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Joint Inventors From:

UW-Milwaukee, Medical University of Vienna, TU Wien,

University of Belgrade, and National Taiwan University

Novel α6GABAAR Subtype-Selective Ligands for CNS Disorders and

Trigeminal Pain(OTT1410)

For further information please contact:

Jessica Silvaggi, PhD, CLP

Senior Licensing Manager

1440 East North Ave. Milwaukee, WI 53202

Tel: 414-906-4654

Jessica@uwmrf.org

Current Problems with CNS Drugs

• Many GABAergic drugs on the market today offer little selectivity towards various subtypes of GABAA receptors, and thus exhibit undesired side effects

• These include sedation, ataxia, amnesia, tolerance, and addiction

• There has been a lack of new drugs developed for CNS disorders, while the social, clinical, and economic needs are growing

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Our Solution: Novel α6GABAAR ligands

Recently, the α6 subunit-containing GABAA receptor (α6GABAAR) has been implicated in neuropsychiatric disorders with sensorimotor gating deficits, migraine, orofacial pain and depression.

• α6GABAAR-selective positive allosteric modulators (α6GABAAR PAMs)

– First-in-Class: First ligands highly selective to α6GABAARs

– Functionally Selective: silent at α1- and α5-GABAARs

• Devoid of sedative, motor-impairing, amnesia side effect

• Should be no tolerance and non-addictive

– Effective: Promising results in animal models for several indications

– Pharmacokinetic profiles: Lead compounds show

• Good metabolic stability (in vitro)

• Excellent bioavailability (oral)

• Appropriate brain concentrations (oral)

– Safe: Lead compounds show a lack of liver or kidney toxicity

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• PCT/US2016/035761 nationalized Fall/Winter 2017

• U.S. Utility Patent Pending

• EP Patent Pending

Looking for a development partner to:

• Provide support for follow up animal testing in lead compounds

• Aid in moving towards the regulatory approval process and IND filing

• License the technology for development of new drugs

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Intellectual Property and Licensing

Market Potential

Potential Applications

• Neuropsychiatric disorders with sensorimotor gating deficits

– Schizophrenia

– Tic disorders (Tourette’s syndrome), Obsessive compulsive disorder, Attention deficit disorders

– Panic disorder, Nocturnal enuresis, Antisocial personality disorder, Mania, Premenstrual dysphoric disorder, Huntington’s disease

• Depression

• Trigeminal pain: Migraine, Trigeminal orofacial pain

Market

• 45% of people will be affected by some type of neuropsychiatric disorder

• Global migraine sales are expected to grow to $8.7 billion by 2026, however current treatments leave a large number of patients undertreated

• The global temporomandibular joint (TMJ) disorders market is expected to reach $1.2B by 2023, while the CNS therapeutic market is expected to reach $128.9 billion by 2025

• Response rate to off-label use of drugs in post-traumatic trigeminal neuropathy is around 11%; the need is urgent for more efficient and tolerable drugs

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GABAA Receptors: Established Drug Targets

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• Chloride ion channel

• Well characterized CNS activity

• Positive modulators work at allosteric sites and increase channel efficacy

• Receptor is readily druggable; approved small molecule drugs in wide-spread clinical use

• α6GABAAR have been found to be located in the cerebellum and trigeminal ganglia

• Compounds targeting α6GABAARs can rescue a disrupted pre-pulse inhibition (PPI), which is a common endophenotype manifestation present in many neuropsychiatric disorders

The GABAA Receptor (GABAAR) α6β3γ2

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• The GABAAR α6β3γ2 subtype is a pentameric ligand-gated chloride channel comprised of two α6, two β3 and one γ2 subunit

• Pyrazoloquinolinones mediate their effects at the α6+β3- interface (PQ Site) as positive allosteric modulators and act at the diazepam insensitive benzodiazepine (DI-Bz) site (α6+γ2-interface) as null modulators

• The PQ site, the DI binding site and the endogenous ligand (GABA) binding sites at the β3+α6- interfaces are displayed

Image modified from Jacob et al., Nature Reviews Neuroscience, 2008.

GABAAR α6β3γ2 subtype selectivity

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Deuterated and N-Hetero analogs retained activity at the α6GABAAR

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Analogs retain activity at the α6GABAAR

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Ligand

Metabolic Stability Pharmacokinetics

t1/2

HLM (h)

t1/2

MLM (h)

CMAX

Plasma (ng / mL)

t1/2

Plasma (h)

CMAX

Brain (ng / g)

t1/2

Brain (h)

PZ-II-029 3.6 ± 0.6 3.2 ± 0.1 5050 ± 770 1.51 ± 0.04 352 ± 17 1.96 ± 0.37

DK-I-56-1 8.7 ± 0.6 10.5 ± 0.9 4430 ± 320 3.54 ± 0.71 589 ± 93 2.28 ± 0.32

RV-I-029 11.1 ± 3.6 14.3 ± 2.8 3740 ± 390 2.46 ± 0.15 657 ± 71 2.60 ± 0.27

DK-I-60-3 13.0 ± 2.9 14.1 ± 3.3 3410 ± 250 2.64 ± 0.54 534 ± 14 3.52 ± 0.71

DK-I-86-1 12.5 ± 3.1 9.2 ± 0.9 1250 ± 80 17.99 ± 15.48 215 ± 23 9.07 ± 5.45

Improvement of Metabolic Stability

• OCD3 and N-hetero Substitution Improved Metabolic Stability and Pharmacokinetic Profile• Deuteration of A or D-ring methoxy group increased the metabolic stability• N-Hetero introduction into D-ring also increased the metabolic stability• Only deuterated compounds exhibited enhanced bioavailability (Cmax, Brain)

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Pre-pulse inhibition of the Startle response (PPI)

Geyer et al., Mol Psychiatry. (2002) 7:1039

PPI: A weaker pre-stimulus (pre-pulse) inhibits the reaction (startle response) of an organism to a subsequent strong startling stimulus (pulse), a measurement of sensorimotor gating function

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• Mouse model mimicking schizophrenia based on the hyper-dopaminergic hypothesis

• Methamphetamine (METH), a hyperdopaminergic agent, significantly disrupted PPI induced by two protocols (71-115 dB and 77-115 dB) in mice

• All α6GABAAR PAMs (10 mg/kg) effectively rescued the METH disruption of PPI

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• Two α6GABAAR PAMs (C6 and C11) significantly rescued METH-induced PPI disruptions

• Furosemide, an α6GABAAR-selective antagonist, blocked their effects, confirming α6GABAAR as the action target

The furosemide-sensitive effects of α6GABAAR PAMs

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Reduction of Trigeminal Neuropathic Pain

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(A) Injections of 10mg/kg DK-I-56-1 or placebo (days 1 to 14) to rats subjected to ligature of the infraorbital nerve or sham surgery, with(B) respective scores of reactivity to von Frey filaments(C) Injections of 10mg/kg DK-I-56-1, 10mg/kg DK-I-87-1 or placebo (days 15 to 28) to rats with the already existing trigeminal neuropathy, with(D) respective scores of reactivity to von Frey filaments

Two-week repeated treatment with DK-I-56-1 in a rat model of trigeminal neuropathycan provide both, a preventive and a therapeutic effect on neuropathic pain

• Capsaicin-induced migraine model; topiramate (TPM) is a clinically effective anti-migraine agent, and was used as a positive control

• Compound 6 significantly decreased the number of c-Fos-ir neurons in the TCC and CGRP-ir in TG induced by capsaicin

• Compound 6 reversed capsaicin-induced depletion of dural CGRP-ir

• Suggests potential for migraine treatment

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Trigemino-cervical complex (TCC) Trigeminal ganglia (TG) Dura mater

Cpd 6 Performs Similar to Clinical Migraine Agent

Toxicity and Drug Potential Assays

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• GABAAR binding >1000 fold higher than all “off target” receptors (46) tested

• Ligands are non-toxic to kidney and liver cells even at 400 µM

• Studies of locomotion on the rotarod confirmed that ligands are not sedating or ataxic in models Rotarod Device

Ligand BZP Rat Brain Site Binding Affinity (nM)

Radioligand % Inhibition Cytotoxicity

BZP Rat Brain Site

hERG Channel

HEK293 (Kidney) LD50 (µM)

HEPG2 (Liver) LD50 (µM)

PZ-II-029 2.7 92.4 7.4 NA NA

DK-I-56-1 2.1 96.2 0.1 >400 >400

RV-I-029 2.0 96.7 6.9 >400 >200

DK-I-60-3 2.3 94.5 8.7 >400 >400

DK-I-86-1 21.7 98.2 8.6 >400 >400

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Next Steps

• ~$700,000 in funding needed to conduct further experiments on animal models mimicking Tourette syndrome, trigeminal pain, depression, and tinnitus.

• In need of collaborations to conduct drug development in preclinical studies in the safety pharmacology, toxicology assessments, and pharmacokinetic studies

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Acknowledgements and Collaborations

Daniel Knutson & Revathi KodaliNicholas Zahn & Alec Huber

Michael Rajesh StephenRanjit Verma, Christopher Witzigmann & Matheus Meirelles

Alexander Arnold & James Cook

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Brian Roth

Pi-Chuan Fan

Lih-Chu ChiouMarco Treven & Zdravko Varagic

Margot Ernst & Werner SieghartBranka Divovic & Vladimir Dobricic

Miroslav Savic

Acknowledgements and Collaborations

Laurin Wimmer & David Siebert

Marko D. Mihovilovic

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