A BIT OF ADME FOR EVERYONE Log D

A BIT OF ADME FOR EVERYONE: Log. D, SOLUBILITY, PERMEABILITY AND THE UNIVERSAL DETECTION SYSTEM 1 CONFIDENTIAL

OUTLINE • ADMET in modern discovery • A brief refresher: – Log. D, Solubility, Permeability – Issues, methods, tradeoffs • More interesting ADME assays: – Caco/PAMPA-less BBB permeability • The Universal Detection System - UDS 2 CONFIDENTIAL

The Miracle of a Drug Stomach Intestine Solubility p. H 3 -8 Stability · p. H 2 · enzymatic Stability Phase I 4 Portal Liver Vein Stability 1 · Phase I 4 · Phase II Blood Protein Binding 4 Stability · p. H 3 -8 · enzymatic Urinary Clearance 2 3 4 3 Inducible Active transport increases permeability of some compounds Intestinal re-uptake of some compounds Drug-drug interaction potential CONFIDENTIAL Cell Distribution Cellular (whole body)Partitioning Stability Enzymatic Permeability 2 Biliary 3 (epithelium) Clearance Passive · p. H 3 -8 · Pgp Efflux 4 1 Organ Kidney Permeability 2 · Passive 4 · Pgp Efflux (Organ Specific) After Susan Petusky Wyeth Research

ADME in Discovery: Many Strategies (1) Sequential Purity • Data integrity • Compound conservation Log. D Solubility • Too slow Permeability 4 CONFIDENTIAL

ADME in Discovery: Many Strategies (2) Weighted Score Purity Log. D 2. 2 0. 85 Solubility 10 Permeability 10 -5 Metabolism Protein binding 5 85% CONFIDENTIAL • Holistic 4 3. 4 0. 5 2 1. 0 30% 0. 75 5 3. 75 99% 0. 9 1 0. 9 9. 05

Log. P/Log. D (1) • Equilibrium partition coefficient between 1 -octanol/buffer • Log. P – non-ionized compound • Log. D – ionized compound – f(p. H) • Good Absorption characteristics: – Log. P ~ 2. 5 6 CONFIDENTIAL

Lipophilicity in Absorption Navia MA, et. al. , DDT 1, (5) May 1996 7 CONFIDENTIAL

Log. P/Log. D (2) • Log. D > 5: – Tough to measure – Promiscuous binders – Poor solubility, oral absorption – Strong CYP 450 interaction • Log. D 0 -3: – Best balance of solubility, permeability • Log. D < 0: – Good solubility, poor permeability 8 CONFIDENTIAL

Measuring Log. D • The real thing: Shake flask method • RP HPLC: – See K. Valko, J. Chrom. Sci, 1037 (2004) • p. H-metric • Microemulsion electrokinetic chromatography • Calculating: – ACD Log. P – CLog. P – Prolog. D 9 CONFIDENTIAL

A Brief Reminder: Log. D is Difficult Compound: Propranolol 1 – Universal buffer is composed of a mixture of acetic, phosphoric, and boric acids with Na. OH 10 CONFIDENTIAL

ADW: Log. D Assay 11 CONFIDENTIAL

Validation: Log. D Accuracy Log. DADW = 0. 002(± 0. 008) + 1. 011(± 0. 005)*Log. Dmanual N = 179; r 2 = 0. 9960; standard error of estimate = 0. 1022 12 CONFIDENTIAL

A Simple Concept • Solubility = Concentration of a dissolved compound in equilibrium with its solid • But: – Which solid? • Equilibrium (most stable form) vs. apparent (other forms) – Which solvent? • Buffers (intrinsic? ) and co-solvents (kinetic) – Which equilibrium? • Time (kinetic) and temperature 13 CONFIDENTIAL

Key Differentiating Factor • Are you measuring the actual concentration? Elemental: Yes, directly Absorbance: Yes, indirectly Turbidity: No, solubility is inferred from dilution factor off a standard 14 CONFIDENTIAL

Can You Tell The Saturated Solution? Saturated solutions Phosphate buffer, p. H 11 1 week incubation Left to Right: Chlorpromazine. HCl Bendroflumethiazide Clofazimine Bifonazole Thioridazine. HCl Triflupromazine. HCl Nifedipine Perphenazine Promazine. HCl 15 CONFIDENTIAL

Assay Effects: Particles • Solid particles are an integral part of the solubility assay – Particles are always present – They must be present for turbidity to work – They are artifacts in absorbance/elemental assays • The effects of particles on the data must be considered when examining the data – Subtle to substantial influence on quality of results 16 CONFIDENTIAL

Permeability: The Real Thing Lipid bilayer (10, 000 X) “The Machinery of Life” David Goodsell Copernicus (Springer-Verlag) 17 CONFIDENTIAL Gap junction (1, 000 X)

Two Choices Measure average concentrations Calculate permeability Bilayer chemistry q Transport physics q Experimental details q q Experimenta l database q 18 Structural parameters CONFIDENTIAL Establish relationship Measure structural parameters Plug into relationship Calculate permeability

PAMPA: Stirring + Acceptor Sink Drug 19 CONFIDENTIAL Scavenger Drug + Scavenger

BBB Permeability: An Alternative Approach* • Instead of: – Trying to find the “ultimate” membrane model – Deciphering permeability from a complex experiment • Use real-world in vivo data + structural descriptors: – What prediction level is necessary? – Which descriptors are useful? *Gulyaeva et. al, EJMC 38 (2003) 21 CONFIDENTIAL

Data for BBB Permeation +: log. BB > 0. 3 --: log. BB < -1. 0 # CNS Compound 1 - Acyclovir 23 - Tiapride 44 + Mequitazine HCl 2 - Acetaminophen 24 + Amitriptiline HCl 45 + Minaprine 3 - Albendazole 25 + Chlorpromazine HCl 46 + Mefexamide 4 - Albuterol 26 + Clomipramine 47 + Naltrexone HCl 5 - Ampicillin 27 + Clonidine HCl 48 + Naloxone 6 - Antipyrine 28 + Desipramine HCl 49 + Nortriptyline 7 - Astemizole 2 HCl 29 + Doxepin 50 + Perphenazine 2 HCl 8 - Atenolol 30 + 51 + Physostigmine 9 - Cimetidine Doxylamine succinate 52 + Progesterone 31 + Estrone 53 + Promazine HCl 32 + Fluoxetine HCl 54 + Promethazine HCl 33 + Flupentixol 2 HCl 55 + Propranolol HCl 34 + Fluphenazine 56 + Protriptyline 35 + Haloperidole HCl 57 + Pyrilamine 36 + Homochlorcyclizine 58 + Thioridazine HCl 37 + Hydroxyzine 2 HCl 59 + Tranylcypromine 60 + Trazodone 61 + Trifluoperazine 62 + Triflupromazine 63 + Trimipramine 10 11 12 13 14 15 -* - Domperidone HCl Ebastine 5 -Fluorouracil Ftorafur Furosemide 5 Hydroxytryptophane 16 Iproniazid 38 + Ibuprofen 17 - Metoclopramide 39 + Imipramine HCl 18 - Metoprolol 40 + Indomethacin 19 - Metronidazole 41 + Lidocaine HCl 20 - Phenelzine 42 +* 21 22 - - Pirenzepine HCl CONFIDENTIAL Loperamide HCl

Structural Descriptors (1) • Basic Premise: – Only structural differences matter – since compounds are predominantly different by structure • Key Methodology: – Ask for minimally acceptable answer: • Must you know a number or will a classification suffice? – Use smallest number of descriptors: • Principle of parsimony 23 CONFIDENTIAL

Structural Descriptors (2) • Log. D(7. 4): – Measure of relative affinity between polar and non-polar media – Obtained from experiments or calculations • N(CH 2): – Measure of relative affinity between two aqueous solvents of different structures – Obtained using aqueous two-phase partitioning experiments (Dex-PEG systems) 24 CONFIDENTIAL

Predictive Equation • Predict what? – Probability of a compound being CNS+ • Predict how? – Logistic regression model using penalized maximum likelihood (nested models) or information criteria (non-nested models) • Predictive accuracy: – 96. 6% using cross validation procedures (one point out of the model for all 63 data points) 25 CONFIDENTIAL

P(CNS+) 26 CONFIDENTIAL

Typical Big Pharma - ANALIZA Collaboration • Log. D (p. H =7. 4) • Solubility (p. H = 6. 5) • Thermodynamic solubility (dry) • Over 21, 000 data points in 2005 • Real data: high throughput, miniaturized shake flask methods 27 CONFIDENTIAL

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1 Year Statistics Log. D Solubility Plate Total Aggreg. rate, % Total failure rate, % Net failure rate, % A 3, 811 5. 8 6. 1 0. 3 0. 7 B 3, 781 6. 4 6. 9 0. 5 0. 7 C 2, 758 2. 5 3. 0 0. 5 D 2, 833 5. 6 6. 3 0. 7 30 CONFIDENTIAL

Universal Detection System – UDS Overview 31 CONFIDENTIAL

The Need • We have automated ID solutions: MS, etc. • Pharmaceutical compounds in discovery/development are impure: – Combichem libraries are 90 -95% pure – Compounds synthesized during LD are 90+% pure – Serious implications for: • Early ADME evaluation (e. g. , solubility) • Late selection and lead optimization • Process development and FDA submissions • Impurity quantification is a vexing problem: – Impurity has to be separated – Standards must be prepared – Very difficult in practice! • Highly desirable: – Automated quantification method for all impurities in a sample w/o standards 32 CONFIDENTIAL

Unique Advantages • Orthogonal detection methodology to Ultra Violet (UV) or Mass Spectrometry (MS) • Equimolar detection for instrument universal calibration curve • Wide applicability to most pharmaceutical compounds (93% +) • Large intrinsic dynamic range (ca. 0. 1 -50, 000 ppm nitrogen) • Complete automation: – On-board separation capabilities with individual peak concentrations – Peak zoom capability to focus on assaying of minor peaks – Automated optimization with variable instrument gain and injection volume 33 CONFIDENTIAL

UDS Architecture Other Agilent 1100 modules PC Agilent Chem. Station 34 CONFIDENTIAL Agilent 35900 E ADC N (a sign na a log l ) Sample Antek 8060 Analiza CTRL N da raw ta PDA data Agilent 1100 PDA N Gain, AZ Analiza NDCS

Development System – Hardware 35 CONFIDENTIAL

Development System – SW 36 CONFIDENTIAL

UDS Operating Modes • Calibration – Construct universal piece-wise linear curve • Quantification – Noise A(gressive) – Noice C(onservative) – P(arent) P(eak) G(ain) – P(arent) P(eak) A(rea) – O(ptimize) V(olume) 37 CONFIDENTIAL

UDS Sequence Table – After Run 38 CONFIDENTIAL

Universal Calibration Example Area Under The Curve # mg/ml ppm. N Inj. #1 Inj. #2 Inj. #3 Avg. 1 0. 0005 0. 144 113 114 96 108 2 0. 001 0. 289 198 163 171 177 3 0. 0025 0. 721 389 393 394 4 0. 005 1. 443 802 803 786 797 5 0. 01 2. 886 1617 1629 1608 1618 6 0. 025 7. 214 4283 4255 4274 7 0. 05 14. 428 8871 8818 8866 8852 8 0. 1 28. 856 18549 18502 18340 18463 9 0. 25 72. 141 47908 47484 47625 10 0. 5 144. 281 97254 96869 95795 96639 11 1 288. 563 193570 193361 193109 193347 12 2. 5 721. 407 460138 462725 466960 463274 13 5 1442. 814 849204 841916 852793 847971 14 10 2885. 627 1494800 1486750 1476160 1485903 39 CONFIDENTIAL

Universal Calibration – UDS Screen 40 CONFIDENTIAL

UDS Performance Tests Sample S mg/ml AC 0. 025: 0. 0 5 A Inj. #2 0. 025 2. 3163 2. 269 2. 267 2. 31 2. 284 2. 28 -1. 5 0. 05 14. 428 1 14. 79 14. 78 14. 89 14. 77 14. 81 2. 6 A 0. 0083 0. 7721 0. 723 0. 668 0. 712 0. 703 0. 70 -9. 1 C 0. 8333 240. 47 240. 7 238. 25 239. 32 238 239. 07 -0. 6 A 0. 09804 9. 0835 9. 362 9. 358 9. 265 9. 26 9. 31 2. 5 C AC 0. 1: 10 Inj. #1 C AC 0. 01: 1 9. 8039 2829. 0 4 2881. 7 2892. 7 2889. 8 2869. 6 2883. 4 1. 9 A – Acetaminophene 41 CONFIDENTIAL C - Caffeine Inj. #3 Inj. #4 Avg. % ppm. N

UDS Report Screen 42 CONFIDENTIAL

Thank You 43 CONFIDENTIAL