Preclinical Development of ALN-AS1, an Investigational RNAi Therapeutic for the Treatment of the Hepatic Porphyrias

Transcription

1 Preclinical Development of ALN-AS1, an Investigational RNAi Therapeutic for the Treatment of the Hepatic Porphyrias Amy Chan, Abigail Liebow, Makiko Yasuda, Scott Barros, Tim Racie, Martin Maier, Satya Kuchimanchi, Don Foster, Stuart Milstein, Klaus Charisse, Muthiah Manoharan, Robert Desnick, Rachel Meyers, Kevin Fitzgerald, Amy Simon, and William Querbes International Conference of Poprhyrins and Porphyrias 15 September

2 Therapeutic Approach to Harnessing RNAi Synthetic sirna dsrna Cleavage dicer Strand separation Natural Process of RNAi Targeted Gene Silencing RISC Complementary pairing mrna Cleavage (A) n mrna degradation (A) n 2

3 Receptor Targeted sirna Conjugates for Delivery to Hepatocytes Lead Selection & Optimization Subcutaneous Delivery Selection Optimization sirna sequence» Specificity» Crossreactivity» Potency sirna chemistry» Stability» Immuno-silence» Potency GalNac-siRNA Conjugates ASGPR Receptor» Highly expressed on hepatocytes» Rapidly recycles» Conserved across species Introduce chemical modifications for drug-like properties Small Scale Gene walks In vitro assays Chemistry, Manufacturing and Controls Medium Scale In vivo biology Large Scale GMP Production Clinical trials 3

4 Clinical Experience with GalNac-siRNA Conjugates GalNAc Conjugate Platform Currently utilized in 6 clinical stage ALNY programs Over 250 patients and >1500 doses administered Generally well tolerated Human Proof of Concept Ex: TTR Lowering in TTR Amyloidosis Program Alnylam Development Pipeline Human Clinical Efficacy Ex: Reduced Bleed Rate in Hemophilia Program 4 Gilmore, ACC, March 2015, Sorenson, ISTH, June 2015

5 ALN-AS1 Therapeutic Hypothesis Targeting Liver ALAS1 to Lower Heme Intermediate Production ALN-AS1 Role of Liver in Disease Liver and domino transplant experience ALA/PBG and symptoms improve or come on rapidly after transplant ALN-AS1 potential to treat all acute hepatic porphyrias including AIP, VP and HCP 5 Brennan et al. Nature 1979, Hermes-Lima et al. Biochem Biophys Acta 1991, Dar et al., Hepatobiliary Pancreat Dis Int.; 9:93-6 (2010), Dowman et al., Ann Intern Med; 154:571-2 (2011)

6 ALN-AS1 Preclinical Development Overview Lead ID Disease Model POC NHP Evaluation Drug Safety and Metabolism Phase 1 Testing Key Data Identify ALAS1 target sequence and optimized chemistry Liver ALAS1 knockdown in rodents Impact of ALN-AS1 on ALA/PBG Define ALAS1 and ALA/PBG relationship Assay Development ALAS1 lowering kinetics Dose and dose regimen exploration Tolerability of ALN-AS1 in different species PK/PD relationships Evaluate any potential on target risks 6

7 ralas1/rgapdh, Relative to pbs=1 Urinary ALA or PBG (mmol/mol creatininine) PBS=1 ALN-AS1 Multidose Prophylaxis in Rat AIP Model D0 D7 D14 D21 D25 (sac) ALAS-GalNAc 3, 1, 0.3mg/kg (QWx4) Phenobarb D18 PBGD LNP ALAS1 mrna 10 8 ALA/PBG ALA PBG sirna PB PBGD LNP 3mg/kg 1mg/kg 0.3mg/kg PBS ALN-AS ALN - AS1 PB PBGD LNP PBS 3 mg/kg 1 mg/kg 0.3 mg/kg

8 ALN-AS1 (µg/g) ALN-AS1 Tissue Distribution in Rats 1000 Single Dose 10mg/kg 4 hours 100 ~12X lower tissue concentration kidney vs. liver 10 Less than 2% of total exposure combined 1 0 Exposure is the highest in the liver, followed by kidney; remaining tissues have <2% of delivered dose deposited 8

9 Normalized ALAS-1 Circulating Extracellular RNA Detection (cerd) Method for Circulating ALAS1 mrna Detection AS1 Transcript by cerd by liver biopsy Monitoring RNAi Activity in Liver mrna or 5 RACE product in tissue Circulating secreted protein Detection of Circulating ALAS1 mrna Exosomes are shed into bodily fluids from many different cell types and contain mrna and mirna derived from tissue of origin Exosomes can be used to monitor ALAS1 mrna levels after ALN-AS1 dosing in serum/urine without need for biopsy PBS ALAS-GalNAc (mg/kg) QDx5, EOD, d15 (not DC) 9 Sehgal et al. RNA 2012

10 Use of cerd Assay to Monitor ALN-AS1 Pharmacology in NHPs QW-weekly 10

11 Liver ALN-AS1 Concentrations and ALAS1 mrna Knockdown in NHP Single Dose 10mg/kg Multiple Dose 2.5mg/kg Weekly x8 ALN-AS1 liver drugs levels correlate with extent and duration of ALAS1 mrna reduction 11

12 Relative ALAS1 mrna Levels (% NHV) NHV 1 NHV 2 NHV 3 NHV cerd Assay Demonstrates ALAS1 Upregulation in Porphyria Patients vs. Healthy Volunteers Serum Urine Normal Healthy Volunteers AIP Patients ~3-fold upregulation of ALAS1 mrna in AIP patients (n=10) vs. NHV (n=4) Good correlation of ALAS1 mrna in urine and serum Chart review suggests trend of ALAS1 upregulation correlating with worse disease severity 12

13 Collection Collection Collection Collection 2 Relative AS1 mrna Levels (% NHV) Day to Day Variability in ALAS1 Levels with cerd Assay in AIP Patient Samples Serum Urine AIP Patient 1 AIP Patient 2 Human cerd Proof of Concept Minimal day to day variability Potential clinical utility ALAS1 changes during attacks Compare patient populations Monitoring PD with ALN-AS1 treatment Exploratory use in our EXPLORE natural history study and in Phase 1 0 Donor ID 13

14 ALN-AS1 Nonclinical Safety and Metabolism CTA-Enabling Studies for Start of Phase I Clinical Trial Exploratory Studies Cytokine screening in vitro (hwba) and in vivo (mouse) No acute pro-inflammatory effects Dose range-finding toxicity studies in mouse, rat, NHP (repeat dose, 3-5 weeks in duration) Establish dose levels for GLP studies GLP-Compliant Safety Studies Safety pharmacology (cardiovascular and respiratory) in telemetered NHPs Genetic toxicology in vitro (Ames, chromosomal aberrations in hpbl) and in vivo (rat BM micronucleus) General Toxicology studies in rat and NHP 13 week studies + 13 week recovery Toxicokinetics and CNS evaluation (NHP) NOAEL 150 mg/kg All negative up to limit doses Rat NOAEL 30 mg/kg NHP NOAEL 150 mg/kg Absorption, Distribution, Metabolism, Excretion (ADME) Studies PK/PD in rat, NHP Toxicokinetics included in all Tox studies (plasma; liver and kidney in rodents) Metabolic profiling in rat, NHP (plasma, liver) Plasma protein binding (mouse, rat, NHP, human) In vitro, ex vivo CYP studies Safety margins of >300x (rat) and >1500x (NHP), based on respective no adverse effect levels (NOAELs) and clinical starting doses (mg/kg) 14

15 ALN-AS1 Impact on P450s No Signs of Heme Deficiency in Preclinical Studies Study/Species Doses CYPs Results In vitro direct CYP inhibition, human liver microsomes In vitro CYP induction, primary human hepatocytes Ex vivo CYP Activity after ALN-AS1 dosing, rat liver microsomes ,000 ug/ml 0.1, 1, 10, 100 ug/ml 30, 100, 300 mg/kg, weekly x5 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4/5 1A2, 2B6, 3A4 1A, 2B, 2D, 2E, 3A No inhibition of any isoform No changes in CYP levels or enzyme activity No biologically significant changes ~75% of total human liver CYPs evaluated in these studies 2A6 4% 2B6 1% 2D6 2% 2E1 7% Other 26% 1A2 13% 3A4,5 29% 2C8,9, 18 18% Ex vivo CYP Activity after ALN-AS1 dosing, NHP liver microsomes In vivo DDI with CYP substrate probes following ALN-AS1 dosing, NHP 30, 100, 300 mg/kg, weekly x5 5 mg/kg, daily x5 1A, 2B, 3A, 2D, 2E 1A, 2D, 2E, 3A No biologically significant changes No reduction in metabolism of any CYP substrate probes No significant reduction in P450 function or signs of heme deficiency detected with ALAS1 lowering 15

16 Conclusions and Next Steps for ALN-AS1 Conclusions ALN-AS1 is a subcutaneous administered RNAi therapeutic targeting ALAS1 in development for the treatment of the hepatic porphyrias Potent silencing of liver ALAS1 mrna and reduction in ALA/PBG in preclinical models Robust safety profile supporting the initiation of Phase 1 The cerd assay is a non invasive method for monitoring circulating ALAS1 mrna ALAS1 mrna upregulated in serum/urine from AIP patients consistent with role in disease pathophysiology Potential utility of the assay to understand ALAS1 levels during attacks, in different patient populations, and following ALN-AS1 dosing in clinical trials ALN-AS1 Clinical Development Plan EXPLORE natural history study (ongoing) Non interventional study in AIP patients with recurrent attacks Phase 1 Study (ongoing) Part A/B- Single ascending dose (SAD) and multiple ascending dose (MAD) study in AIP asymptomatic high excreter patients (ASHE) Part C- Multiple dose study in AIP patients with multiple recurrent attacks Pivotal study in patients with recurrent attacks 16

17 Acknowledgements ALN-AS1 Core Team Bill Querbes (PL) Tanya Sengupta (PM) Amy Simon Craig Penz Amy Chan Scott Barros Lauri Binne Jason Costigan Satya Kuchimanchi Anshul Gupta Rena Denoncourt ALN-AS1 Advisors and Extended Team Abigail Liebow Nate Taneja Tim Racie Jon O Shea Brian Bettencourt Sarfraz Shaikh Kirsten McCarthy Mano Manoharan Siddharth Jain Rajeev Kallanthottathil Don Foster Jeff Rollins Stu Milstein Lubo Nechev Rick Duncan John Frenz Jared Gollob Jolly Bhatia Martin Maier Patrick Igwenagu Klaus Charisse Husain Attarwala Matthias Kretschmer Renta Hutabarat Lauren Melton Ju Liu Akshay Vaishnaw Krishna Aluri Jeff Cehelsky Chris Tran Kevin Fitzgerald Yongli Gu Rachel Meyers Minggeng Gao Dave Watkins Qianfan Wang Oved Amitay Xuemei Zhang Sara Nochur Mary Carioto Carol Pitcher Towner Julia Hettinger Donna Mackey Garvin Warner Ligang Zhang Mike Placke Lubomir Tchangov Matt Algarin Karen Dobson Dave Mosher Jeff Kopacz Svetlana Shulga Morskaya Jennifer Pena Meghan Collins Kelley Hanson Tim Mooney Preclinical Collaborators Robert Desnick Makiko Yasuda John Phillips APF Desiree Lyon Jessica Hungate Natalia Sturza APC Coordinator Hetanshi Naik Phase 1 Investigators Eliane Sardh Nabil AL-Tawil Pauline Harper David Rees Penelope Stein Manisha Balwani Karl Anderson Monty Bissell Joseph Bloomer EXPLORE Investigators Karl Anderson Herb Bonkovsky Montgomery Bissel John Phillips Charles Parker Manisha Balwani Joseph Bloomer Pauline Harper Eliane Sardh David Rees Mike Badminton Penelope Stein Raili Kauppinen Ulrich Stölzel Jorge Frank Elisabeth Minder Jean Charles Deybach Laurent Gouya Pavel Martesek Janne Langendonk Sverre Sandberg Delia D Avola Maria Ivanova Paulo Ventura Maria Capellini Jerzy Wingyga Peter Meissner Joanne Marsden 17