ACCELERATING THE DEVELOPMENT OF PRECISION PAIN MEDICINE IMMPACT

ACCELERATING THE DEVELOPMENT OF PRECISION PAIN MEDICINE, IMMPACT XIX Friday, June 3 rd, 2016 Rare vs. common gene variants as guides to pain mechanisms and drug development Alban Latremoliere, Ph. D. Kirby Neurobiology Center Boston Children’s Hospital Harvard Medical School, Boston, MA

New treatments have mostly failed Most attempts to develop new drugs to treat neuropathic pain have failed -- mostly due to lack of efficacy Gabapentin/pregabalin Antidepressants Opioids Local anesthetics Need novel targets

New treatments have mostly failed How to increase translational success? 2 Target pathway 1 1 - Select a pathway from patients 2 - Confirm its validity in rodent models 3 - Determine new drug target 3 4 4 - Go back to patients

Human genetics to identify better targets Human genetics studies: Congenital insensitivity to pain Trk. A loss-of-function Na(v)1. 7 loss-of-function patient Ideal Potent analgesics outcome: Nociceptive pain Effect size Population frequency

Human genetics to identify better targets Human genetics studies: Effect size Population frequency Congenital insensitivity to pain Trk. A loss-of-function Na(v)1. 7 loss-of-function Chronic pain modulation GCH 1, COMT, SERT, MOR Chronic pain patient Ideal outcome: polymorphisms Disease-specific / mechanism-based Target maladaptive pain only

What is GCH 1 BH 4? What is GCH 1? De novo pathway Recycling pathway Salvage pathway 6 -pyruvoyltetrahydropterin GTP GCH 1 AR/CR 7, 8 -dihydroneopterintriphosphate 4α-hydroxytetrahydrobiopterin 6 -lactoyl tetrahydropterin PCBD 6 -lactoyl-7, 8 Dihydropterin PTPS 6 -pyruvoyltetrahydropterin SPR Quinonoiddihydrobiopterin SPR/CR 7, 8 -dihydrobiopterin QDPR DHFR BH 4 4α-hydroxy-tetrahydrobiopterin (tetrahydrobiopterin) GCH 1: GTP cyclohydrolase I AR: Aldose reductase n. NOS i. NOS PTPS: Pyruvoyl tetrahydropterin synthase e. NOS Tyr. OH CR: Carbonyl reductase Neuronal NOS (NOS 1) SPR: sepiapterin reductase Inducible NOS (NOS 2) Endothelial NOS (NOS 3) Tyrosine hydroxylase PCBD: Pterin-carbinolamine dehydratase Trp. OH Phe. OH QDPR: quinoid dihydropteridine reductase Tryptophan hydroxylase DHFR: Dihydrofolate reductase Phenlyalanine hydroxylase

GCH 1 and BH 4 are relevant clinically Genetic association studies identified a ‘protective haplotype’ for neuropathic pain within GCH 1 locus Chromosome 14: Haplotype: Frequency: GCH 1 locus O/O ≈ 80% O/X ≈ 18% X/X ≈ 2% BH 4 levels: high low Pain score: Very Basal painful Nerve injury Low pain

Where is the BH 4 pathway engaged? GCH 1 Promoter DRG: e. GFP Sciatic nerve: Naive Sciatic nerve naive Sciatic nerve SNI 3 d Axons Non-neuronal cells DAB anti-GFP Macrophages SNI 7 d DAB anti-GFP CD 68/GFP

Where is GCH 1 expressed GCH 1 Activity Sciatic nerve GCH 1 Activity DRG BH 4 levels: * 20 15 10 5 0 20 (m. U/mg prot) 25 N SNI * 15 10 5 0 N SNI Biopterin concentration pg/mg tissue Sciatic nerve DRG * 250 * 200 150 100 50 0 N SNI Identification of 2 target tissues and 2 cell types involved

GCH 1 loss of function lox. P 1 2 4 5 6 3 Sensory neuron GCH 1 KO: Spared nerve injury model of neuropathic pain (SNI) GCH 1 Activity: Mechanical allodynia: 60 40 20 * 0 DRG neurons (culture) 1. 00 * * 0. 50 0. 01 * 0 5 10 15 * 20 von Frey threshold (g) 80 von Frey threshold (g) (m. U/mg prot) DRG Inducible KO Constitutive KO 100 1. 00 0. 50 0. 05 Days after SNI * * * Tamoxifen 0 5 10 15 20 25 Days after SNI Sensory neuron-specific BH 4 blockade reduces neuropathic pain *: compared to SNI WT GCH 1 KO mice provided by Pr. Keith Channon WT GCH 1 KO

GCH 1 loss of function But nociceptive pain remains unaffected Time spent licking (s) Time to withdraw (s) 30 25 20 15 10 5 0 49 *: compared to SNI WT 52 Temperature (C) 55 von Frey responses (withdrawals out of 10) 40 35 30 20 10 0 1 µg in 20 µl Calibrated forceps: Von Frey: Capsaicin: 5 Pressure to withdraw (g) Contact heat: 4 3 2 1 0 0 0. 5 GCH 1 KO mice provided by Pr. Keith Channon 1 1. 5 2 Pressure (g) 250 200 150 100 50 0 WT GCH 1 KO

Pharmacological inhibition of BH 4 production – select a target & drug design BH 4 De novo pathway GTP Salvage pathway Structure-based approach 6 -pyruvoyl. Tetrahydropterin GCH 1 7, 8 -dihydroneopterintriphosphate AR/CR 1’-oxo-2’-OH/1’-OH-2’-oxo propyltetrahydropterin PTPS 6 -pyruvoyl. Tetrahydropterin SPR BH 4 6 -lactoyl-7, 8 dihydropteridin CR GCH 1 Active pocket SPR 7, 8 dihydrobiopterin Rate-limiting enzyme DHFR Directly affects BH 4 production SPRi 3 Compound designed by Pr. Julian Blagg

Pharmacological inhibition of BH 4 production – in vitro SPRi 3 Cell-free assay: Cell-based assays: IC 50 NAS: 1. 9 µM IC 50 SPRi 3: 74 n. M 0. 3 0. 2 0. 1 0 10 -9 10 -6 [Compound] (M) *: compared to BSL 10 -3 100 Biopterin synthesis levels (%) FRET RATIO 0. 4 50 0 IC 50 NAS: 54 µM IC 50 SPRi 3: 5. 2 µM 10 -8 10 -6 DRG neurons Sepiapterin reductase Activity (U/mg prot) SK-N-BE(2) neuroblastoma cells 10 -4 [Compound] (M) 150 120 90 60 0 * * 0. 1 0. 3 1 3 [SPRi 3] (μM) 10 * 30 0

Pharmacological inhibition of BH 4 production – in vivo *: compared to SNI veh Mechanical allodynia: von Frey threshold (g) Dose (mg/kg): 1. 00 * 0. 50 * 100 200 * 300 SPRi 3 -1 0 4 BH 4 levels: DRG 300 SN 0 500 0 Dose (mg/kg) 300 150 * [biopterin] pmol/mg protein 1000 300 0 Dose (mg/kg) 3 0 25 300 0 * 0 4 SN 50 [SPRi 3] pmol/mg protein * 0 [SPRi 3] pmol/mg protein DRG 2 [biopterin] pmol/mg protein SPRi 3 levels in target tissues 8 1 Time after injection (h) SN BS L I 6 d 0. 10

SPR as a new target for neuropathic pain Clinically relevant pathway: Mouse genetics: Validate pathway GCH 1 BH 4 SPR Novel drug target PAIN↘ Possible new treatment? SPRi 3 Tool-compound

Current treatment of pain Current Medicine Gabapentin/pregabalin Antidepressants High variability Side-effects Opioids Local anesthetics Poor tolerability

Precision Medicine ”Patient-powered research (…) to accelerate biomedical discoveries and provide clinicians with new tools, knowledge, and therapies to select which treatments will work best for which patients. ” The White House, Office of the Press Secretary, 2015

Precision Medicine – how to use BH 4 Patients suffering from chronic pain Diagnostic: - Questionnaire - QST - biomarkers Activation of BH 4 pathway: - Injured sensory neurons - Activated macrophages - T-cells Peripheral neuropathic pain Inflammatory bowel disease “Cancer pain” Diseases likely to involve BH 4

Precision Medicine – how to use BH 4 Diagnostic: nerve injury/macrophages activation Patients suffering from chronic pain Genetics: ! African-American: GCH 1 protective GCH 1 aggravating haplotype (2%) Too much BH 4; Other haplotype? likely responsive COMT? MOR? SERT? To treatment GCH 1 protective haplotype het (18%) Poor responder; Higher doses? ‘standard’ GCH 1 (80%) Too much BH 4; likely responsive To treatment GCH 1 haplotype can define which patients are BH 4++

Precision Medicine – how to use BH 4 Diagnostic: nerve injury/macrophages activation Genetics: GCH 1 active – BH 4 levels too high Patients suffering from chronic pain Treatment: What dose to reduce pain SPR no side effects Drug target Need a biomarker for treatment efficacy ?

Sepiapterin as a biomarker for SPR inhibition BH 4 de salvage novopathways: pathway: Sepiapterin: - stable SN 5 300 0 15 0 Dose (mg/kg) *: compared to SNI veh 300 10 * 30 0 * [sepiapterin] pmol/mg protein 15 0 [sepiapterin] pmol/mg protein DRG

Sepiapterin as a biomarker for SPR inhibition BH 4 salvage pathways: Sepiapterin: - Secreted by cells Supernatant from DRG neurons in culture [sepiapterin] *, §: compared to resp. veh

Sepiapterin as a biomarker for SPR inhibition BH 4 salvage pathways: Sepiapterin: - Detectable in plasma Supernatant from DRG neurons in culture [sepiapterin] Sepiapterin Pain relief No pain relief [SPR inhibitor]

Conclusion – Precision Medicine Diagnostic: nerve injury/macrophages activation Genetics: GCH 1 active – BH 4 levels too high Patients suffering from chronic pain Treatment: sepiapterin as a biomarker of efficacy [sepiapterin] Blood SPR Urine Drug target PAIN↘ Individualized treatment Drug dose

Acknowledgements Thank you! F. M. Kirby Neurobiology Program Clifford J Woolf Michael Costigan Alexandra Latini Nick Andrews Shane J Cronin Masahide Fujita Carla Lionela Giulia Miracca Michio Painter Olusegun Babaniyi Kelly Duong Aline Pertile Remor Priscilla Rivia Lee B Barrett École Polytechnique Fédérale de Lausanne, Switzerland Kai Johnsson Katarzyna Gorska Ruud Hovius University of Oxford, U. K. Keith Channon Surawee Chuaiphichai University of Frankfurt, Germany Irmgard Tegeder Nerea Ferreiros The Institute of Cancer research, U. K. Julian Blagg Burke Medical Research Institute Jian Zhong Solace Pharma Alasdair Naylor Sage Partner International Jinbo Lee

Acknowledgements Thank you! Funding: Disclosure: R 01 NS 58870 R 37 NS 039518 R 01 DE 022912 R 01 NS 074430 R 01 DE 022912 Clifford J Woolf, Kai Johnsson, Katarzyna Gorska, Michael Costigan, Nick Andrews, Alban Latremoliere have equity shares in Quartet Medicine