The Structure/Function Relationship: What do we know? What would we like to know?

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Madeline Stephanie Collins
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1 The Structure/Function Relationship: What do we know? What would we like to know? Brian R. Berridge, DVM, PhD, DACVP GlaxoSmithKline Research Triangle Park, NC

2 Outline A (very) brief overview of the structure and function of the cardiovascular system? What data are we integrating and where do we get it? Connecting the dots What do we still need to learn?

We know a fair bit about the cardiovascular system as

conduct blood to the heart itself as well as the rest

Cardiomyocytes are contractile cells with immense

ensure coordinated contraction of different regions

3 We know a fair bit about the cardiovascular system as an integration of form and function Blood vessels conduct blood to the heart itself as well as the rest of the body. Cardiomyocytes are contractile cells with immense energy needs Rhythmic waves of electrical activity ensure coordinated contraction of different regions of the heart. A muscular pump and its delivery system. Heart valves ensure unidirectional flow of blood.

We know something about what cardiovascular toxicity looks

cardiomyocyte injury vascular injury Adverse CV events

valvulopathy ultrastructural injury cellular biochemistry

cardiac mass function QT prolongation Non-QT arrhythmias QT

4 We know something about what cardiovascular toxicity looks like! Nonclinical CV Toxicity Clinical CV Toxicity cardiomyocyte injury vascular injury Adverse CV events (Vioxx, Darvocet) Congestive Heart Failure (TZDs, TKIs) valvulopathy ultrastructural injury cellular biochemistry morphology Decreased EF (TKIs) Valvulopathy (Fen-Phen) cardiac mass function QT prolongation Non-QT arrhythmias QT prolongation/tdp risk BP HR contractility Increased blood pressure (Torcetrapib)

5 Possible Cardiac Adverse Effects of Drugs Primary functional effects ± morphological consequences Primary effect on cardiac structures ± secondary functional effects Effects on vascular motricity Adverse effects on cardiac function Direct effect on cardiac structure Exaggerated vasodilation and reflex activation of heart Arrhythmias ± conduction impairment Effect on contractile protein or Ca channels Myocardial degeneration Valvular proliferation e.g. QT prolongation Acute Chronic Myocardial necrosis Cardiac fibrosis Cardiac hypertrophy Decreased/impaired cardiac contractility Impairment of atrioventricular and/or arterial flows e.g. isoproterenol e.g. minoxidil e.g. doxorubicin e.g. anorexigenic drugs Exacerbation of pre-existing dz- 1 o, 2 o Not in the Hanton scheme Adapted from Hanton, G. Drugs R D 2007: 8(4):213

6 Possible Cardiac Adverse Effects of Drugs Primary functional effects ± morphological consequences Primary effect on cardiac structures ± secondary functional effects Effects on vascular motricity Adverse effects on cardiac function Direct effect on cardiac structure Exaggerated vasodilation and reflex activation of heart Arrhythmias ± conduction impairment Effect on contractile protein or Ca channels Myocardial degeneration Valvular proliferation e.g. QT prolongation Acute Chronic Myocardial necrosis Cardiac fibrosis Cardiac hypertrophy Decreased/impaired cardiac contractility Impairment of atrioventricular and/or arterial flows e.g. isoproterenol e.g. minoxidil e.g. doxorubicin e.g. anorexigenic drugs Exacerbation of pre-existing dz- 1 o, 2 o Not in the Hanton scheme Adapted from Hanton, G. Drugs R D 2007: 8(4):213

7 We know we generate a lot of relevant data in relatively sensitive ways?

8 In vitro screening for ion channel and receptor binding activity Prospective opportunity for identifying putative risks Retrospective opportunity to define pathogenesis/mechanism

9 In vivo studies Current paradigms in preclinical safety assessment Acute/Single Dose Safety Pharmacology Studies Functional endpoints Multiple species In vitro/ex vivo Rodents Non-Rodents Early Development CV Fxn FTIH-enabling GLP CV Study Patch clamp ion channel assays Rabbit wedge assay Translatable biomarkers Telemetered rat Heart rate ECG (quantitative) No QT Blood pressure ± surrogate measures of contractility- LVP, QA Telemetered dog or monkey Heart rate ECG (quantitative) Blood pressure ± surrogate measures of contractility- LVP, QA

10 Current paradigms in preclinical safety assessment In Vivo Repeat-Dose General Toxicity Studies Rodents Multiple species Non-Rodents 4 days Repeat-dose studies of increasing length at super-pharmacologic 2 years doses Morphologic endpoints Blood/urine biomarkers Functional endpoints Organ weight Gross Microscopic - light microscopy - ultrastructure Serum chemistry Hematology Hemostasis Urinanalysis Heart rate ECG (qualitative) Translatable biomarkers * Repeat dose studies biased toward morphologic endpoints

11 Morphologic Evaluation- Gross to Microscopic Gross examination of the intact rodent heart Light microscopy of myocardium Ultrastructure of myocardium Evaluation of atrioventricular valve of opened non-rodent (dog) heart Light microscopy of atrioventricular valve

12 Standard clinical pathology endpoints in repeat-dose toxicity studies Serum chemistry Biomarkers of hepatobiliary injury- ALT, AST, Tbili, ALP, GGT Biomarkers of muscle injury- CK, AST, Potassium Biomarkers of renal injury- BUN, Cr, Urine Sp Gr, Urine Total Protein Biomarkers of metabolic health- glucose, triglycerides, cholesterol, electrolytes, total protein, albumin, globulins Biomarkers of calcium-phosphorus balance calcium, inorganic phosphorus Hematology Biomarkers of inflammation, hematologic dyscrasia, hematopoiesis CBC, reticulocyte count, blood smear review Biomarkers of coagulation- PT, APTT, fibrinogen, platelet count Urinanalysis Biomarkers of renal health, metabolic health - ph, protein, glucose, blood, bilirubin, ketones, urobilinogen, urine sediment, urine specific gravity, renal electrolytes Endpoints with particular relevance to CV injury Compliments of A. Eric Schultze

13 Novel CV Biomarker Interests Cardiac troponins Natriuretic peptides H-FABP Novel contexts of use for troponins or natriuretic peptides Imaging mirnas

14 We know what we re looking for! Changes in serum chemistry Increases in ctn, NTproANP/BNP, CK, K+ Altered coagulation parameters Changes in function Inotropy, chronotropy, rhythmicity Changes in heart mass with our without proportional changes in chamber volumes Cellular injury Vacuolation Degeneration Necrosis Apoptosis Hypertrophy Myocardial injury Hemorrhage Inflammation Fibrosis Valvular injury Vascular injury *Important to recognize that the heart, like many target organs, has a finite number of ways to respond to altered physiology or noxious stimuli.

15 And what it looks like! 4 mg/kg s.c. in F344 rats 2-4 hr 24 hr 48 hr Temporal pathogenesis of isoproterenolinduced injury in the rat heart 2 weeks

16 Vascular responses to injury Vasoconstrictor/hypertension injury Medial hypertrophy/hyperplasia Periarterial fibrosis Fibrinoid necrosis Compliments of H. Thomas

17 We know what to do with the data when we get it: Integrated Interpretation of Histopathology Data Light microscopy Vascular injury Myocardial injury Valvulopathy Is there a correlative change in blood pressure? Is the injury reversible or irreversible (e.g. necrosis, fibrosis) Generally not monitorable = Dose and development limiting NO YES reversible irreversible Possible direct vasculotoxin and more difficult development challenge Possible translational biomarker or screening parameter NO May be dose &/or development limiting Is there a correlative functional change? YES Translational biomarkers Is there a correlative increase in ctni? NO Re-examine time of sampling YES Reporting translational biomarker Is the molecule an ergot alkaloid or have ALK 5 inhibitory activity? SAR screen Monitorability and exposure margins significantly impact progression opportunity!

We know functional changes can lead to structural injuries: Minoxidil as a model cardiovascular toxicant in rats Effect of Minoxidil on Arterial Blood Pressure and Heart Rate in Conscious Rat

7%) Multifocal myocardial necrosis Single dose rat CV study Arterial injury in the mesentery Minoxidil (mg/kg) MAP HR 3 Maximal decrease of 22 mmhg Effect last ~22 h Maximal increase of 70

18 We know functional changes can lead to structural injuries: Minoxidil as a model cardiovascular toxicant in rats Effect of Minoxidil on Arterial Blood Pressure and Heart Rate in Conscious Rat Minoxidil is a vasodilating K+ channel blocker 7d repeat-dose rat CV study Mean absolute (relative to BW) heart weight Gp % (+3.6%) Gp % (+13.4%) Gp % (+6.7%) Multifocal myocardial necrosis Single dose rat CV study Arterial injury in the mesentery Minoxidil (mg/kg) MAP HR 3 Maximal decrease of 22 mmhg Effect last ~22 h Maximal increase of 70 beats/min Effect last ~6 h 30 Maximal decrease of 23 mmhg Effect last ~24 h Maximal increase of 181 beats/min Effect last ~24 h 100 Maximal decrease of 33 mmhg Effect last ~24 h Maximal increase of 138 beats/min Effect last ~24 h

19 PDE4 inhibitors can do it too! Subendocardial necrosis Vascular injury *no HW reported

20 What I would like to know? Structure Every Study Function Logistical challenges Technological challenges How do we get these relationships from the right studies?

21 We need to understand the complexity of our target patients better! Attributes of a diabetic patient population Concurrent medications Ischemic heart dz Stroke Heart failure Renal disease Hyperglycemia Hypertension Dyslipidemia How do we model diabetic patients preclinically? Baseline characteristics of patients with diabetes studied in the RECORD trial. Lancet 373: , 2009

22 We need to know how to better model our target patients!

23 Drug-induced vascular injury- place for a significant safety pharmacology contribution??

24 Gap = there are no biomarkers of vasoactivity on these lists!

25 Thank you! Questions?

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