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.

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The role of nitric oxide (NO) produced by adherent spleen cells in the systemic immunosuppression developing in tumor-bearing hosts was investigated. After therapeutic immunization of rats carrying an intrahepatic colon carcinoma, H1D2, the spleen cell antitumor immune responsiveness was analyzed. Compared to parallel immunized tumor-free rats, tumor-bearing rats (TB rats) had a greatly reduced proliferative T-cell response to wild-type tumor stimulator cells. The TB rats had a depressed proliferative response to anti-CD3 and to the superantigen SEA. TB rats with small tumors had a stronger response to IL-18-producing H1D2 stimulator cells than to wild type H1D2 cells. This was not the case with TB rats carrying larger tumors. Also the IFN-gamma production and cytotoxicity against the wild-type tumor cells and the NK sensitive YAC cells were depressed in spleen cells of TB rats after 5-day restimulation with wild-type tumor cells. A part of this immunosuppression was mediated by adherent spleen cells, mostly consisting of macrophages. An important mode of action appears to involve their production of an enhanced level of nitric oxide, since the competitive nitric oxide synthase (NOS) inhibitor L-NAME could partially counteract the suppression in vitro. We conclude that NOS inhibitors in combination with immunostimulatory cytokines, such as IL-18, could be useful tools to enhance anti-tumor immune responses in TB rats and therefore to increase the efficiency of immunotherapies.

Interferon gamma (IFN‐γ) has successfully been used in immunotherapy of different experimental tumours. Mechanistically, IFN‐γ has extensive effects on the immune system including release of nitric oxide (NO) by upregulation of the inducible nitric oxide synthase (iNOS). NO has putative immunosuppressive effects but could also play a role in killing of tumour cells. Therefore, the aim of the present study was to clarify whether inhibition of iNOS in rats immunized with glioma cells (N32) producing IFN‐γ (N32‐IFN‐γ), could enhance the anti‐tumour immune response. Initially, both a selective iNOS, l‐N6‐(1‐Iminoethyl)‐l‐lysine (l‐NIL), and non‐selective, N‐nitro‐l‐arginine methyl ester (l‐NAME), inhibitor of NOS were tested in vitro. After polyclonal stimulation with LPS and SEA, both l‐NIL and l‐NAME enhanced proliferation and production of IFN‐γ from activated rat splenocytes and this effect was inversely correlated to the production of NO. However, l‐NIL had a broader window of efficacy and a lower minimal effective dose. When rats were immunized with N32‐IFN‐γ, and administered NOS inhibitors by intraperitoneal (i.p.) mini‐osmotic pumps, only splenocytes of rats treated with l‐NIL, but not l‐NAME, displayed an enhanced proliferation and production of IFN‐γ when re‐stimulated with N32 tumour cells. Based on these findings, l‐NIL was administered concurrently with N32‐IFN‐γ cells to rats with intracerebral (i.c.) tumours resulting in a prolonged survival. These results show that inhibition of iNOS can enhance an IFN‐γ‐based immunotherapy of experimental i.c. tumours implying that NO released after immunization has mainly immunosuppressive net effects.

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