PAGE 2013; Glasgow

Towards the prediction of cardiovascular effects in human

Nelleke Snelder, Bart Ploeger, Olivier Luttringer, Dean Rigel, Fumin Fu, Michael Beil, Donald Stanski and Meindert Danhof

[email protected] 1 fingolimod

• Fingolimod is effective in the treatment of multiple sclerosis • In 2010 fingolimod was approved at a dose of 0.5 mg

Freedoms trial: patients with MS Event fingolimod 0.5 mg placebo • A dose of 0.5 mg resulted in: (N=425) (N=418) • A small increase in blood pressure Hypertension 26 (6.1 %) 16 (3.8 %) • 1-2 mm Hg after 2 months Bradycardia 9 (2.1 %) 3 (0.7 %) • A transient bradycardia • 8 bpm at 5 hours after the 1st dose (attenuation after 6 hours)

• Are cardiovascular effects of relevance for fingolimod? • Is the effect reversible? • What are the long term effects?

• Can early selection of follow-up compounds be improved?

Introduction 2 Effect of fingolimod on the CVS

Vasocontriction/dilation • Sphingosine-1-phosphate (S1P) Angiogenesis is a major regulator of vascular Airway resistance S1P1 S1P3 / and immune systems S1P1? Barrier Vascular Endothelium function S1P1/ Bronchial smooth S1P3 muscle S1P3 (S1P1, S1P2?)

S1PS1P S1P3 (S1P1, S1P3 (S1P1 in S1P2?) humans?) • Fingolimod is a S1P agonist Vascular smooth Atrial myocytes • Subtypes S1P1, S1P3, muscle S1P4 and S1P5 Heart-rate Vascular tone AV Conduction

• The mechanism of action needs further investigation

Introduction 3 Hypothesis: a better understanding of the mechanism of action of compounds can result from a quantitative understanding of the system

• Physiological principles of BP regulation Mean Arterial Total Peripheral Cardiac Output Pressure (MAP) = Resistance (TPR) x (CO)

Cardiac Output (CO) = Heart Rate (HR) x Volume (SV) • Several feedback mechanism: • Baroreflex system • Renin-- System

• Preclinical research may contribute to a quantitative understanding of the cardiovascular system (CVS)

Introduction 4 Objectives

1) Development of a systems pharmacology model characterizing the effects of drugs on the interrelationship between BP, TPR, CO, HR and SV ⇒ Drug-independent CVS model

2) Characterization of the effect of fingolimod on the CVS using the developed systems pharmacology model ⇒ A better understanding of mechanisms leading to cardiovascular effects following administration of fingolimod

Objectives 5 CVS model

PAGE 2013 Glasgow 6 Collect preclinical data from different drugs acting on CVS to identify system specific parameters

• Compounds: Effect on HR Effect on TPR Effect on SV atropine (M2 receptor antagonist) () (diuretic) propanolol (non-selectiveβ blocker) enalapril (ACE-inhibitor) enalapril (ACE-inhibitor) fasudil (rho-kinase inhibitor) HCTZ (diuretic) (selective α1 blocker) 2-3 rats per compound

Electical swivel • Study design: Baseline measurements A different dose each day Washout arterial Ascending radiotransmitter aortic flow probe Day 1 Day 2-5 Days 6-7 BP,BP, COCO andand TPRHR are measured measured

CVS model 7 System-specific Model

• Linked turnover model with SV = SVT * (1-HR_SV *LN(HR/BSL_HR)) differential equations for HR, HR CO = HR⋅SV SV and TPR linked by negative Kin_HR kout_HR MAP = CO ⋅TPR feedback through MAP FB - • Direct inverse relationship between HR and SV MAP FB - FB -

• Circadian rhythm: two cosine SVT TPR functions, one influencing HR Kin_SV kout_SV Kin_TPR k and one influencing TPR out_TPR

• Handling effect: exponentially decreasing functions influencing Kin_HR and Kin_TPR

• Differences between hypertensive and normotensive rats: • Different baseline • The feedback was found to be dependent on the baseline blood pressure

CVS model 8 Negative effect on HR MAP mmHG 120 140 HR beats/min 150 250 CO mL/mi n 0506070 0 40 4 SV mL/beat ) 0.22 0.26 0.3 TPR mHG/(mL/mi n 2.3 2.5 2.7 m 0122436 Time (h) 9 Negative effect on TPR MAP mmHG 100 130 0 HR beats/min 320 360 40 CO mL/mi n 70 80 90 SV mL/beat 0.22 0.24 ) TPR mHG/(mL/mi n 1.2 1.8 m 0122436 Time (h) 10 Drug-specific Model

EFF_HR

SV = SVT * (1-HR_SV *LN(HR/BSL_HR)) HRC CO = HR⋅SV • PKPD modelling approach (NONMEM): Kin_HR kout_HR MAP = CO ⋅TPR PK models from literature FB - MAP FB - FB -

• Drug effects on HR, SV or TPR SVC T TPR Kin_SV kout_SV Kin_TPR kout_TPR

EFF_SV EFF_TPR • Best drug effect models: Compound Effect site Drug effect model

amiloride SV Emax with Emax fixed to 1

amlodipine TPR Emax with Emax fixed to 1 atropine HR Linear

enalapril TPR and SV Emax with Emax fixed to 1

fasudil TPR Emax with Emax fixed to 1

HCTZ SV Emax with Emax fixed to 1 prazosin TPR Power HR No effect

• The effect of ACE inhibitors is delayed. This was described by an effect compartment model

CVS model 11 Adequate description of the effect of amlodipine hypertensive rats

Vehicle 0.3 mg/kg 1 mg/kg 3 mg/kg 10 mg/kg

Effect on all five endpoints MAP

mmHG can be described by a single drug effect on TPR 100 140 180 HR beats/min 250 400 CO mL/mi n Measured 50 70 90 SV mL/beat 0.20 0.30

Derived Individual prediction TPR Population prediction (n=38) mHG/(mL/mi n) 1.0 2.0 3.0 m 0 12 24 36 48 60 72 84 96 108 132 156 Observations (colored per rat) Ti me (h) • CVS model 12 Adequate description of the effect of amlodipine normotensive rats

Vehicle 0.3 mg/kg 1 mg/kg 3 mg/kg 10 mg/kg MAP mmHG Effect of amlodipine 80 100 described adequately in normotensive rats HR beats/min 250 400 CO mL/mi n Measured 120 160 SV mL/beat 0.35 0.45

Derived Individual prediction TPR

mHG/(mL/mi n) Population prediction (n=38) m 0.4 0.7 1.0 0 12 24 36 48 60 72 84 96 108 132 156 Ti me (h) • Observations (colored per rat) CVS model 13 Conclusions and Future perspective CVS model

•A systems pharmacology model was developed to describe the interrelationship between MAP, CO, HR, SV and TPR

• The developed systems pharmacology model can be used to quantify cardiovascular drug effects of novel compounds

• Can the CVS model be used to elucidate the mechanism of action of novel compounds?

CVS model 14 Application of the developed drug- independent CVS model to fingolimod

PAGE 2013 Glasgow 15 Prediction of the effect of fingolimod on the CVS

Experimental design: MAP • Dose: 0, 0.1, 0.3, 1, 3 and 10 mg/kg 160 • MD administration (4 weeks) 150 • Hypertensive and normotensive rats 140 • 32 rats mmHg 130 • Measured: MAP, HR and CO 120 0 400 800 1200 Time (h) TPR HR 1.8 360 1.6 340 1.4 320

1.2 beats/min 300 mmHG/(mL/min) 1.0 280 0 400 800 1200 0 400 800 1200 Time (h) Time (h) CO SV 0.50 120 0.45 110 0.40 100 0.35 mL/min mL/beat 90 0.30 80 0.25 0 400 800 1200 0 400 800 1200 Time (h) Time (h)

Example of a typical hyeprtensive rat (10 mg/kg)

Effect of fingolimod on the CVS 16 Prediction of the effect of fingolimod on the CVS

• The CVS model was used to Hypothesis 1: effect is on HR MAP predict the effect of fingolimod 160 • System-specific parameters were fixed 150 140 mmHg 130mmHg 120 0 400 800 1200 Time (h) TPR HR 1.8 EFF_HR 450360 1.6 1.5 400340 SV = SVT * (1-HR_SV *LN(HR/BSL_HR)) CO = HR⋅SV 1.4 HRC 350320 K k MAP = CO ⋅TPR 1.2 beats/min in_HR out_HR beats/min 1.2 300 mmHG/(mL/min) mmHG/(mL/min) 300 0.91.0 280 FB - 0 400 800 1200 0 400 800 1200 MAP Time (h) Time (h) FB FB CO SV - - 0.50 140120 0.45 125110 0.40 SVC T TPR 0.40 Kin_SV kout_SV Kin_TPR k 110100 out_TPR 0.35 mL/min mL/min mL/beat mL/beat 9590 0.30 80 0.25 0 400 800 1200 0 400 800 1200 Time (h) Time (h)

The CVS model indicates that it is not likely that the primary effect of fingolimod is on HR

Effect of fingolimod on the CVS 17 Prediction of the effect of fingolimod on the CVS

• The CVS model was used to Hypothesis 2: effect is on TPR MAP predict the effect of fingolimod 160 • System-specific parameters were fixed 150 140 mmHg 130mmHg 120 0 400 800 1200 1200 Time (h) TPR HRHR 1.8 450 350 1.5 400 SV = SVT * (1-HR_SV *LN(HR/BSL_HR)) 1.5 330 CO = HR⋅SV 310 HRC 350 K k MAP = CO ⋅TPR 1.2 290 in_HR beats/min

out_HR beats/min 270 mmHG/(mL/min) mmHG/(mL/min) 300 0.9 FB 0.9 250 - 0 400 800 1200 00 400 400 800 800 1200 1200 MAP Time (h) TimeTime (h)(h) FB FB CO SVSV - - 140 0.500.50 140 0.450.45 125 SVC TPR 0.400.40 K T k K k 110 in_SV out_SV in_TPR out_TPR 110 0.35

mL/min 0.35 mL/min mL/beat mL/beat 95 0.300.30 80 0.250.25 EFF_TPR 0 400 800 1200 00 400 400 800 800 1200 1200 Time (h) TimeTime (h)(h)

The CVS model indicates that the primary effect of fingolimod is on TPR There may be a secondary effect on HR Effect of fingolimod on the CVS 18 Model to describe the effect of fingolimod

Transient • PKPD modelling approach: negative • PK of the pro-drug fingolimod and its EFF_HR SV = SVT * (1-HR_SV *LN(HR/BSL_HR)) active metabolite were described HRC CO = HR⋅SV K k simultaneously by a compartmental model in_HR out_HR MAP = CO ⋅TPR FB - MAP FB - FB -

SVC T TPR Kin_SV kout_SV Kin_TPR kout_TPR

Fast positive SlowSlow positivepositive • The effect of fingolimod on the CVS was EFF_TPR diseaseEFF_TPR altering described by a combination of 3 effects: EFF_TPR • Fast positive effect on TPR: log-linear model • Slow positive effect on TPR: linear model K ·M Kin R out • Slow structural effect only in hypertensive rat at high doses • Transient negative effect on HR: power model • Tolerance was described by feedback model - type 1 K ·M Ktol·R M tol

Effect of fingolimod on the CVS 19 Adequate description of the effect of fingolimod on the CVS in hypertensive rat

MAP

200 Example: Dose 10 mg/kg 180 Individual prediction 160 mmHg • Observations (colored per rat) 140 120 0 600 1200 1800 2400 Time (h) CO HR 120 350 100 330 310 80

mL/min 290 60 beats/min 270 250 40 0 600 1200 1800 2400 0 600 1200 1800 2400 Time (h) Time (h) TPR SV 4.00 0.5

3.25 0.4

2.50 0.3

1.75 mL/beat 0.2 mmHG/(mL/min) 1.00 0.1 0 600 1200 1800 2400 0 600 1200 1800 2400 Time (h) Time (h) Effect of fingolimod on the CVS 20 Adequate description of the effect of fingolimod on the CVS in normotensive rat

MAP 140

Example: Dose 10 mg/kg 125

Individual prediction 110 mmHg • Observations (colored per rat) 95 80 0 600 1200 1800 2400 Time (h) CO HR 140 350 120 330 100 310

mL/min 290 80 beats/min 270 60 250 0 600 1200 1800 2400 0 600 1200 1800 2400 Time (h) Time (h) TPR SV 2.0 0.5

0.4 1.5 0.3

1.0 mL/beat 0.2 mmHG/(mL/min) 0.5 0.1 0 600 1200 1800 2400 0 600 1200 1800 2400 Time (h) Time (h) Effect of fingolimod on the CVS 21 Conclusions Effect of fingolimod on the CVS

• The effect of fingolimod on MAP, CO, HR, SV and TPR was characterized adequately using the developed CVS model

• The CVS model was used to elucidate the mechanism of action of the effect of fingolimod on the CVS • The long term effect of fingolimod on BP resulted from an effect on TPR • The primary effect was reversible • The secondary effect, which is relevant in hypertensive rat for extreme high doses, was structural • A transient effect on HR was quantified

Effect of fingolimod on the CVS 22 General Conclusions and Future Perspectives

•A systems pharmacology model was developed to describe the interrelationship between MAP, CO, HR, SV and TPR in rat

• The CVS model can be used to elucidate the mechanism of action of novel compounds

• Future research: prediction of the clinical response based on preclinical data for fingolimod and other (follow-up) compounds

Conclusions 23 Acknowledgements

Novartis-Leiden collaboration

24 Further reading

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