A BIT OF ADME FOR EVERYONE: LogD, SOLUBILITY, PERMEABILITY AND THE UNIVERSAL DETECTION SYSTEM

1. A BIT OF ADME FOR EVERYONE:LogD, SOLUBILITY, PERMEABILITYANDTHE UNIVERSAL DETECTION SYSTEM

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

3. Blood Organ Cell Stomach Intestine Kidney Portal Vein Liver • Stability1 • Phase I4 • Phase II Stability Phase I4 Solubility pH 3-8 Protein Binding4 Urinary Clearance Distribution (whole body) Cellular Partitioning • Stability • pH 2 • enzymatic • Stability • pH 3-8 • enzymatic Stability Enzymatic Biliary Clearance3 • Permeability2 • Passive • Pgp Efflux4 • (Organ • Specific) • Permeability2 • Passive • Pgp Efflux4 • Permeability2 • (epithelium) • Passive • pH 3-8 • Pgp Efflux4 The Miracle of a Drug 1 Inducible 2 Active transport increases permeability of some compounds 3 Intestinal re-uptake of some compounds 4 Drug-drug interaction potential After Susan PetuskyWyeth Research

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

5. ADME in Discovery: Many Strategies (2) • Weighted Score • Holistic  85% Purity 0.85 4 3.4 2.2 LogD  10 Solubility 9.05 10-5 0.5 2 1.0 Permeability 30% 0.75 5 3.75 Metabolism 0.9 1 0.9 99% Protein binding

6. LogP/LogD (1) • Equilibrium partition coefficient between 1-octanol/buffer • LogP – non-ionized compound • LogD – ionized compound – f(pH) • Good Absorption characteristics: • LogP ~ 2.5

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

8. LogP/LogD (2) • LogD > 5: • Tough to measure • Promiscuous binders • Poor solubility, oral absorption • Strong CYP450 interaction • LogD 0-3: • Best balance of solubility, permeability • LogD < 0: • Good solubility, poor permeability

9. Measuring LogD • The real thing: Shake flask method • RP HPLC: • See K. Valko, J. Chrom. Sci, 1037 (2004) • pH-metric • Microemulsion electrokinetic chromatography • Calculating: • ACD LogP • CLogP • PrologD

10. A Brief Reminder: LogD is Difficult Compound: Propranolol1 – Universal buffer is composed of a mixture of acetic, phosphoric, and boric acids with NaOH

11. ADW:LogD Assay

12. Validation: LogD Accuracy LogDADW = 0.002(± 0.008) + 1.011(± 0.005)*LogDmanualN = 179; r2 = 0.9960; standard error of estimate = 0.1022

13. 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

14. 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

15. Can You Tell The Saturated Solution? Saturated solutionsPhosphate buffer, pH 111 week incubation Left to Right: ChlorpromazineHCl Bendroflumethiazide Clofazimine Bifonazole ThioridazineHCl TriflupromazineHCl Nifedipine Perphenazine PromazineHCl

16. 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

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

18. Two Choices Measure average concentrations Calculate permeability • Bilayer chemistry • Transport physics • Experimental details Measure structural parameters Plug into relationship • Experimental database • Structural parameters Establish relationship Calculate permeability

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

20. 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. Data for BBB Permeation +: logBB > 0.3--: logBB < -1.0

22. 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. Structural Descriptors (2) • LogD(7.4): • Measure of relative affinity between polar and non-polar media • Obtained from experiments or calculations • N(CH2): • Measure of relative affinity between two aqueous solvents of different structures • Obtained using aqueous two-phase partitioning experiments (Dex-PEG systems)

24. 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. P(CNS+)

26. Typical Big Pharma - ANALIZA Collaboration • LogD (pH =7.4) • Solubility (pH = 6.5) • Thermodynamic solubility (dry) • Over 21,000 data points in 2005 • Real data: high throughput, miniaturized shake flask methods

29. 1 Year Statistics

30. Universal Detection System – UDSOverview

31. 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. 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. UDS Architecture Other Agilent 1100 modules Agilent 1100 PDA Antek 8060 Sample N signal (analog) Agilent 35900E ADC PDA data Analiza CTRL N raw data PC Agilent ChemStation Analiza NDCS N Gain, AZ

34. Development System – Hardware

35. Development System – SW

36. 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. UDS Sequence Table – After Run

38. Universal Calibration Example

39. Universal Calibration – UDS Screen

40. UDS Performance Tests A – Acetaminophene C - Caffeine

41. UDS Report Screen

42. Thank You