The use of membrane-free OFM for studies into the PK-PD of ... · The use of membrane-free OFM for studies into the PK-PD of large molecules Case Study Secukinumab Papers reporting

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

ContentsWishful thinking: The ‚ideal‘ sampling technique

Open-Flow Microperfusion

Sampling of LARGE MOLECULES –The Secukinumab PK-PD case study

Next challenges

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Wishful thinkingThe ideal sampling technique

Patients: tolerable, min. invasive, no pain/scarsResearcher: multiple probesrepeatedly, contin. long-term, stable, physiological informationDrugs: Small & large, hydro- & lipophilicAnalyst: clean sample, undilutedcost effective

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The jack of all trades

Microdialysis andOpen Flow Microperfusion(OFM)

OFM is like Microdialysis, but …

Probe: no filtering membrane all analytesPump: versatile multiple & long samplingAccessories for standardization

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Pumps

Skin stabilisation, covers, ..

OFM MaterialsOFM probe

Fully permeable sampling mesh(0.5 x 15 mm)anti-adsorptive inner coatinghighly flexible & robustluer connector

OFM pumpbattery drivenwearablepush-pull0.1 – 10 µl/min3 - 6 probes per pump

5CE certified for clinical use

OFMbasic properties

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Basic evaluationdOFM is tolerable

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

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VAS graph3.3 1.4 vs. 1.9 1.2 , p<0.001, n=141 probes

Basic evaluationdOFM sampling is stable

RR sodium 25h = 71.2 3.8% (mean SD)

RR without drift – i.e. stable sampling

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Bodenlenz et al. in Skin Res Technol (2013)

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dOFM in drug studiesWhich drugs are accessible?

Small molecules – YES, no challenge!

Lipophilic molecules - feasible? If applied topically in low strength?Does probe depth matter?

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PK-PD of lipophilic drugsThe Galderma study

8 patients with stable plaque psoriasis

CP-17 cream (0.05%) daily for 14 days

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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

PK-PD of lipophilic drugsFeasible, and depth matters!11

Probe depth matters!

0

0.5

1

1.5

2

1 2 3 4 5 6 7 8

ofm10ofm11ofm12

Subj. 001, non lesional

0.62mm0.72mm0.83mm

24h

CP

-17

in n

g/m

l

It‘s feasible!

Mixed effect modelling:

Modell: Clearly distinguished

YES - depth matters!

Better plot AUC vs. depth!

Variability: Mainly inter-subject!

SC is a trap for lipophilic drugs

healing! skinatrophy!

dOFM propertiesWhich drugs are accessible?

Small molecules - YES

Lipophilic molecules - feasible? – YES! Also if low topical strength? – YES!Does probe depth matter? – YES!

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Large molecules?Can we also quantify the concentration?

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

of large moleculesCase 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

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dOFM - Case Study SecukinumabmAB Secukinumab

Secukinumab (Novartis Pharma AG)A fully human monoclonal antibody (MW 150.000 Da)Treatment of moderate-to-severe psoriasisSelectively targets (i.e. binds) IL-17A

Rapid and significant efficacy in phase 3 trials70% of subjects show PASI 90 response within 16 weeks

Secukinumab marketed as Cosentyx®dOFM data used for drug filing

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from pharmacodia.com

dOFM - Case Study SecukinumabObjectives

Primary Aim (PK)To measure the absolute concentration of secukinumab in the dermis of healthy controls & psoriatic patients on Day 8 and 15

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Secondary Aims (PD)Investigate postulated IL17a signaling pathways (Days 1-8-15)Investigate postulated mode of action down-stream IL17a markersInvestigate effect on mediators for keratinocyte proliferation and angiogenesis and keratinocyte mobility…by comparing lesional, non-lesional, healthy skin on Days 1,8,15

Overall ObjectiveTo characterize the mechanism of action of secukinumabin psoriatic skin following a single s.c. dose of 300 mg secukinumab

dOFM - Case Study SecukinumabDesign& Quantification Approch

Exploratory, single-center, open-label study (NCT01539213)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 serumShowed that inulin dISF = inulin serum

Quantified secukinumab in dISF using OFM & inulinQuantified secukinumab in dISF using dOFM - independently of inulinCompared results & established inulin/secukinumab relationshipTo be sure: Quantified secukinumab in dermis also by biopsies, suction blisterCharacterized biomarkers in healthy

Part 2- Aim: To study the mechanism >> 8 plaque psoriasis patientsQuantified secukinumab using dOFM – sufficient to bind all IL-17A?Characterized biomarkers in patients – compare lesion-unaffected-healthy

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0 100 200 300 400 500-150

-100

-50

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50

100

150

Perfusate Cin (µg/ml)

288 µg/ml

Healthy Subjects - Day 8

Linear regression line 95% CI of linear regression line

Sam

ple

Cou

t - Pe

rfus

ate

Cin (µ

g/m

l)

dOFM - Case Study SecukinumabInulin NNF in healthy skin on Day8

Primed-cont. inulin infusionmean 277µg/ml & stable!

No Net Flux with dOFMNNF quality was fine (R2)104% (90%CI: 96-113%)RRI = 16 ± 3%

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R²=0.66

dOFM - Case Study SecukinumabSecukinumab NNF in healthy skin on Day15

mAB given s.c. on Day1

mAB NNF on Day15

Results:CISF= 23% Cserum

RRs = 6.5%

RR comparison:RRI/RRs = 2.45

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R2=0.59

dOFM - Case Study Secukinumab Validation of dOFM results using standards19

Sucion Blister …causes pigment changes

Biopsy …anaestesia, causes small scars, done on the back

dOFM - Case Study SecukinumabSummary PK- healthy

RRI = 16 ± 3%

Secukinumab:

Serum: 35µg/mldISF: 22/23%

dOFM = biopsy= blister fluid

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23%22%

dOFM - Case Study SecukinumabSummary PK - patients

RRI = 15 ± 3% healthy: 16 ± 3%

Secukinumab:

serum: 21µg/ml healthy: 35µg/ml

dISF: 28-39%, sufficient to neutralize IL17A!healthy: 22/23%

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Done in addition: No Net Flux…

R2=0.03 R2=0.41

IC at ~40%

dOFM - Case Study SecukinumabSecukinumab NNF in patients

NNFs may fail in the delivery section

Problem: No or slow removal of large molecules?

Solution: Stay in gain and approach IC stepwise!

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Secondary aimsPharmacodynamics

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dOFM - Case Study SecukinumabPD Results 1 – IL17 family24

IL-17A, but not IL-17F, is significantly higher in psoriatic lesional skin compared with non-lesional skin or skin of healthy volunteers.

dOFM - Case Study SecukinumabPD Results 2 – IL17 downstream BD-225

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.

dOFM - Case Study SecukinumabPD Results 3 – protein levels26

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)

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dOFM - Case Study SecukinumabConclusions

PharmacokineticsSecukinumab distributes into healthy skin.Secukinumab distributes into psoriatic skin to a similar or higher degreeSecukinumab concentration is sufficient to neutralize IL-17a in psoriasis.

PharmacodynamicsKey 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.

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Summary - Where are we with OFM?

OFM …is safe, tolerated and stableis tissue-specific with depth-resolutionshowed utility for a wide range of drugsenables quantitative sampling

Quantitative sampling: Challenging but possible!

Quality & Standardization - a continuous effort

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OFM The next challenges

Topical BioequivalenceUnderstand topical variabilityFind best clinical setting

Animal models ofskin inflammation

Cell Sampling & FACS characterization of immune cells

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Generic

Reference Generic

Reference

HEALTH - an interdisciplinary teamWe link technology and medicine

Chemists, biologists, engineers, statisticians, data managers, Q-experts, IT-experts, writers, technicianssi

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Medical University Graz CRC - Clinical Research Center31

MDs, study nurses, GCP quality experts, monitors, project managers, lab staff

Thank you for your attention

Contact:Dr. Frank Sinner

JOANNEUM RESEARCH Forschungsgesellschaft mbH

HEALTH – Institute for Biomedicine and Health Sciences

Neue Stiftingtalstrasse 2, 8010 Graz+43 316 876-4000

[email protected]/health

(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 dilutedbut otherwise unfiltered interstitial fluid for subsequent bioanalysis. Hence, OFM probes facilitate theinvestigation of large molecules, highly protein bound drugs and rather lipophilic drugs. Moreover, ourwearable multi-channel OFM pumps support multi-probe settings in mobile volunteers as well ascalibration approaches in studies aiming for quantitative sampling. The combination of these fully-permeable probes with versatile pumps has enabled informative pharmacokinetic and –dynamic drugstudies in healthy volunteers as well as patients.

The talk will briefly introduce the principles of OFM, describe the current OFM devices and outline theirperformance in typical research applications. Thereafter the talk will focus on a clinical trial that wasable to fully utilize the strengths of OFM when investigating the intradermal PK-PD of a large IL-17Aantibody in the skin in healthy volunteers and psoriatic patients (Secukinumab, MW 150.000Da). In thiscomprehensive trial dermal OFM was able to demonstrate (i) that Secukinumab distributes into dermalinterstitial fluid of healthy and psoriatic skin reaching intradermal levels sufficient to completelyneutralize IL-17A in skin and (ii) that Secukinumab treatment rapidly leads to positive proteomic andtranscriptional changes in psoriatic skin. The presentation of this successful trial will also provide theopportunity to give special attention to the calibration and validation strategies that should be of utilityfor OFM as well as Microdialysis.

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(Publikum)Publikum

PhDs & Supervisors bekannter MD-GruppenCMA, 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 …

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Preclinical applicationsInsulin 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

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