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

1 CONFIDENTIALCONFIDENTIAL

A BIT OF ADME FOR EVERYONE:

LogD, SOLUBILITY, PERMEABILITY

AND

THE UNIVERSAL DETECTION SYSTEM


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


The Miracle of a Drug

1 Inducible2 Active transport increases permeability of some compounds3 Intestinal re-uptake of some compounds4 Drug-drug interaction potential

Stomach Intestine

Stability pH 2 enzymatic

Solubility pH 3-8

Stability pH 3-8 enzymatic

StabilityPhase I4

Organ Cell

Permeability2

Passive Pgp Efflux4

(Organ Specific)

Permeability2

Passive Pgp Efflux4

CellularPartitioning

Distribution(whole body)

Blood

Permeability2

(epithelium) Passive pH 3-8 Pgp Efflux4

StabilityEnzymatic

ProteinBinding4

Stability1

Phase I4

Phase II

Biliary Clearance3

UrinaryClearance

Portal Vein

LiverKidney

After Susan PetuskyWyeth Research


ADME in Discovery: Many Strategies (1)

Purity

LogD

Solubility

Permeability

Sequential

•Data integrity

• Compound conservation

• Too slow


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


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


Lipophilicity in Absorption

Navia MA, et.al., DDT 1, (5) May 1996


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


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


A Brief Reminder: LogD is Difficult

Buffer Composition pH LogD0.01 M Universal Buffer1 7.4 0.015 0.0090.01 M K-Phosphate Buffer 7.4 0.010 0.0070.01 M Na-Phosphate Buffer + 0.15 M NaCl 7.4 0.632 0.0090.01 M Universal Buffer1 + 0.15 M NaCl 7.4 0.697 0.0110.10 M K-Phosphate Buffer 7.4 0.981 0.0080.01 M Universal Buffer1 11.0 1.147 0.0040.10 M K-Phosphate Buffer 11.0 1.530 0.0020.07 M Na-Bicarbonate Buffer 11.0 2.440 0.010

Compound: Propranolol1 – Universal buffer is composed of a mixture of acetic, phosphoric, and boric acids with NaOH


ADW:LogD Assay


Validation: LogD Accuracy

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

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


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


Key Differentiating Factor

Elemental Concentration:CarbonNitrogen

...

Static Light Scattering

Absorbance

Evaporative LightScattering

No standards / calibration

No standards / calibration

Compound specific

• Are you measuring the actual concentration?

Elemental:Yes, directly

Absorbance:Yes, indirectly

Turbidity:No, solubility is inferred from dilution factor off a standard


Saturated solutionsPhosphate buffer, pH 111 week incubation

Left to Right:

ChlorpromazineHCl Bendroflumethiazide Clofazimine Bifonazole ThioridazineHCl TriflupromazineHCl Nifedipine Perphenazine PromazineHCl

Can You Tell The Saturated Solution?


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


Permeability: The Real Thing

Lipid bilayer (10,000,000 X)

“The Machinery of Life”David GoodsellCopernicus (Springer-Verlag)

Gap junction (1,000,000 X)


Two Choices

Measure average

concentrations

Bilayer chemistry Transport physics Experimental details

Calculate permeability

Experimental database

Structural parameters

Establish relationship

Measure structural

parameters

Plug into relationship

Calculate permeability


PAMPA: Stirring + Acceptor Sink

Drug 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)


Data for BBB Permeation

# CNS Compound

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

# CNS Compound

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

# CNS Compound

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


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


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)


• 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

215.04 12.56*log 0.4* * ( )

( ) exp( ) /(1 exp( ))

A D LogD N CH

P CNS A A

=- + -

+ = +


P(CNS+)

-20

0

20

40

60

80

100

120

0

1

2

3

4

5

05

1015

2025

P(C

NS

+)

LogD

(7.4

)

N(CH2)


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


1 Year Precision Data: Solubility Controls

0

100

200

300

400

500

600

12/29/2004 2/17/2005 4/8/2005 5/28/2005 7/17/2005 9/5/2005 10/25/2005 12/14/2005

Assay Date

uM

harmine imipramine sulfamethizole


1 Year Precision: LogD Controls

-3

-2

-1

0

1

2

3

4

1/3/2005 2/22/2005 4/13/2005 6/2/2005 7/22/2005 9/10/2005 10/30/2005 12/19/2005

log

D

harmine imipramine sulfamethizole


1 Year Statistics

LogD Solubility

Plate TotalAggreg. rate, %

Total failure rate, %

Net 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


Universal Detection System – UDS

Overview


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


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


UDS Architecture

Agilent 1100PDA

Antek 8060

AgilentChemStation

AnalizaNDCS

Agilent 35900EADC

Sample

N Gain, AZ

N s

igna

l(a

nalo

g)

N ra

w

data

PD

A

data Analiza CTRL

PC

Other Agilent1100 modules


Development System – Hardware


Development System – SW


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)


UDS Sequence Table – After Run


Universal Calibration Example

Area Under The Curve

# mg/ml ppmN 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 0.721 389 399 393 394

4 0.005 1.443 802 803 786 797

5 0.01 2.886 1617 1629 1608 1618

6 0.025 7.214 4284 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 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

ppm Nitrogen

0.1 1 10 100 1000 10000

Are

a U

nder

the

Cur

ve

1e+1

1e+2

1e+3

1e+4

1e+5

1e+6

1e+7


Universal Calibration – UDS Screen


UDS Performance Tests

Sample S mg/ml ppmN Inj. #1

Inj. #2

Inj. #3

Inj. #4

Avg. %

AC 0.025:0.0

5

A 0.025 2.3163 2.269 2.267 2.31 2.284 2.28 -1.5

C 0.05 14.4281

14.79 14.78 14.89 14.77 14.81 2.6

AC 0.01:1 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

AC 0.1:10 A 0.09804

9.0835 9.362 9.358 9.265 9.26 9.31 2.5

C 9.8039 2829.04

2881.7 2892.7 2889.8 2869.6 2883.4 1.9

A – Acetaminophene C - Caffeine


UDS Report Screen


Thank You

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

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