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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
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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