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

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Presentation on theme: "1 CONFIDENTIAL A BIT OF ADME FOR EVERYONE: LogD, SOLUBILITY, PERMEABILITY AND THE UNIVERSAL DETECTION SYSTEM."— Presentation transcript:

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

2 2 CONFIDENTIAL 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 3 CONFIDENTIAL 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 StomachIntestine Stability  pH 2  enzymatic Solubility pH 3-8 Stability  pH 3-8  enzymatic Stability Phase I 4 OrganCell Permeability 2  Passive  Pgp Efflux 4 (Organ Specific) Permeability 2  Passive  Pgp Efflux 4 Cellular Partitioning Distribution (whole body) Blood Permeability 2 (epithelium) Passive  pH 3-8  Pgp Efflux 4 Stability Enzymatic Protein Binding 4 Stability 1  Phase I 4  Phase II Biliary Clearance 3 Urinary Clearance Portal Vein Liver Kidney After Susan Petusky Wyeth Research

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

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

6 6 CONFIDENTIAL 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 7 CONFIDENTIAL Lipophilicity in Absorption Navia MA, et.al., DDT 1, (5) May 1996

8 8 CONFIDENTIAL 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 9 CONFIDENTIAL 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 10 CONFIDENTIAL A Brief Reminder: LogD is Difficult Compound: Propranolol 1 – Universal buffer is composed of a mixture of acetic, phosphoric, and boric acids with NaOH

11 11 CONFIDENTIAL ADW: LogD Assay

12 12 CONFIDENTIAL Validation: LogD Accuracy LogD ADW = 0.002(± 0.008) + 1.011(± 0.005)*LogD manual N = 179; r 2 = 0.9960; standard error of estimate = 0.1022

13 13 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

14 14 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

15 15 CONFIDENTIAL Saturated solutions Phosphate buffer, pH 11 1 week incubation Left to Right: ChlorpromazineHCl Bendroflumethiazide Clofazimine Bifonazole ThioridazineHCl TriflupromazineHCl Nifedipine Perphenazine PromazineHCl Can You Tell The Saturated Solution?

16 16 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

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

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

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

20 20 CONFIDENTIAL The “BCS” for Permeability Experiments HIGH DIFFUSIVITY HIGH PERMEABILITY HIGH DIFFUSIVITY LOW PERMEABILITY LOW DIFFUSIVITY HIGH PERMEABILITY LOW DIFFUSIVITY LOW PERMEABILITY GOOD ? BAD * Diffusivity = Bulk Transport

21 21 CONFIDENTIAL 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)

22 22 CONFIDENTIAL Data for BBB Permeation #CNSCompound 1-Acyclovir 2-Acetaminophen 3-Albendazole 4-Albuterol 5-Ampicillin 6-Antipyrine 7-Astemizole 2HCl 8-Atenolol 9-Cimetidine 10-*Domperidone HCl 11-Ebastine 12-5-Fluorouracil 13-Ftorafur 14-Furosemide 15-5- Hydroxytryptophan e 16-Iproniazid 17-Metoclopramide 18-Metoprolol 19-Metronidazole 20-Phenelzine 21-Pirenzepine HCl #CNSCompound 23-Tiapride 24+Amitriptiline HCl 25+Chlorpromazine HCl 26+Clomipramine 27+Clonidine HCl 28+Desipramine HCl 29+Doxepin 30+Doxylamine succinate 31+Estrone 32+Fluoxetine HCl 33+Flupentixol 2HCl 34+Fluphenazine 35+Haloperidole HCl 36+Homochlorcyclizine 37+Hydroxyzine 2HCl 38+Ibuprofen 39+Imipramine HCl 40+Indomethacin 41+Lidocaine HCl 42+*Loperamide HCl #CNSCompound 44+Mequitazine HCl 45+Minaprine 46+Mefexamide 47+Naltrexone HCl 48+Naloxone 49+Nortriptyline 50+Perphenazine 2HCl 51+Physostigmine 52+Progesterone 53+Promazine HCl 54+Promethazine HCl 55+Propranolol HCl 56+Protriptyline 57+Pyrilamine 58+Thioridazine HCl 59+Tranylcypromine 60+Trazodone 61+Trifluoperazine 62+Triflupromazine 63+Trimipramine +: logBB > 0.3 --: logBB < -1.0

23 23 CONFIDENTIAL 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

24 24 CONFIDENTIAL Structural Descriptors (2) LogD(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)

25 25 CONFIDENTIAL 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) Predictive Equation

26 26 CONFIDENTIAL P(CNS+)

27 27 CONFIDENTIAL 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

28 28 CONFIDENTIAL

29 29 CONFIDENTIAL

30 30 CONFIDENTIAL 1 Year Statistics LogDSolubility PlateTotal Aggreg. rate, % Total failure rate, % Net failure rate, % A3,8115.86.10.30.7 B3,7816.46.90.50.7 C2,7582.53.00.5 D2,8335.66.30.7

31 31 CONFIDENTIAL Universal Detection System – UDS Overview

32 32 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

33 33 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

34 34 CONFIDENTIAL UDS Architecture Agilent 1100 PDA Antek 8060 Agilent ChemStation Analiza NDCS Agilent 35900E ADC Sample N Gain, AZ N signal (analog) N raw data PDA data Analiza CTRL PC Other Agilent 1100 modules

35 35 CONFIDENTIAL Development System – Hardware

36 36 CONFIDENTIAL Development System – SW

37 37 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)

38 38 CONFIDENTIAL UDS Sequence Table – After Run

39 39 CONFIDENTIAL Universal Calibration Example Area Under The Curve #mg/mlppmNInj. #1Inj. #2Inj. #3Avg. 10.00050.144 11311496108 20.0010.289 198163171177 30.00250.721 389399393394 40.0051.443 802803786797 50.012.886 1617162916081618 60.0257.214 4284428342554274 70.0514.428 8871881888668852 80.128.856 18549185021834018463 90.2572.141 4790847484 47625 100.5144.281 97254968699579596639 111288.563 193570193361193109193347 122.5721.407 460138462725466960463274 1351442.814 849204841916852793847971 14102885.627 1494800148675014761601485903

40 40 CONFIDENTIAL Universal Calibration – UDS Screen

41 41 CONFIDENTIAL UDS Performance Tests SampleSmg/mlppmNInj. #1 Inj. #2 Inj. #3 Inj. #4 Avg. %% AC 0.025:0.05 A0.0252.31632.2692.2672.312.2842.28-1.5 C0.0514.428114.7914.7814.8914.7714.812.6 AC 0.01:1A0.00830.77210.7230.6680.7120.7030.70-9.1 C0.8333240.47240.7238.25239.32238239.07-0.6 AC 0.1:10A0.098049.08359.3629.3589.2659.269.312.5 C9.80392829.042881.72892.72889.82869.62883.41.9 A – Acetaminophene C - Caffeine

42 42 CONFIDENTIAL UDS Report Screen

43 43 CONFIDENTIAL Thank You


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