The role of pharmacokinetics inThe role of pharmacokinetics in drug discovery:Where are we now? How did we

t h ? Wh i ?get here? Where are we going?

P t W bbPeter Webborn

RSC February 2013

PK studies in Drug DiscoveryWhy conduct PK studies in animals?

“The primary purpose of pre-clinical pharmacokinetic studies is to validate the tools that will be used to predict human kinetics”

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The role of pharmacokinetics in drug discoveryOverview

1. The past - The 4 key steps that got us here

2. PK studies / data / tactics in a modern Drug Discovery programme

3. The future – When free plasma concentrations don’t tell us everythingdon t tell us everything

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How did we get here?Four key Developments

1. A Bioanalytical breakthrough2. A Pharmacokinetic breakthrough3 An Experimental breakthrough3. An Experimental breakthrough4. A Conceptual change

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Bioanalytical breakthroughLC-MS : The Thermospray interface

Thermospray Source designThermospray Source design, Blackley et al (1978)

Historically Gas chromatography (+derivatisation)Thin layer chromatographyThin layer chromatographyuv HPLC

LC-MS (triple quadrupole)

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LC MS (triple quadrupole)Key for analysis in drug discovery - Sensitive, Selective, Generic, Fast

Pharmacokinetic breakthroughThe Introduction of “clearance concepts”

int.. CLfuQCL b

The ‘well stirred’ liver model

int.CLfuQCL

b

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Pharmacokinetic breakthroughClearance – Six cornerstones of understanding

“The Clearance is the volume of blood cleared of drug per unit time”

1. Clearance (not half-life) is the best measure of the efficiency of an elimination process

“The Clearance is the volume of blood cleared of drug per unit time”

( ) y p

2. Clearance is the scaling factor between the iv dose you give and the AUC you get!

3 Clearance relates the rate of elimination (ng/min) to the substrate concentration (ng/ml)3. Clearance relates the rate of elimination (ng/min) to the substrate concentration (ng/ml)V = CL x S

4. Determined from CL = Dose/AUCiv (units of flow)

5. Is influenced by plasma protein binding and by blood flow

6. “Intrinsic clearance” or CLint - relates free drug concentration to the rate of eliminationelimination…….

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Experimental breakthroughPrediction of clearance from in vitro dataPrediction of clearance from in vitro data

Also: Rane A, Wilkinson GR and Shand DG, Prediction of hepatic extraction ratio from in vitro measurement of intrinsic , , pclearance. J Pharrnacol Exp Ther 200: 420- 424, 1977.

Also– No need to monitor appearance of metabolites, can be derived from loss of parent compound - and - From V/S not Vmax / Km

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loss of parent compound and From V/S not Vmax / Km

Conceptual breakthroughPK properties are predictable and amenable to optimisationPK properties are predictable and amenable to optimisation

Amounts and routes of Rates and affinitieselimination. Types of

metabolism.Rates and affinities

“The compound” The Chemical seriesSAR

Dose and half-life Concentration and clearanceand clearance

N d t t Physicochemical

Predictions don’t always have to be right

Name and structure Physicochemical properties

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Predictions don’t always have to be right“If you know what to expect – you are more likely to spot the unexpected”

SAR Ionisation and Volume of Distribution (Vss)Conceptual breakthrough - ExampleSAR - Ionisation and Volume of Distribution (Vss)

Acid

Base20

4060

Neutral468

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• Vss tends to be acid < neutral < base

0.60.8

1

2

• Little influence of log D7.4

• Why do we see this?0 1

0.2

0.4

logD

-4 -3 -2 -1 0 1 2 3 4 50.080.1

Understand drug behaviour – anticipating risks/issuesExamples: Acidic drugsExamples: Acidic drugs

Poor Absorption

Low permeability

prisk

Uptake Transporter substrate?

Renal CL Interspecies differences

Interspecies differences

Biliary CL

differences

Enterohepatic recirculation

Interpretation of PK data

InterspeciesDifficult to

Acids

Interspecies differences

High Albumin affinity

measure very high ppb

Low Vss Risk of short t½Intrinsic

clearance must be low

Interspecies differencesbe o

AcylGlucuronidereactivity risk

Interspecies differences

Glucuronidation Gut metabolism Interspecies differences

Standard microsomemicrosome

assays no use

PK studies in Drug DiscoveryWhere are we now?Why conduct PK studies in animals?

- How to get most value from studies? - How to use optimally to progress projects?

What have we learnt about experimental systems?- Accuracy, reproducibility - Optimal protocolsOptimal protocols- Cassette dosing

Specific issuesSpecific issues - Plasma protein binding- Formulation choice

H ti t k t t- Hepatic uptake transporters

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PK studies in Drug DiscoveryWhy conduct PK studies in animals?

“The primary purpose of pre-clinical pharmacokinetic studies is to validate the tools that will be used to predict human kinetics”

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PK studies in Drug DiscoveryWhy conduct PK studies in animals?

Why do you want to know the......

ClearanceHalf-lifeBioavailabilityBioavailabilityVolume of distribution*

..........of a compound in animals?

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PK studies in Drug DiscoveryAssessing risk

What you really want to know how well you were able to predict in vivo kinetics- Clearance - microsome /hepatocyte CLint - in silico

Volume of distribution Physicochemical properties Vssu across species- Volume of distribution – Physicochemical properties – Vssu across species- Bioavailability - Permeability /solubility – 1st pass metabolism

“I understand why this compound behaves the way it does”Or

“I have no idea why this compound behaves the way it does”

“If I can predict the kinetics in rat and dog, I have a reasonable case to ask you to believe I can predict human kinetics”to believe I can predict human kinetics

Or“If I can’t predict the kinetics in rat and dog, why should anyone think I can predict human kinetics”

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predict human kinetics

Predicting hepatic metabolic clearance

R Scaled Predicted

Lab specific correction

Predicted

In vitro scaling factors,fuP, Rb, fuinc

Well stirred model (WSM)

Raw CLint

Scaled CLint

Predicted In vivo CLint

Predicted in vivo clearance

b)

• A regression approach adjusts for systematic under-predictions observed when scaling in vitro CLint directly

Qh*

CL b

)/(Q

h-C

L b using the well stirred model, unbound fractions in blood and the in vitro matrix, and physiological scaling factors.

Riley, McGinnity and Austin (2005)

Log(CLint*SF*fub/fuinc)

Log(

Q

• This is commonly seen, and is not understood• Correction factor is associated with the assay, not the compound

g( b inc)

y, p

Setting criteria for an acceptable IVIVEPK studies in Drug DiscoverySetting criteria for an acceptable IVIVE.

2-sided 80% prediction interval1-sided 90% upper prediction limit

Project 3

2 sided 80% prediction interval

l/min

/kg)

Project 2 Project 3

Lint

in v

ivo

(ml

Having an optimised, standardised

Obs

erve

d C

L

Allows a common understanding of

method puts the focus on the compound, not the scaling method

Rat reference set

Project 1

Log 1

0O Allows a common understanding of

“scaling” and “non-scaling” compounds, and uncertainty in predictions.

Log10 Predicted CLintin vivo (ml/min/kg)

Project example - IVIV correlationsPK studies in Drug DiscoveryProject example - IVIV correlations

RAT DOG

• Compounds in general scaled well in rats if LogD was kept below 3

Clarity of message / ”rules” all can understandAllows focus on compound – not scaling method

PK studies in Drug DiscoveryEffective use of PK data - focus on prediction validation

Re-enforce positive project behaviours• Predict - measure - learn• Ensure correct use of in vitro data• Ensure correct use of in vitro data• Build trust in in silico /in vitro systems

S ti di ti ti I ili i it PK PKPDSupporting prediction continuum: In silico - in vitro - PK - PKPD • Understanding relative risk/uncertainty in extrapolations

Efficient projects work in chemical series that are “predictable”

Candidate drugs that are understood, are less risky

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PK studies in Drug DiscoveryA Problem...... Projects like having compounds tested

Less beneficial behaviours• Overriding belief that more data = better informed• Rigid screening cascades• More “success” in testing cascade validates molecules

P j t lik t d t t• Projects like to demonstrate progress • “It’s our best compound to date –let’s get a full data package”

ConsequencesConsequences• Many measurements that fit with predictions• Many results we learn nothing from• Much data gathered on incrementally “better” compoundsMuch data gathered on incrementally better compounds• Too much data to manage –unfocussed optimisation strategies

Management of study requestsManagement of study requests“I’ll run any test you are prepared to make a decision on”

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A PK i t t “E bli th t d i i ”PK studies in Drug DiscoveryA PK screening strategy –“Enabling the next decision”

HI LO

Number of studiesstudies

Fold under-prediction ofprediction of

rat CL

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PK studies in Drug DiscoveryThe Power of DatabasesThe Power of Databases

What can we learn from having PK data on 1000’s of compounds?

SAR• SAR• Cassette dosing studies – Yielding a better understanding of Variability

(inter-animal /inter-study)P l h di i• Protocol enhancement diagnosis- N=3 v n=2- First time point after iv bolus- Cannulated v non-cannulated animals

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Renal Clearance Model

Key descriptors:Lipophilicity– Lipophilicity

– Ability of compound to carry a positive charge

PK studies in Drug DiscoveryUse of Reference compounds to track assay performance

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20

25

30

g

Mean Clearance over 5 months

10

15

20

CL m

l/m

in/k

g

Non-cannulated Animals

Cannulated Animals

0

5

10

06/05/2013 25/06/2013 14/08/2013 03/10/2013 22/11/2013 11/01/2014Date of study

Reference compounds - Key advantage of cassette studies

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How many animals to use? n=2 vs n=3PK studies in Drug DiscoveryHow many animals to use? n=2 vs n=3

Impact on Vss estimates

Vsswithin Count % of valueswithin Count % of values0-20% 490 80

20-30% 65 1130-40% 24 4>40% 32 5

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PK studies in Drug DiscoveryImpact on CL - 2min or 5 min first sample

in A

UC

Cha

nge

i%

C

300

N=2750

30 3300~Clearance (ml/min/Kg)

Charnwood rat iv PK data2min or 5 min first sample - Impact on CL

in A

UC

73% of TV within 10%Cha

nge

i

73% of TV within 10%83% of JVC within 10%

90% of TV within 20%

% C

300

90% of TV within 20%93% of JVC within 20%

N=2750

30 3300Clearance (ml/min/Kg)

PK studies in Drug DiscoverySpecific Issues

•Plasma protein binding•Formulation choiceFormulation choice•Transporters

28 Author | 00 Month Year Set area descriptor | Sub level 1

Only one of these statements is mechanistically tcorrect..................

• “Because of the high plasma protein binding, free plasma concentrations will be very low”

• “Because of the high plasma protein binding, total plasma concentrations will be very high”total plasma concentrations will be very high

PK studies in Drug DiscoveryProtein Binding - Don’t let it trip you up!

In vitro systems In vivo systems

• A closed system

F l l d i b

• An open system

F l l d i b• Free levels driven by binding

• Free levels driven by elimination rate

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Smith et al (2010) Nature Drug Discovery Dec;9(12):929-39

PK studies in Drug DiscoveryChoice of formulation

“ f f“The formulation should be appropriate for the conclusion that will be drawn from the study”

e.g.

If you want to draw conclusions about likely human bioavailability /absorption, a clinically relevant y p yformulation should be used.

If you are assessing exposure prior to an efficacy studyIf you are assessing exposure prior to an efficacy study, ensure the formulation is tolerated for the duration of study + does not affect the PD endpoint

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PK studies in Drug DiscoveryHepatic Uptake transporters

Uptake and efflux transporters have made understanding drug clearance more complicated.....

some simple concepts are no longer valid..some simple concepts are no longer valid

“Additi it f l ” l li t ll l“Additivity of clearance” - only applies to parallel processes

Transporter modelling requires “barriers”, serial processes and concentration gradients across membranesg

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“Uptake is the rate-limiting step in the overall hepatic elimination of pravastatin at steady-state in rats” p yYamazaki, M., Akiyama, S., Nishigaki, R., Sugiyama, Y. 1996 Pharmaceutical Research 13 (10), 1559

What is this really saying?What is this really saying?

Consider this example: in a 3 step reaction:

For the formation of D (excretion into bile) - The rate determining step is always the slowest step in the process. y p p

For loss of A, the rate determining step is always k1 (plasma clearance)

In this scenario:In this scenario:

The rate of conversion of A to B depends on k1, k2 and k4.

For poorly permeable compounds uptake is the rate determining step in the plasma clearance of active uptake substrates (because the back-rate is insignificant)back-rate is insignificant)

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PK studies in Drug DiscoveryThe future

More Chemical diversity- Oligonucleotides- Extremely polar molecules - Intravenous antibioticsExtremely polar molecules Intravenous antibiotics- Drug – Antibody conjugates- Nanotechnology delivery systems

I t t tiInstrumentation- New interfaces – More sensitive- Higher throughput - No chromatography

More reliance on predictions- Cost- Trust- Trust

Moving beyond plasma- Mass Spectrometry Imaging (MSI)Mass Spectrometry Imaging (MSI)

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PK studies in Drug DiscoveryWhen (free) plasma concentrations can’t tell the whole story• Free drug hypothesis

• It is the unbound drug that is in equilibrium with the target• At equilibrium the free drug concentration in plasma and tissues are the

same•Problem areas

• Poorly perfused tissues• Hypoxic regions• Hypoxic regions• Substrates for drug transporters• Active / toxic metabolites• Low target off-rates• Local administration (eg lung, skin)

•Mass spectrometry Imaging – Key points• Drug / metabolite / biomarkers / metabonomics in tissues• Not “free drug”• Not free drug• Not all compounds/ not all studies – a targeted approach• Resolution is not at the cellular level ~100µm (10µm)• Several rapidly evolving technologies (eg MALDI, DESI, LESA, SIMS)

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PK studies in Drug DiscoveryMass Spectrometry Imaging

A unique insight into PK and PKPD – MALDI MSIA unique insight into PK and PKPD MALDI MSI

If you can get the y gsame answer through tissue homogenisation – Don’t do MSI

Ch ll S iti it d ti l l ti36

Challenges – Sensitivity and spatial resolution

PK studies in Drug DiscoveryDrug localisation as a driver of toxicity

Polymyxin nephrotoxicity – Aiding compound design

PMB1 AZ1PMBPMB1 AZ1PMB

•The technique can meet speed/volume requirements of discovery Programmes

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•Need to translate results to man

Summary

•PK is a well established component of drug discovery

•There is now a big opportunity to exploit PK databases

•PK resource management remains a challenge

• The future will be PKPD and translation

MSI i idl d l i t h i ith l t ti l t•MSI is a rapidly developing technique with real potential to solve both efficacy and toxicity related problems

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