (C) and 24h (D) time points in the T cell stimulation assay. The gene expression profiles in (A) is a composite of PBMCs from n = 3 donors, while the individual gene expression profiles in (B-D) are mean±SEM of mRNA expression from n = 3 PBMC donors.</p

The effect of small molecule inhibitors on reactive oxygen species (ROS) production by human phagocytes.

<p>Ten minutes following stimulation with opsonized bacteria, the production of ROS in neutrophils and monocytes from human whole blood was evaluated using flow cytometry. Small molecule inhibitors inhibit ROS production neutrophils (A) and monocytes (B) in human whole blood following stimulation with opsonized bacteria. For each compound, the potency (red circles) and the percentage maximal inhibition achieved in the assay (black squares) are plotted along the X-axis. The potencies of the evaluated compounds in these assays are displayed as IC₅₀ values. A maximum of 100% inhibition is possible for small molecule inhibitors in these assays. The reported potency and % max inhibition values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound. The positive control used in this assay (Diphenyliodonium chloride [DPI], an NADPH oxidase inhibitor), can inhibit ROS production from neutrophils (C) or monocytes (D) in a dose dependent manner. A BTK inhibitor consistently inhibited ~50% neutrophil ROS production (E) across multiple donors evaluated in this assay. Each point in the dose response curve indicates mean±SEM of % inhibition at that dose from n = 6–8 donors.</p

Evaluating clinical relevance of the immune function impact of a SYK/ZAP-70 inhibitor.

<p>(A–D) Relationship between the <i>in vitro</i> immune function impact and hypothetical clinical exposure levels of a SYK/ZAP-70 inhibitor. The potency (red circles) of SYK/ZAP-70 inhibitor in the individual assays are overlaid on the hypothetical clinical exposure levels of SYK/ZAP-70 inhibitor (grey box). The potencies of the evaluated compounds in these assays are displayed as an IC₅₀ in the dose response experiments. A maximum of 100% inhibition is possible for small molecule inhibitors in these assays. The reported potency and % max inhibition values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound. The immune function impact of the SYK/ZAP-70 inhibitor on NK cell killing (E), neutrophil ROS production (F), monocyte ROS production (G), response to TLR3 agonist (H), response to TLR7 agonist (I), response to TLR9 agonist (J), T cell IL-2 production (K), CD4⁺ T cell proliferation (L) and CD8⁺ T cell proliferation (M) are depicted as mean±SEM of % inhibition of that response from n = 6–8 donors for each assay. The Neutrophil and monocyte phagocyte responses were evaluated in human whole blood, while all the other responses were evaluated in PBMCs. The potencies depicted in A are corrected for plasma protein binding, using the potencies obtained from dose-response experiments depicted in E-M.</p