The use of membrane-free OFM for studies into the PK-PD of large molecules

Transcription

1 The use of membrane-free OFM for studies into the PK-PD of large molecules Manfred Bodenlenz & Frank Sinner JOANNEUM RESEARCH Forschungsges. mbh HEALTH Inst. for Biomedicine and Health Sciences, Graz, Austria 8th International Symposium on Microdialysis, Uppsala, May 2016

2 2 Contents Wishful thinking: The ideal sampling technique Open-Flow Microperfusion Sampling of LARGE MOLECULES The Secukinumab PK-PD case study Next challenges

3 Folie 2 sim1 ist das absieht? Selma Mautner;

4 3 Wishful thinking The ideal sampling technique Patients: tolerable, min. invasive, no pain/scars Researcher: multiple probes repeatedly, contin. long-term, stable, physiological information Drugs: Small & large, hydro- & lipophilic Analyst: clean sample, undiluted cost effective The jack of all trades

5 4 OFM is like Microdialysis, but Microdialysis and Open Flow Microperfusion(OFM) Probe: no filtering membrane all analytes Pump: versatile multiple & long sampling Accessories for standardization Pumps Skin stabilisation, covers,..

6 OFM Materials OFM probe Fully permeable sampling mesh (0.5 x 15 mm) anti-adsorptive inner coating highly flexible & robust luer connector OFM pump battery driven wearable push-pull µl/min 3-6 probes per pump CE certified for clinical use 5

7 6 OFM basic properties

8 7 Basic evaluation dofm is tolerable VAS vs. 1.9 graph 1.2, p<0.001, n=141 probes Multiple insertion is tolerated (VAS<3.3). Cooling with ice is sufficient! Long protocols are tolerated (VAS=0). Wearability ensures mobility! Most common protocol: 12 probes and 24 h sampling

9 8 Basic evaluation dofm sampling is stable RR sodium 25h = % (mean SD) RR without drift i.e. stable sampling Bodenlenz et al. in Skin Res Technol (2013)

10 Folie 8 sim [2]1 kein abstand bei %, kein abstand bei / - ist anders als im deutschen Selma Mautner;

11 9 dofm in drug studies Which drugs are accessible? Small molecules YES, no challenge! Lipophilic molecules - feasible? If applied topically in low strength? Does probe depth matter?

12 10 PK-PD of lipophilic drugs The Galderma study 8 patients with stable plaque psoriasis CP-17 cream (0.05%) daily for 14 days Day1 + 14: dofm for 24 h 3 probes into lesional skin 3 probes into healthy skin Perf: Saline + 1%HSA Probe depth by ultrasound Further 6 probes for cytokines

13 11 2 CP-17 in ng/ml ofm10 ofm11 ofm12 Subj. 001, non lesional It s feasible! PK-PD of lipophilic drugs Feasible, and depth matters! 0.62mm 0.72mm 0.83mm Mixed effect modelling: healing! skin atrophy! h Probe depth matters! Modell: Clearly distinguished YES - depth matters! Better plot AUC vs. depth! Variability: Mainly inter-subject! SC is a trap for lipophilic drugs

14 12 dofm properties Which drugs are accessible? Small molecules - YES Lipophilic molecules - feasible? YES! Also if low topical strength? YES! Does probe depth matter? YES! Large molecules? Can we also quantify the concentration?

15 13 The use of membrane-free OFM for studies into the PK-PD of large molecules Case Study Secukinumab Papers reporting the results: PK Results: Christian Dragatin, Florine Polus, Manfred Bodenlenz, Claudio Calonder, Birgit Aigner, Katrin Irene Tiffner, Julia Katharina Mader, Maria Ratzer, Ralph Woessner, Thomas Rudolf Pieber, Yi Cheng, Christian Loesche and Frank Sinner and Gerard Bruin. Experimental Dermatology 2016 PD Results: Frank Kolbinger, Christian Loesche, Marie-Anne Valentin, Xiaoyu Jiang, Yi Cheng, Philip Jarvis, Thomas Peters, Claudio Calonder, Gerard Bruin, Florine Polus, Birgit Aigner, David M Lee, Manfred Bodenlenz, Frank Sinner, Thomas R. Pieber and Dhavalkumar D Patel. Journal of Allergy and Clinical Immunology in revision

16 14 dofm - Case Study Secukinumab mab Secukinumab Secukinumab (Novartis Pharma AG) A fully human monoclonal antibody (MW Da) Treatment of moderate-to-severe psoriasis Selectively targets (i.e. binds) IL-17A Rapid and significant efficacy in phase 3 trials 70% of subjects show PASI 90 response within 16 weeks from pharmacodia.com Secukinumab marketed as Cosentyx dofm data used for drug filing

17 15 dofm - Case Study Secukinumab Objectives Overall Objective To characterize the mechanism of action of secukinumab in psoriatic skin following a single s.c. dose of 300 mg secukinumab Primary Aim (PK) To measure the absolute concentration of secukinumab in the dermis of healthy controls & psoriatic patients on Day 8 and 15 Secondary Aims (PD) Investigate postulated IL17a signaling pathways (Days ) Investigate postulated mode of action down-stream IL17a markers Investigate effect on mediators for keratinocyte proliferation and angiogenesis and keratinocyte mobility by comparing lesional, non-lesional, healthy skin on Days 1,8,15

18 16 dofm - Case Study Secukinumab Design& Quantification Approch Exploratory, single-center, open-label study (NCT ) In vitro checks of adsorption, 2 clinical parts Part 1 - Aim: To establish the methodology! >> 8 healthy volunteers Validated a calibrator in dofm (inert inulin) Showed that inulin is stable in serum Showed that inulin disf = inulin serum Quantified secukinumab in disf using OFM & inulin Quantified secukinumab in disf using dofm - independently of inulin Compared results & established inulin/secukinumab relationship To be sure: Quantified secukinumab in dermis also by biopsies, suction blister Characterized biomarkers in healthy Part 2- Aim: To study the mechanism >> 8 plaque psoriasis patients Quantified secukinumab using dofm sufficient to bind all IL-17A? Characterized biomarkers in patients compare lesion-unaffected-healthy

19 17 dofm - Case Study Secukinumab Inulin NNF in healthy skin on Day8 Primed-cont. inulin infusion mean 277µg/ml & stable! Healthy Subjects - Day 8 No Net Flux with dofm NNF quality was fine (R 2 ) 104% (90%CI: %) RR I = 16 ± 3% Sample C out - Perfusate C in (µg/ml) R²= µg/ml Linear regression line 95% CI of linear regression line Perfusate C in (µg/ml)

20 18 dofm - Case Study Secukinumab Secukinumab NNF in healthy skin on Day15 mab given s.c. on Day1 mab NNF on Day15 Results: R 2 =0.59 C ISF = 23% C serum RRs = 6.5% RR comparison: RR I /RR s = 2.45

21 19 dofm - Case Study Secukinumab Validation of dofm results using standards Sucion Blister causes pigment changes Biopsy anaestesia, causes small scars, done on the back

22 20 dofm - Case Study Secukinumab Summary PK- healthy RR I = 16 ± 3% Secukinumab: Serum: 35µg/ml disf: 22/23% 22% 23% dofm = biopsy = blister fluid

23 21 dofm - Case Study Secukinumab Summary PK - patients RR I = 15 ± 3% healthy: 16 ± 3% Secukinumab: serum: 21µg/ml healthy: 35µg/ml disf: 28-39%, sufficient to neutralize IL17A! healthy: 22/23% Done in addition: No Net Flux

24 22 dofm - Case Study Secukinumab Secukinumab NNF in patients R 2 =0.03 R 2 =0.41 IC at ~40% NNFs may fail in the delivery section Problem: No or slow removal of large molecules? Solution: Stay in gain and approach IC stepwise!

25 23 Secondary aims Pharmacodynamics

26 24 dofm - Case Study Secukinumab PD Results 1 IL17 family IL-17A, but not IL-17F, is significantly higher in psoriatic lesional skin compared with non-lesional skin or skin of healthy volunteers.

27 25 dofm - Case Study Secukinumab PD Results 2 IL17 downstream BD-2 Skin β Defensin 2 in Psoriasis Subjects and Healthy Volunteers (Secukinumab 300 mg s.c. at Day 1 after baseline samples were obtained) Baseline (Day 1) ß-Defensin-2 protein levels are elevated in psoriasis lesional skin and serum do decrease rapidly in response to secukinumab treatment.

28 26 dofm - Case Study Secukinumab PD Results 3 protein levels Amphiregulin (AREG) Epiregulin (EREG) Collagenase (MMP-1) Gelatinase B (MMP-9) Reduction of protein levels of amphiregulin and epiregulin within 7 days (drivers of autocrine keratinocyte proliferation). Downregulation of expression of gelatinase B (MMP-9) protein (implicated in angiogenesis and tissue destruction)

29 Folie 26 sim [4]1?? Selma Mautner; sim [5]1 da hat es etwas mit der text formatierung Selma Mautner;

30 27 Pharmacokinetics dofm - Case Study Secukinumab Conclusions Secukinumab distributes into healthy skin. Secukinumab distributes into psoriatic skin to a similar or higher degree Secukinumab concentration is sufficient to neutralize IL-17a in psoriasis. s Pharmacodynamics Key molecular factors were positively affected in psoriatic skin within 7 days. Expression of pro-inflammatory cytokines were positively affected. Key mediators reduced (keratinocyte proliferation, angiogenesis, keratinocyte mobility) dofm proved useful for the study of this mab at the target in vivo. Inulin (sinistrin) proved useful as calibrator. No Net Flux protocol proved useful as independent method for quantification.

31 Folie 27 sim [6]1 sim [7]1 kein eigenname Selma Mautner; ganze Sätze mit Punkt abschliessen - in der ganzen praesentation Selma Mautner;

32 28 Summary - Where are we with OFM? OFM is safe, tolerated and stable is tissue-specific with depth-resolution showed utility for a wide range of drugs enables quantitative sampling Quantitative sampling: Challenging but possible! Quality & Standardization - a continuous effort

33 29 OFM The next challenges Topical Bioequivalence Understand topical variability Find best clinical setting Generic Reference Reference Generic Animal models of skin inflammation Cell Sampling & FACS characterization of immune cells

34 HEALTH - an interdisciplinary team We link technology and medicine Chemists, biologists, engineers, statisticians, data managers, Q-experts, IT-experts, writers, technicians si

35 Folie 30 sim [8]1 director passt hier nicht Selma Mautner;

36 31 Medical University Graz CRC - Clinical Research Center MDs, study nurses, GCP quality experts, monitors, project managers, lab staff

37 Contact: Dr. Frank Sinner JOANNEUM RESEARCH Forschungsgesellschaft mbh HEALTH Institute for Biomedicine and Health Sciences Neue Stiftingtalstrasse 2, 8010 Graz Thank you for your attention

38 33 (Abstract) The use of membrane-free open flow microperfusion for studies into the PK-PD of large molecules Manfred Bodenlenz and Frank Sinner, HEALTH Joanneum Research Open-flow microperfusion (OFM) utilizes membrane-free probes for sampling, thus providing diluted but otherwise unfiltered interstitial fluid for subsequent bioanalysis. Hence, OFM probes facilitate the investigation of large molecules, highly protein bound drugs and rather lipophilic drugs. Moreover, our wearable multi-channel OFM pumps support multi-probe settings in mobile volunteers as well as calibration approaches in studies aiming for quantitative sampling. The combination of these fullypermeable probes with versatile pumps has enabled informative pharmacokinetic and dynamic drug studies in healthy volunteers as well as patients. The talk will briefly introduce the principles of OFM, describe the current OFM devices and outline their performance in typical research applications. Thereafter the talk will focus on a clinical trial that was able to fully utilize the strengths of OFM when investigating the intradermal PK-PD of a large IL-17A antibody in the skin in healthy volunteers and psoriatic patients (Secukinumab, MW Da). In this comprehensive trial dermal OFM was able to demonstrate (i) that Secukinumab distributes into dermal interstitial fluid of healthy and psoriatic skin reaching intradermal levels sufficient to completely neutralize IL-17A in skin and (ii) that Secukinumab treatment rapidly leads to positive proteomic and transcriptional changes in psoriatic skin. The presentation of this successful trial will also provide the opportunity to give special attention to the calibration and validation strategies that should be of utility for OFM as well as Microdialysis.

39 34 (Publikum) Publikum PhDs & Supervisors bekannter MD-Gruppen CMA, andere Microdialyse Hersteller Interesse am Weg zum Ziel, weniger Interesse an konkreten Results Material, Methoden, Geräte, Settings Was geht beim Sampling, Was geht nicht, Worauf achten

40 35 Preclinical applications Insulin studies in rats Skin inflammation models in rats Drug penetration into pig skin in vivo Drug penetration into fresh/frozen human skin Drug metabolism into fresh human skin