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ADME SOLUTIONSADME SOLUTIONS

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CORE TECHNOLOGIES/PRODUCTS

ADME • Line of automated workstations for drug screening in discovery and development.

• Installed base at big pharma, strong current interest, clear product expansion route.

StructuralSignature

Technology

• Breakthrough technology for rapid assessment of structural changes to proteins.

• Very significant potential application base in both biotech and pharma. Initial commercial feasibility underway.

Proteomics • First true adjustable third dimension.

• Studies in clinical marker discovery underway.

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Technologies

University of Alabama at Birmingham

Macromolecular Crystallization Center 1918 University Avenue South Birmingham, AL 35206-0005 USA Telex (205) 934 - 3841 Fax (205) 934 - 0480

Analiza Nano Crystallization System (ANCS) Operational Manual

World Leader in Structure Based Drug Design Version 1.3

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Spectrum of Solutions

Automated Discovery Workstation

Automated Solubility Workstation

Automated LogD Workstation

Solubility

LogD

Metabolic Stability

Plasma Protein Binding

Permeability

Blood-Brain Barrier Permeability

Solubility LogD

Permeability

Included

Option

Available

R&D Completed

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ADME in Discovery and Development (1)

• Discovery:– Objective: Fail hits early

– Samples: Dry and DMSO-dissolved

– Throughput: Up to 200 samples/day (typically)

– Accuracy: (Supposedly) not high – rank order is sometime sufficient

• Development:– Objective: Optimize lead (better ADME parameters),

salt & excipient selection, QSAR, etc.

– Samples: Dry

– Throughput: Typically up to 100/day, but increasing

– Accuracy: High, must use gold-standard validated assays

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ADME in Discovery and Development (2)

• State-of-the-art today:– Discrete instruments, each performing a single assay– Data consistency between different assays non-existent– Significant interaction required from trained personnel– No process engineering methodologies:

• When assay is not required, the system is idle• When a system is down, there is no replacement• To double the capacity, each system must be duplicated

• Automated Discovery Workstation:

The modern approach to the entire lab

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Automated Discovery Workstation - ADW

• Not an instrument: It is an integrated solution (lab-on-a-bench)

• Machine work – people think

• Turnkey, automated, multi-assay workstation

• Performs (presently) discovery/development assays:– True LogD/LogP vs. pH

– True aqueous solubility vs. pH

– Identity and purity of compound

– Compound concentration w/o standard

• Scalable, Flexible, Extendable technology

• Standardized, validated data

same sample

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ADW - Automated Discovery Workstation

ANALIZA’sIn-house DevelopmentSystem (2002)

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Two Key ADME Assays1. LogD/LogP

• P is the partition coefficient between octanol and a buffer, that is obtained under equilibrium

• Important for multiple ADME processes in discovery and development, and for QSAR lead optimization

• Very extensive database exists well validated method

• Direct LogP assay (shake flask = gold standard):

Octanol sat. w/buffer

Buffer sat. w/octanol

Phases

Add solute

Mix SettleSample andanalyze

Ctop

Cbot

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ADW:LogD/LogP Assay

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Two Key ADME Assays2. Solubility

• Solubility is the concentration of the compound in solution in equilibrium with its solid

• Important for bioavailability and other ADME processes

• Very sensitive to measurement methodology

• Current profiling solubility indicators are frequently different from the true values

• Direct solubility assay (shake flask = gold standard)

• Unique features:– No sample weighting

– No assay calibration required to calculate solubility as mass/volume

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ADW:Solubility Assay

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Validation: LogD Accuracy

LogDADW = 0.002( 0.008) + 1.011( 0.005)*LogDmanual

N = 179; r2 = 0.9960; standard error of estimate = 0.1022

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Validation: Solubility Accuracy

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Validation: Precision

Run #

0 5 10 15 20

Sol

ubili

ty,

mg/

ml

0.0

0.1

0.2

0.3

0.4

0.5

Average = 0.264Upper Limit = 0.283

Lower Limit = 0.245

Allopurinol in 0.15M NaCl in 0.01M Universal buffer, pH 6.6

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ADW Control and Analysis Software

ADWSoftware

Turnkey software

Com prehensive

M ouse-clickhardware control

Graphical assaysetup

Autom ation dataanalysis

Graphical datarepresentation tools

Technician-levelcom plexity

ADWData

Viewer

Archived raw data

Browsing capabilities

Network connectivity

Stand-alone code

Re-analyzecapabilities

Graphical datarepresentation and

m anipulation

Query original assaysetup param eters

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LogD Assay: Main screen with optional information windows

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Solubility Assay: Setup, imported data

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Data Viewer: Full featured archival data re-display andre-analyze GUI, which runs from any computer on the network

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Data Viewer: Display of archived raw PDA data – sample spectraYou Never Lose Your Data!

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ADW Hardware

• Robotic liquid handler (Hamilton 4200, four computer-controlled syringes, custom valves).

• Custom computer-controlled valve system (Rheodyne).• UV/VIS photo-diode array spectrophotometer (Groton

Technologies)• Chemiluminescence total nitrogen detector (Antek

Instruments)• Custom electronics, bar-code reader, and high-end control

computer• Vacuum and HPLC pumps• Deckware: plate and filter holders, buffer racks, etc.

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LogD• Customized plate vortexer• Customized plate sealer• Refrigerated plate centrifuge

Solubility• Custom filter plate holders• Customized filter plate presser• Customized shaker• Buffer containers holder• Temperature option (heated shaker, on-deck water bath )

ADW Hardware: Assay Specific

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ADW SuppliesConsumables:• LogD systems:

– Loaded in 96 deep well plates, bar-coded, QC’ed– Ready-to-use in 6 standard pH levels

• Solubility:– Filter vials

Optional/Maintenance:• Nitrogen-detector calibration plates• Extra filter plates• Repair kits, partsAlways Available:A direct hot-line to our scientists and system developers – you

always get a timely answer, advice, and dedicated service

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ADW Key Specifications

ATTRIBUTE ASSAY VALUESample Requirement LogD

Solubility

Dry DMSO-dissolved

150 µl

0.1 mg*

2 µl*

Range LogD

Solubility

-3<LogD<+4**

1 µg/ml lower rangeNo upper range

Throughput LogD

Solubility

~ 100/day**

~250/day**

* Typical for 1-50 µg/ml range. Proportional with required upper range** Depending on user-selected settings and compound

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The ADW Solution Vs. CompetitionAttribute ADW Competition

Turnkey solution Yes Typically No

Number of Assays 5 - small incremental cost per additional assay retains capital investment

1

Assay Methodology Direct gold standard assaysTrue data only

Typically indirect – limited applicability range and

potential incompatibility with development data

Degree of Automation High – Typical user interface is about 1 hr. per 24 hr. operation

Varied – Typically requires extensive interaction

Operator Level Technician Varied – Typically a scientist

Archival Database Unique, enterprise-based, web-shared, graphical raw data and analysis tool set

Typically final data reporting

Flexibility Yes – Reconfigurable easily between assays = full workstation utilization

No – When the specific assay is not required, the

workstation is idle

Process EngineeringMethodology

Yes – Modular, extendable,production-line approach

No – Every instrumentperforms one assay

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ADW: The Integrated ADME EnvironmentAssay Description Estimated Time to Market

Library Integrity(Discovery)

• Identity (LC/MS)• Purity (PDA, LC/MS)• Concentration (N-det)

• 6 months• Available, 6 months• Available

Solubility• True (dry)• Kinetic (DMSO)

• Available• Available

LogD True (DMSO) Available

Metabolic Stability Standard LC/MS assay 6 months

Permeability Novel high throughput assay • LogP & PSA based 3 months• Surface tension based 1 year

Plasma Protein Binding• Microdialysis • Novel direct free/bound assay

• 12 months• 18 months

Blood-Brain Barrier• LogD/LogK• Novel high throughput assay

• Available (software 3 months)• 12 months

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AUTOMATED SOLUBILITY WORKSTATION

ASolW in Pre-Formulation/Development

All Slides Confidential

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Solubility

• Solubility is the concentration of the compound in solution in equilibrium with its solid

• Solubility takes several forms and is a function of multiple parameters (crystalline form, temperature, pH, co-solvent, etc.)

• Important for bioavailability• Very sensitive to measurement methodology• Current profiling solubility indicators in early discovery are

frequently different from the true values obtained during development

• A direct solubility assay is defined by the shake flask method and is recognized as the gold standard

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• Discovery:– Obtain a measure of solubility of relevance to HTS and

early ADMET• Can we determine if the compound will crash out of solution?

– Obtain ADMET information of relevance for selecting amongst hits

• Development:– Early lead characterization

– Lead optimization

– Formulation excipient selection

Who Are The Users?

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Who Are The Users? (2)

• Will the compound crash out? solubility

• Everyone else needs good data:

– For promoting a compound: binning may be sufficient

– For lead optimization: accurate data

– For formulation development: accurate data

– For anything regulatory: accurate data

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Alternative Techniques for Measuring SolubilityA Comparison (1/2)

Feature Absorbance Light Scattering ASolW

True Solubility – dryYes/No

Kinetic Solubility – DMSO Yes/No

Shake Flask Method Yes/No

Automation Yes/No

Measure Solubility:Direct/Indirect

Standard Curve:Required/Not required

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Alternative Techniques for Measuring SolubilityA Comparison (2/2)

Feature Absorbance Light Scattering ASolW

Multiple pH Levels Yes/No

Multiple TemperaturesYes/No

Multiple Co-SolventsYes/No

Timed Sampling Yes/No

Additional Steps:Pre-concentration,Dilution curves

Rely on:Chromophores

Rely on:Scattering definition

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The ASolWAssay

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ASolW with Whatman’s MiniPrep Filter

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Options for Measuring Solubility

ASolW:

Dedicated Instrument

ADW:

Multi-Assay Instrument

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ASolW – Automated Solubility Workstation

ANALIZA’s ASolW

In-house development system

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ASolW: Assay setup

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ASolW: Data Check

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ASolW: Runtime Panel

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ASolW: Accuracy

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ASolW: Precision

Run #

0 5 10 15 20

Sol

ubili

ty,

mg/

ml

0.0

0.1

0.2

0.3

0.4

0.5

Average = 0.264Upper Limit = 0.283

Lower Limit = 0.245

Allopurinol in 0.15M NaCl in 0.01M Universal buffer, pH 6.6

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Total Nitrogen Detection

• Exceptions:– N2 – nothing is detected!

– Nitrosamine (for GC)

– Azides (1/3 response)

– Double bonds (partial response)

)900600(2*23

1050

2

nmhONOONO

productsNOONR

Separate

calibration

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ASolW: Assay Using Nitrogen Content

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Basic Issue:What is the Effect of DMSO?

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Basic Issues:What is the Effect of DMSO?

Solubility(Kinetic)= 0.0605+/-0.012 + 1.017+/-0.034*Solubility(true)Compounds: Bendroflumethiazide, Butamben, Nitrofurazone, Perphenazine, Phenacetin

"True" experimental solubility, Sexp, mg/ml

0.0 0.2 0.4 0.6 0.8 1.0Kin

etic

So

lub

ility

in th

e p

rese

nce

of 1

% D

MS

O, m

g/m

l

0.0

0.2

0.4

0.6

0.8

1.0

Trimethoprim

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Basic Issue:What is the Effect of DMSO?

Typically:• DMSO is a fact of life during screening• DMSO solubility is > or >> than true solubility• But just because we relaxed one experimental

parameter does not mean that we can dispense with everything else

• Make sure you know what you are using the data for – especially in lead promotion decisions

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ASolW Hardware

• Robotic liquid handler (Hamilton 4000, two computer-controlled syringes, custom valves).

• Custom computer-controlled valve system (Rheodyne).• Chemiluminescence total nitrogen detector (Antek

Instruments CLND 8600)• Custom electronics, bar-code reader, and high-end control

computer.• Custom filter plate compressor• 4-filter plate shaker/heater incubator• Miscellaneous accessories: Vacuum pumps, HPLC pump• Deck accessories: Filter plates, buffer racks

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ASolW Options, Kits, and Consumables

Options:

• Temperature option (to 37 deg.C)

Kits:

• Starter filter pack (included)

• Maintenance kit

• Additional filter plates, buffer/co-solvent bottles, etc.

Consumables:

• Filter packs

• Nitrogen-free buffers

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Why ADME with ANALIZA?

• We are experienced• We provide innovations with gold-standard assays• We don’t just automate• We report archival-quality data while minimizing

material consumption and at high throughput• We guard your investment and offer flexibility and

extendibility to our workstations• We pride ourselves with a personal, responsive, first-

class organization before and after the installation

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Thank You