IPR-179, a inhibitor, is able to mitigate epileptogenesis in the rat

A Bertran1,*, D W M Broekaart2,*, E Aronica2,3, T Tarragó1, E A van Vliet2, R Prades1 1 Iproteos, S.L., Barcelona, Spain; 2 Academic Medical Center, Department of (Nuero)Pathology, University of Amsterdam, The Netherlands; 3Stichting Epilepsie Instellingen Nederland (SEIN), The Netherlands; * Contributed equally INTRODUCTION Matrix (MMPs) have been highly desired therapeutic targets due to their involvement in a number of physiological process and pathological conditions. In this regard, elevated expression of MMPs, in particular MMP2 and MMP9 has been observed in animals models of epilepsy, where the expression of these two MMPs play a role in the development of epilepsy (epileptogenesis) through the control of neuronal death, synaptic plasticity and neuroinflammation [1,2]. The discovery of a selective inhibitor for gelatinase would be of high interest for treatment of epilepsy. The lack of selective MMP inhibitors has hampered the development of pharmacological therapies, as unspecific MMPs inhibitors are well known to have severe side effects in humans. IPROTech: engine for searching novel drugs Potency, selectivity and BBB permeability

IC50 (nM) MMP9 MMP2 MMP1 MMP3 MMP7 ADAM10 ADAM17 IPR-179 334 589 >10000 >10000 >10000 >50000 >15

In vitro permeability PAMPA (transport) BBB 12,70% GIT 9% (pH 7.4) 9.7% (pH6.3) 10% (pH5)

Brain exposure (mice)

AUChippocampus/plasma 0.28 IPR-179 AUC 0.35 (Small molecule with anticipated capacity to cross the BBB) cortex/plasma Proof of Concept: rapid kindling model in rats

Experiment outline:

• Electrical stimulation via electrodes implanted intracranially in the angular • Minocycline, a BBB permeable broad spectrum MMP inhibitor, was bundle (50 Hz, 12/day for 3 days) use as reference control (ip, 45 mg/kg/day) • Stimulation started 1 h after IPR-179 administration (ip, 6 mg/kg/day) • Seizure scoring using Racine scale • Re-stimulation after one week washout (IPR-179 not present any more in • MMP9 proteolytic activity assessment in brain tissue at the end of plasma) period to analyze the anti-epileptogenic effect of IPR-179 the experiment (Nectin-3 cleavage monitoring by western blot)

Ratio nectin-3 full /SPF

Vehicle Minocycline IPR-179 scale scale Relative expression Racine (normalized to vehicle) Racine Vehicle IPR-179 Minocycline ↑ Western blot analysis. Relative expression of Nectine-3 ratio of full protein over SPF (small proteolytic fragment). **P<0.01 Mann-Whitney test. kindling Re-test ← Racine score after electrical stimulation. Vehicle n= 8, IPR-179 n=8, Minocycline = 10. Mixed-effects ordinal regression *P<0.05. Stimulation number CONCLUSIONS IPR-179 is a small molecule generated with the technological platform IPROTech. This compound shows selectivity for , in particular for MMP9, a highly desired target for the treatment of epilepsy. IPR-179 also shows relevant BBB permeability. In vivo data in the rapid kindling model in rats indicates that IPR-179 has anti-ictogenic and anti-epileptogenic effects.This finding matches with a decrease in MMP9 activity induced by IPR-179, as demonstrated in the western blot analysis of brain tissue from IPR-179 treated animals.

References: [1] Wilczynski et al. J. Cell biol. 2008, 180(5): 1021-1035; [2] Yin et al. Med. Hypotheses. 2011, 76(2):184-186

Disclosure: A Bertran, T Tarragó, and R Prades are employees of Iproteos SL. T Tarragó is co-founder of Iproteos SL. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642881 and the Dutch Epilepsy Foundation, project number 642881.