Discovery of an Oral, Rule of 5 Compliant, Interleukin 17A Protein-Protein Interaction Modulator for the Potential Treatment of Psoriasis and Other Inflammatory Diseases.

Interleukin 17A (IL-17A) is an interleukin cytokine whose dysregulation is implicated in autoimmune disorders such as psoriasis, and monoclonal antibodies against the IL-17A pathway are now well-established and very effective treatments. This article outlines the work that led to the identification of 23 as an oral, small-molecule protein-protein interaction modulator (PPIm) clinical development candidate. Protein crystallography provided knowledge of the key binding interactions between small-molecule ligands and the IL-17A dimer, and this helped in the multiparameter optimization toward identifying an orally bioavailable, Rule of 5 compliant PPIm of IL-17A. Overlap of early ligands led to a series of benzhydrylglycine-containing compounds that allowed the identification of dimethylpyrazole as a key substituent that gave PPIm with oral bioavailability. Exploration of the amino acid portion of the structure then led to dicyclopropylalanine as a group that gave potent and metabolically stable compounds, including the development candidate 23.

Characterizing Cutaneous Drug Delivery Using Open-Flow Microperfusion and Mass Spectrometry Imaging.

Traditionally, cutaneous drug delivery is studied by skin accumulation or skin permeation, while alternative techniques may enable the interactions between the drug and the skin to be studied in more detail. Time-resolved skin profiling for pharmacokinetic monitoring of two Janus Kinase (JAK) inhibitors, tofacitinib and LEO 37319A, was performed using dermal open-flow microperfusion (dOFM) for sampling of perfusate in an ex vivo and in vivo setup in pig skin. Additionally, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) was performed to investigate depth-resolved skin distributions at defined time points ex vivo in human skin. By dOFM, higher skin concentrations were observed for tofacitinib compared to LEO 37319A, which was supported by the lower molecular weight, higher solubility, lipophilicity, and degree of protein binding. Using MALDI-MSI, the two compounds were observed to show different skin distributions, which was interpreted to be caused by the difference in the ability of the two molecules to interact with the skin compartments. In conclusion, the techniques assessed time- and depth-resolved skin concentrations and were able to show differences in the pharmacokinetic profiles of two JAK inhibitors. Thus, evidence shows that the two techniques can be used as complementary methods to support decision making in drug development.

Macrolide Inspired Macrocycles as Modulators of the IL-17A/IL-17RA Interaction.

Interleukin 17 (IL-17) cytokines promote inflammatory pathophysiology in many autoimmune diseases, including psoriasis, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Such broad involvement of IL-17 in various autoimmune diseases makes it an ideal target for drug discovery. Psoriasis is a chronic inflammatory disease characterized by numerous defective components of the immune system. Significantly higher levels of IL-17A have been noticed in lesions of psoriatic patients, if compared to non-lesion parts. Therefore, this paper is focused on the macrolide inspired macrocycles as potential IL-17A/IL-17RA modulators and covers the molecular design, synthesis, and in vitro profiling. Macrocycles are designed to diversify and enrich chemical space through different ring sizes and a variety of three-dimensional shapes. Inhibitors in the nM range were identified in both target-based and phenotypic assays. In vitro ADME as well as in vivo PK properties are reported.

Evaluating Dermal Pharmacokinetics and Pharmacodymanic Effect of Soft Topical PDE4 Inhibitors: Open Flow Microperfusion and Skin Biopsies.

PURPOSE: To investigate the difference in clinical efficacy in AD patients between two topical PDE4 inhibitors using dermal open flow microperfusion and cAMP as a pharmacodynamic read-out in fresh human skin explants. METHODS: Clinical formulations were applied to intact or barrier disrupted human skin explants and both skin biopsy samples and dermal interstitial fluid was sampled for measuring drug concentration. Furthermore, cAMP levels were determined in the skin biopsies as a measure of target engagement. RESULTS: Elevated cAMP levels were observed with LEO 29102 while no evidence of target engagement was obtained with LEO 39652. In barrier impaired skin the dISF concentration of LEO 29102 was 2100 nM while only 33 nM for LEO 39652. For both compounds the concentrations measured in skin punch biopsies were 7-33-fold higher than the dISF concentrations. CONCLUSIONS: Low unbound drug concentration in dISF in combination with minimal target engagement of LEO 39652 in barrier impaired human skin explants supports that lack of clinical efficacy of LEO 39652 in AD patients is likely due to insufficient drug availability at the target. We conclude that dOFM together with a pharmacodynamic target engagement biomarker are strong techniques for establishing skin PK/PD relations and that skin biopsies should be used with caution.

Discovery and Early Clinical Development of Isobutyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (LEO 39652), a Novel "Dual-Soft" PDE4 Inhibitor for Topical Treatment of Atopic Dermatitis.

We describe the design of a novel PDE4 scaffold and the exploration of the dual-soft concept to reduce systemic side effects via rapid elimination: introducing ester functionalities that can be inactivated in blood as well as by the liver (dual-soft) while being stable in human skin. Compound 40 was selected as a clinical candidate as it was potent and rapidly degraded by blood and liver to inactive metabolites and because in preclinical studies it showed high exposure at the target organ: the skin. Preclinical and clinical data are presented confirming the value of the dual-soft concept in reducing systemic exposure.

Identification of natural products using HPLC-SPE combined with CapNMR.

Two major development areas in HPLC-NMR hyphenation are postcolumn solid-phase extraction (HPLC-SPE-NMR) and capillary separations with NMR detection by means of solenoidal microcoils (CapNMR). These two techniques were combined off-line into HPLC-SPE-CapNMR, which combines the advantage of high loadability of normal-bore HPLC columns with high mass sensitivity of capillary NMR probes with an active volume of 1.5 microL. The technique was used for rapid identification of complex sesquiterpene lactones and esterified phenylpropanoids present in an essentially crude plant extract (toluene fraction of an ethanolic extract of Thapsia garganica fruits). Elution profiles of 10 x 1 mm i.d. SPE cartridges filled with poly(divinylbenzene) resin were found to be only marginally broader than those observed upon direct injection of 6-microL samples into the probe. Thus, the technique focuses analytes emerging in the HPLC elution bands of 0.5-1 mL into volumes of approximately 10 microL, compatible with the CapNMR probe. Using this technique, nine natural products (1-9) present in the plant extract in amounts varying from 0.1 to 20% were identified by means of 1D and 2D NMR spectra, supported by parallel HPLC-ESIMS measurements. Therefore, HPLC-SPE-CapNMR should be regarded as an attractive alternative to other applications of CapNMR for mixture analysis.

Stereoelectronic effects on 1H nuclear magnetic resonance chemical shifts in methoxybenzenes.

Investigation of all O-methyl ethers of 1,2,3-benzenetriol and 4-methyl-1,2,3-benzenetriol (3-16) by 1H NMR spectroscopy and density-functional calculations disclosed practically useful conformational effects on 1H NMR chemical shifts in the aromatic ring. While the conversion of phenol (2) to anisole (1) causes only small positive changes of 1H NMR chemical shifts (Delta delta < 0.08 ppm) that decrease in the order Hortho > Hmeta > Hpara, the experimental O-methylation induced shifts in ortho-disubstituted phenols are largest for Hpara, Delta delta equals; 0.19 +/- 0.02 ppm (n = 11). The differences are due to different conformational behavior of the OH and OCH3 groups; while the ortho-disubstituted OH group remains planar in polyphenols due to hydrogen bonding and conjugative stabilization, the steric congestion in ortho-disubstituted anisoles outweighs the conjugative effects and forces the Ar-OCH3 torsion out of the ring plane, resulting in large stereoelectronic effects on the chemical shift of Hpara. Conformational searches and geometry optimizations for 3-16 at the B3LYP/6-31G** level, followed by B3LYP/6-311++G(2d,2p) calculations for all low-energy conformers, gave excellent correlation between computed and observed 1H NMR chemical shifts, including agreement between computed and observed chemical shift changes caused by O-methylation. The observed regularities can aid structure elucidation of partly O-methylated polyphenols, including many natural products and drugs, and are useful in connection with chemical shift predictions by desktop computer programs.

5alpha-cardenolides from Kanahia laniflora inhibit ionotropic acetylcholine receptors.

5alpha-cardenolides isolated from Kanahia laniflora are inhibitors of muscle-type nicotinic acetylcholine receptors expressed in TE671 cells with IC (50) values in the range of 27 - 60 microM, as determined by whole-cell patch-clamp electrophysiological experiments.

Rapid extract dereplication using HPLC-SPE-NMR: analysis of isoflavonoids from Smirnowia iranica.

A novel hyphenated technique, HPLC-SPE-NMR, was used for accelerated identification of isoflavonoids from the roots of Smirnowia iranica. The extract constituents eluted from a HPLC column were automatically trapped on solid-phase extraction (SPE) cartridges, and NMR spectra were acquired with concentrated solutions after solvent change. The structures of 10 new isoflavonoids (1, 4, 5, 7-10, 12, 13, 16) and of seven previously described constituents (2, 3, 6, 11, 14, 15, 17) were elucidated from NMR spectra acquired in the HPLC-SPE-NMR mode. Multiple peak trapping on the same SPE cartridge increased analyte amounts and provided access to 2D NMR data. It was demonstrated that linear accumulation of material is possible in up to seven repeated trapping steps. The use of HPLC-SPE-NMR speeded up dereplication of the S. iranica extract considerably by providing detailed information about the constituents of a complex, essentially crude extract prior to their preparative-scale isolation or extract pre-fractionation, and the information obtained could be used to direct preparative isolation work. In connection with structure elucidation of isoflavonoids containing O-methylated 1,2,3-benzenetriol moieties as the B-ring, O-methylation-induced changes of chemical shifts of aromatic hydrogens were found to depend on the conformation of the resulting methoxy group, i.e., on the number of its ortho substituents. The recognized regularities will be useful in structure determination of partially O-methylated polyphenols based on 1D (1)H NMR spectra obtainable from HPLC-SPE-NMR experiments, diminishing dependence on 2D NMR data and (13)C NMR chemical shifts.

HPLC-SPE-NMR hyphenation in natural products research: optimization of analysis of Croton membranaceus extract.

The HPLC-SPE-NMR technique was used for the analysis of a root-bark extract of Croton membranaceus. The components of the extract were separated on an analytical-size reversed-phase HPLC column, the chromatographic peaks were trapped on SPE (solid-phase extraction) cartridges after post-column dilution of the eluate with water and the compounds were eluted from the cartridges with acetonitrile-d(3) into a 30 microl 600 MHz NMR probe in a fully automated procedure. The trapping efficiency of scopoletin (1), the major extract constituent, was much higher on a GP (general phase, a polystyrene-type polymer) SPE phase than on a C18 phase. Thus, under the conditions used, up to 100 microg of scopoletin per cartridge could be accumulated linearly after repeated trappings. The maximum achievable NMR signal-to-noise ratio using the GP cartridges was at least four times higher than that achievable with the C18 cartridges. It was shown that excessively long T(1) relaxation times may compromise experiments in which acetonitrile-d(3) is used as the cartridge eluent. Nevertheless, the sensitivity gain provided by the HPLC-SPE-NMR technique through repeated peak trappings allowed the acquisition of good-quality proton-detected 2D NMR spectra without the need for solvent suppression.

Labdanes and isopimaranes from Platycladus orientalis and their effects on erythrocyte membrane and on Plasmodium falciparum growth in the erythrocyte host cells.

Six labdanes (1-6) and four isopimaranes (7-10), including three new natural products (7, 9, and 10), were isolated from Platycladus orientalis, and their structures determined using 1D and 2D NMR methods, ion-cyclotron resonance HRMS, and optical rotation data. Relative configurations of all chiral centers in the isopimaranes were determined using NOESY experiments at 600 and 800 MHz. Specific optical rotation data were used to correlate absolute configurations. Compounds 1-9 and aframodial (11) were tested for their in vitro antiplasmodial activity and for their ability to induce changes of erythrocyte shape in order to obtain data about possible correlation between the two effects. All compounds tested exhibited weak (IC(50) > 25 microM) in vitro antiplasmodial effects against Plasmodium falciparum strain 3D7. At the same time, the compounds caused echinocytic or stomatocytic changes of the erythrocyte membrane curvature, indicative of their incorporation into the lipid bilayer, in the concentration region where the antiplasmodial activity was observed. The antiplasmodial effect of these compounds thus appears to be an indirect effect on the erythrocyte host cell. Weak or moderate antiplasmodial activity observed with many other apolar natural products, in particular those with amphiphilic structures, is also likely to be an indirect effect.