automation of transil assays outperforms traditional ... · pdf fileautomation of transil...

Automation of TRANSIL assays outperforms traditional methods in preclinical research in traditional methods in preclinical research in terms of speed, cost-effectiveness and reproducibility

Dr. Hinnerk Borisshbo@sovicell [email protected]

07/06/2011

AgendaAgenda

• TRANSIL Core Technology• TRANSIL Core Technology

• Protocol and workflow

• ApplicationsB ain Tiss e Binding– Brain Tissue Binding

– Brain-to-Plasma Distribution– Liver Microsomal Binding– Plasma Binding

2

Benefits of TRANSIL AssaysBenefits of TRANSIL Assays

• Matrix free method:• Matrix free method:– Enables rapid separation of the drug from the

biological phase = fast quantificationR i 15 i t ilib ti ti• Requires 15 minutes equilibration time– in comparison dialysis requires 4-6 hours

• Requires only 5 minutes manual handling time– In comparison dialysis requires 100 minutes

• Fully automatable • No need for analytical techniques with very high • No need for analytical techniques with very high

sensitivity• Overcomes analytical limitations for highly bound drugs • Reduces laboratory animal consumption• Reduces laboratory animal consumption

3

TRANSIL Binding Assays:TRANSIL Binding Assays:

• Protein Binding– HSA, RSA– AGP (AAG)AGP (AAG)– PPB (HSA + AGP)

• Membrane Binding = Membrane Affinity– Phosphatidylcholine (intestinal)– BrainBrain– Liver microsomes (coming Q4/2011)– Custom membranes

4

TRANSIL TechnologyTRANSIL Technology

Q tifi ti l ti t f • Quantification relative to references = no calibration curve needed

• Immobilized matrix f t tifi ti

5

= fast quantification• Ready-to-use

= minimal labor requirement

TRANSIL Membrane affinity (MA)A P i i lAssay Principle

• Consists of silica beads d ith i b i li id covered with porcine brain lipid

or other bilayers suspended in PBS buffer

• Assess the drugs brain membrane affinity by incubating fixed concentration

B i h h li id bilg

of drug with varying concentrations (six) of lipids immobilized on the silica beads

Brain phospholipid bilayer

Silica bead

and analyzing the drug free fraction

• Prediction of tissue absorption

6

• Prediction of tissue absorption via specific statistical models using MA as key parameter

M b Affi it E ti tiMembrane Affinity Estimation

[ ]llipid cdrug[ ]

[ ] b

l

buffer

lipid

VcVcn

cc

drugg

MA

+

==

lb

lb

b

t

llbbt

VccV

cn

VcVcn

⋅+=

⋅+⋅=

1010

blb

t VVMAcn

+⋅=

410510610710810910

1010ta

l)/c(

buffe

r)

slope

10110210310410

0.00 0.50 1.00 1.50

n(to

t

lipid volume [µl]

7

Natural Membrane Fluidity on TRANSIL B dTRANSIL Beads

0.0

nits

]

gel state liquid crystal phase

t d-1.0x106

-5.0x105

supported bilayer

capa

city

[arb

. un

supported DPPCd8-bilayer

DPPCd8-MLV10 15 20

-1.5x106

vesiclesheat

c

Dynamics and Orientation: 2H-NMRtemperature [°C]

Thermodynamics: DSC

Natural fluidity and orientation preserved after reconstitution8

TRANSIL Protein binding A P i i lAssay Principle• Consists of silica beads covered with human plasma albumin

(HSA) and human α1-acid glycoprotein (AGP) suspended in PBS buffer

• Assess the drugs proteins affinity by incubating fixed concentration of drug with varying concentrations (six) of HSA or AGP immobilized on silica beads and analyzing the drug free fraction

• Drugs plasma fu is measured in terms of dissociation constant to HSA (KD

HSA ) and to AGP (KDAGP ) assuming HSA

and AGP concentration corresponding to the mean b d f b h h l h d d labundance of both proteins in healthy individuals

u AGPHSAf ][][1

=

PD ][][ ×

AGPD

HSAD K

AGPKHSA ][][1 ++

[HSA] = 40 g/L (588 µMol/L)

9

i

iD DP

PDK][][][

⋅×

= [HSA] = 40 g/L (588 µMol/L)[AGP]= 0.8 g/L (20 µMol/L)

Protein Binding:Fit t F d t l M d lFit to Fundamental Model

[ ] [ ][ ]AP

PAKD⋅

=

[ ] [ ] [ ]( )APAfA u +⋅=

[ ] [ ]( ) [ ][ ]

[ ] [ ]( )[ ] P

ffPf

APAPA

APPAPAfK

b

uu

f

uD

b

⋅=⋅⋅+

=⋅+⋅

=

= /143421

PKf

f

Du

b ⋅=1

slopef Du slope

10

A I t i i QCAssay Intrinsic QC

• Data consistency• Data consistency– Declining conc– Signal < reference

• Reference quality– Predicted vs measured

• Intercept of nt/cb vs[lipid] plot: Vb

• Outliers based on regression model– Robust regression

V

11

Vb

TRANSIL WorkflowTRANSIL Workflow

1. Prepare compound (5 minutes)2. Add compound3. Mix4. Incubate and mix (12 minutes)5. Centrifuge (10 minutes)6. Transfer supernatant

12

7. Quantify drug in supernatant8. Paste data into spreadsheet for analysis

M i F D F tiMeasuring Free Drug Fractions

Plasma Binding• Ultrafiltration• Ultracentrifugation• Protein Columns

Brain Binding• Brain microdialysis• Brain slice method

Equilibrium Dialysis• Pros:

– accurate method for estimating the unbound fraction of drugs– HT method: 96-well format and sample pooling approaches

• Cons:• Cons:– Throughput is limited by long equilibration times (4-6 hours)– Animal consumption (plasma and brain homogenate)– Requires highly sensitive analytical techniques– The resolution is limited for highly bound drugs

13

TRANSIL vs DialysisTRANSIL vs Dialysis

Features TRANSIL DialysisFeatures TRANSIL Dialysis

Format 96 (384) 24 - 48well

Test items Small and large molecules Small molecules

Incubation time 12 minutes 5-16 h

Biological matrix immobilized full tissue extract

Assay principleSeparation by removal of

particles; Surface area > 1m2

Separation by diffusion across membrane

Surface area < 1cm2Surface area > 1m2 Surface area < 1cm2

Analytics

for 6 replicates:8 samples with 1 min gradient in LC/MS/MS (total/12 cmps: 1.5 h)

for triplicates:10 samples with 5 min gradient in LC/MS/MS (total/12 cmps: 10 h)( / p ) ( / p )

Compounds per day 192 / 768 29

Issues metabolism, instrument down-time

Labor requirement 5 min 100 minLabor requirement 5 min 100 min

14

ApplicationsApplications

1 Brain Tissue Binding1. Brain Tissue Binding

2. Brain-to-Plasma Distribution

3. Liver Microsomal Binding

4. Plasma Protein Binding

15

Application:B i Ti Bi diBrain Tissue Binding

• Efficacy of drugs depends on potency and drug i i iconcentration in tissue

• Pharmacodynamics of CNS drugs only related to free drug in brain

• Free drug in brain depends on total brain concentration and • Free drug in brain depends on total brain concentration and fraction unbound in brain

16

B i F F ti d l BBBrain Free Fraction and logBBin

tissu

eou

ndto

brai

ion

drug

bofr

acti

logBB17

Ti Bi di P di tiTissue Binding Prediction

( ) ( ) 63.0log89.010 +⋅−= brainMAu brainf

• Parameterized with 25 highly diverse structures

• Prediction of brain free fractions on >1000 k d t >1000 unknown compounds greater than r2>0.92

18

P di ti f B i F F tiPrediction of Brain Free Fraction

1

0.1

1

thra

t

r2=0.94n=67

0.01

dial

ysis

wit

nex

trac

t

0.001

fu(b

rain

)by

dbr

ai

0.00010.0001 0.001 0.01 0.1 1

fu(brain) by TRANSIL

19

GSK V lid ti D tGSK Validation Data

40

GSK

aly

sis

GSK

Marketed

Longhi et al. (2011)DMD 39: 312 321

4

Eq

uil

ibri

um

dia

DMD 39: 312-3210.4

fub

rain

% E

0.040 04 0 4 4 40

20

0.04 0.4 4 40

fubrain% TRANSIL

Brain Free Fraction in PigE l F F ti i R tEquals Free Fractions in Rat

21

Brain Free Fractions are the same i ll T S i in all Tox Species

Di et al. (2011)DMD 39: 1270 77DMD 39: 1270-77

22

M lti l i C bilit f TRANSILMultiplexing Capability of TRANSIL

1

0.1

1TRANSIL single incubationTRANSIL cassette incubation

(Bra

in)

0.01

f u

0.001

GSK

14G

SK13

GSK

16G

SK17

GSK

1G

SK12

Clo

zapi

nem

itrip

tylin

eH

alop

erid

olro

pran

olol

GSK

8G

SK10

GSK

4M

idaz

olam

GSK

5G

SK11

GSK

3G

SK15

GSK

7G

SK2

amaz

epin

eup

ivac

aine

GSK

9B

upro

prio

nG

SK6

0.0001

23

CA

m Ha

Pr M

Car

ba Bu B






24

Application:B i T Pl Di t ib tiBrain-To-Plasma Distribution

l d h b f• Blood Brain Barrier protects the brain from xenobiotics while maintaining metabolic functions

• Significant hurdle for CNS targeted drugs

I t t t ti f d id ff t• Important protection from drug side-effects

• CNS-accessible chemical space much smaller in CNS accessible chemical space much smaller in comparison to other organs (2 to 20%)

25

Blood-Brain-Barrier MorphologyBlood-Brain-Barrier Morphology

pericyte

tight junctions

B t bastrocyte feet Basement membrane20-50 nm

astrocyte feet

26

B i t Pl Di t ib tiBrain-to-Plasma Distribution[ ][ ] l

brain

drugdrugBB ⎟

⎟⎠

⎞⎜⎜⎝

⎛= loglog

Validation Data2

p=2 2*10-12

[ ]( ) df

fcPSAbbrainMAa

plasma

ub

drug

+⎟⎠⎞⎜

⎝⎛⋅+⋅+⋅

=

⎠⎝loglog

10

B

1

Variable p-value

p=2.2 10

viv

o Lo

gB

0

logMAbrain 6.9*10-7

logKB/F 2.1*10-4

in

-1PSA 1.2*10-8

TRANSIL LogBB-2 -1 0 1 2

-2

27

CNS Cl ifi tiCNS Classification

28

Classification by Rate & Extent:PAMPA BBB TRANSILPAMPA BBB vs TRANSIL

PAMPA BBB TRANSIL50% to 60% correct 87% correct

29






30

Application:Li Mi l Bi diLiver Microsomal Binding

• Liver microsomes are used to assess metabolic • Liver microsomes are used to assess metabolic stability of drugs

• Stability is assessed via disappearance of compound

• Compound binding to microsomal membranes confounds stability estimates

• Affinity to liver microsomal membranes can be used to correct for membrane binding

31

TRANSIL Li Bi di Di l iTRANSIL Liver Binding vs Dialysis

100

%] 80

100

r2=0.91n=23

dial

ysis

[%

60

f u(m

ic)

20

40

0 20 40 60 80 1000

fu(mic) TRANSIL [%]

32






33

Application:Pl Bi diPlasma Binding

• Only the unbound drug fraction penetrates • Only the unbound drug fraction penetrates tissues

• High population differences in plasma binding

• Plasma composition variable• Plasma composition variable

34

TRANSIL HSA BindingTRANSIL HSA Binding

35

TRANSIL HSA BindingTRANSIL HSA Binding

36

TRANSIL HSA & AGP Di l iTRANSIL HSA & AGP vs Dialysis

100Li

tera

ture

100

N=71r2=0.8

ound

[%] -

10

ctio

n un

bo

1

fraction unbound in TRANSIL [%]0.1 1 10 100

fra

0.1

37

fraction unbound in TRANSIL [%]

Physiological Variation in AGP C t tiAGP Concentration

3 - Severe Trauma3 - Severe Trauma

Major SurgeryTrauma

2 - Mycaridal Infarction

Lung CancerRenal Failure

1 -

norm

al

Renal Failure

0 -n

Neonate Hepatic Cirrhosis

U t 15 f ld i

Piafsky et al. (1978): New England J. of Medicine 299:1435-1439

Up to 15-fold increase

38

TRANSIL vs DialysisTRANSIL vs Dialysis

Features TRANSIL Dialysis

Test items Small and large molecules Small moleculesTest items Small and large molecules Small molecules

Incubation time 12 minutes 5-16 h

Biological matrix immobilized full tissue extract

Compounds per day 192 / 768 29Compounds per day 192 / 768 29

Issues metabolism, instrument down-time

Labor requirement 5 min 100 min

39

SSummary

• TRANSIL assays are ready-to-use assay kits fory y y– Brain tissue binding– Brain-to-plasma distribution prediction

Liver microsomal binding– Liver microsomal binding– Plasma protein binding

• All TRANSIL Assays are highly defined and reproducible

• Intrinsic quality control• Matrix free fast & easy high-throughput assay• Matrix free, fast & easy high throughput assay

– Minimal labor requirements through automation– Multiplexing capability

Hi hl t ff ti– Highly cost effective

40

Contact Details

Sovicell GmbHSovicell GmbHDeutscher Platz 5b04103 LeipzigGermanyGermanyTel: +49-341-52044-0Fax: +49-341-52044-12hboriss@sovicell [email protected]

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automation of transil assays outperforms traditional ... · pdf fileautomation of transil...

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