The yin-yang of drug discovery: side effects create opportunities
Psoriasis
Psoriasis is a common skin disease hallmarked by well-demarcated and erythemous plaques and excessive scaling caused by the hyperproliferative state of skin cells. Treatment of the affected skin with ointments or creams is often sufficient in patients who are mildly or moderately affected.
No effect without side effect
When developing a new drug against the malaria parasite, we tested compounds for undesirable side effects against human cells. An unexpected activity of some of these compounds was that they appeared to be able to inhibit cell division of human skin cells, without causing cell death. We realized that this side effect can have a positive effect for psoriasis patients if we applied
these substances to their affected skin. The hyperproliferative cell division of the skin can be prevented, resulting in the disappearance of the scaly plaques. This hypothesis was investigated in this project.
Study design
Compounds were first tested on submerged keratinocytes. Subsequently, the most active compounds were tested in a 3D reconstructed skin model. Finally, the best compound was tested in an in vivo psoriasis model. The chemical structures of the proprietary compounds used in the study cannot be disclosed.
Results
We have tested more than 500 compounds for their effect on proliferating skin cells. We then tested the most promising compounds in a so-called human 3D skin model. This is a reconstructed skin model that is grown in the laboratory. Finally, we tested the most active compound in a psoriasis animal model. Unfortunately, we did not see a significant positive effect of this compound on the proliferative skin.
Conclusion
The initial finding that our compounds have an inhibitory effect on proliferating skin cells has unfortunately not yet led to a new therapy. It may be difficult for the compound to penetrate through the skin to the dividing cells. Another explanation would be that the compounds are not active enough to show an effect in this model. In this case, chemical optimization of the compounds could be beneficial. The research described here could then be used as preliminary work to determine a so-called structure activity relationship (SAR), from which targeted optimization can take place.
Abbreviations
PCK: Population Control Keratinocytes
This dataset contains the following files:
Submerged.pdf
Growth inhibiting effect of compounds on primary human keratinocytes at a concentration of 10 uM. Primary human keratinocytes were grown in triplicate in 96 well plates until they reach a confluency of 30-40%. Compounds are added at a concentration of 10 uM and after 72 hours the confluency was measured using CyQuant Direct Cell Proliferation Assay Kit.
Cytotoxicity was measured using a LDH cytotoxicity assay kit. No cytotoxicity was observed. Growth effect was divided in three groups: no effect (70-100% confluency), small effect (50-70% confluency) or strong effect (30-50% confluency).
Submerged_dilution_range.pdf
Concentration (uM) of compounds that inhibit growth of three different primary human keratinocyte donors. Primary human keratinocytes were grown in triplicate in 96 well plates until they reach a confluency of 30-40%. Compounds are added at in a concentration range of 2-10 uM and after 72 hours the confluency was measured using CyQuant Direct Cell Proliferation Assay Kit. Cytotoxicity was measured using a LDH cytotoxicity assay kit. No cytotoxicity was observed. The concentration at which each compound inhibits cell growth for each donor was put in the table.
HE_2_donors_IL4,_2_compounds_concentration_range.pdf
Effect of a dilution range (30 uM – 300 nM) of two compounds (CXP18.6-017 and CXP18.6-064) on 3D reconstructed skin after stimulation with IL-4 (10 ng/ml). Constructs are stained with H/E.
HE_3_donor_IL4,_12,_17_en_64_100_uM.pdf
Effect of three compounds (CXP18.6-012, CXP18.6-017 and CXP18.6-064) at a concentration of 100 uM on 3D reconstructed skin after stimulation with IL-4 (10 ng/ml). Constructs are stained with H/E.
Ki-67_donors_IL4,_2_compounds-concentration_range.pdf
Effect of a dilution range (30 uM – 300 nM) of two compounds (CXP18.6-017 and CXP18.6-064) on 3D reconstructed skin after stimulation with IL-4 (10 ng/ml). Constructs are stained with Ki-67.
Ki-67_2_donors,_2_compounds,_3_stimuli.pdf
Effect of two compounds (CXP18.6-017 and CXP18.6-064) on 3D reconstructed skin after stimulation with IL-4 (20 ng/ml), IL-17/IL-22 (30/30 ng/ml) and IL-4/IL-13 (20/20 ng/ml). Constructs are stained with Ki-67.
IMQ_model_with_compound.pdf
Imiquimod model to test in vivo efficacy of compound CXP18.6-017 on proliferative skin. The protocol is explained in de file. Hyperproliferation of the back skin of mice was induced by Imiquimod. Treatment with CXP18.6-017 (1% in gel) was initiated two days before the Imiquimod treatment.
