Discovery and early clinical development of 2-{6-[2-(3,5-dichloro-4-pyridyl)acetyl]-2,3-dimethoxyphenoxy}-N-propylacetamide (LEO 29102), a soft-drug inhibitor of phosphodiesterase 4 for topical treatment of atopic dermatitis.

Development of orally available phosphodiesterase 4 (PDE4) inhibitors as anti-inflammatory drugs has been going on for decades. However, only roflumilast has received FDA approval. One key challenge has been the low therapeutic window observed in the clinic for PDE4 inhibitors, primarily due to PDE4 mediated side effects. Here we describe our approach to circumvent this issue by applying a soft-drug concept in the design of a topically acting PDE4 inhibitor for treatment of dermatological diseases. We used a fast follower approach, starting from piclamilast. In particular, simultaneous introduction of 2'-alkoxy substituents and changing an amide to a keto linker proved to be beneficial when designing potential soft-drug candidates. This effort culminated in identification of LEO 29102 (20), a potent, selective, and soft-drug PDE4 inhibitor with properties suitable for patient-friendly formulations giving efficient drug delivery to the skin. Compound 20 has reached phase 2 and demonstrated clinically relevant efficacy in the treatment of atopic dermatitis.

Calcipotriol delivery into the skin with PEGylated liposomes.

The D-vitamin analogue calcipotriol is commonly used for topical treatment of psoriasis, but skin penetration is required for calcipotriol to reach its pharmacological target: the keratinocytes in the lower epidermis. Liposomes can enhance the delivery of drugs into the skin, but a major challenge for the development of dosage forms containing liposomes is to maintain the colloidal stability in the formulation. The purpose of this study was to investigate the effect of stabilising liposomes with the lipopolymer poly(ethylene glycol)-distearoylphosphoethanolamine (PEG-DSPE) on the physicochemical properties of the liposomes and the ability to deliver membrane-intercalated calcipotriol into the skin. Inclusion of 0.5, l and 5 mol% PEG-DSPE in the membrane enhanced the colloidal stability of the liposomes without compromising the delivery of calcipotriol from the vehicle into excised pig skin. Calcipotriol-loaded liposomes with 1 mol% PEG-DSPE did even provide for a significantly increased deposition of calcipotriol into the stratum corneum. The size of the liposomes affected the penetration of calcipotriol into the stratum corneum since small unilamellar vesicles enhanced calcipotriol penetration as compared to large multilamellar vesicles, indicating that the liposomes to some extent migrate as intact vesicles into the stratum corneum. However, calcipotriol penetrated the skin better than the lipid component of the liposomes, suggesting that at least a fraction of the drug is released from the liposomes during skin migration. In conclusion, PEGylation is therefore a promising approach for stabilising calcipotriol-containing liposomal dispersions without compromising their favourable skin accumulation properties.

Different secondary structure elements as scaffolds for protein folding transition states of two homologous four-helix bundles.

Comparison of the folding processes for homologue proteins can provide valuable information about details in the interactions leading to the formation of the folding transition state. Here the folding kinetics of 18 variants of yACBP and 3 variants of bACBP have been studied by Phi-value analysis. In combination with Phi-values from previous work, detailed insight into the transition states for folding of both yACBP and bACBP has been obtained. Of the 16 sequence positions that have been studied in both yACBP and bACBP, 5 (V12, I/L27, Y73, V77, and L80) have high Phi-values and appear to be important for the transition state formation in both homologues. Y31, A34, and A69 have high Phi-values only in yACBP, while F5, A9, and I74 have high Phi-values only in bACBP. Thus, additional interactions between helices A2 and A4 appear to be important for the transition state of yACBP, whereas additional interactions between helices A1 and A4 appear to be important for the transition state of bACBP. To examine whether these differences could be assigned to different packing of the residues in the native state, a solution structure of yACBP was determined by NMR. Small changes in the packing of the hydrophobic side-chains, which strengthen the interactions between helices A2 and A4, are observed in yACBP relative to bACBP. It is suggested that different structure elements serve as scaffolds for the folding of the 2 ACBP homologues.

Backbone dynamics of the first, second, and third immunoglobulin modules of the neural cell adhesion molecule (NCAM).

The neural cell adhesion molecule (NCAM) is a cell surface multimodular protein, which plays an important role in cell-cell adhesion by homophilic (NCAM-NCAM) and heterophilic (NCAM-non-NCAM molecules) binding. In the present study, the backbone dynamics of the first three immunoglobulin-like (Ig) modules of NCAM have been investigated by NMR spectroscopy. Ig1, Ig2, and Ig3 share low sequence identity but possess the same fold and have very similar three-dimensional structures. (15)N longitudinal and transverse relaxation rates and heteronuclear NOEs have been measured and subsequently analyzed by the axial symmetric Lipari-Szabo modelfree formalism to characterize fast (pico- to nanosecond) and slow (micro- to millisecond) motions in the three protein modules. We found that backbone motions of residues located in the beta-strand regions are generally restricted, while increased flexibility is observed in turns and loops. In all three modules, residues located in the segments connecting the C- and D-strand plus residues located in the segment connecting the E- and F-strand show significant chemical exchange on the micro- to millisecond time scale. In addition, a number of residues with small chemical exchange contribution seem to form contiguous regions in the beta sheets, suggesting that these motions might be correlated. Only few residues in the homophilic binding sites in the NCAM Ig1 and Ig2 modules show increased flexibility, indicating that the Ig1-Ig2-mediated NCAM homophilic binding does not depend on the local backbone mobility of the interacting modules.