Low Quality Predictions (Or Measurements) Can Lead to ...€¦ · Low Quality Predictions (or...

Low Quality Predictions (or Measurements) Can Lead to

Erroneous Decisions

John ComerTechnical Director

Sirius Analytical Ltd.

Talk given at ACD Users Meeting, Obernai, France, 25 October 2007

/412© 2007

Who are Sirius?

UK-based instrument company We make instruments for measuring physicochemical properties– pKa

– logP– Solubility

We analyse samples sent by customersWe support these activities with scientific research– Over 50 publications– Many new methods introduced

We specialise in accurate measurement

www.sirius-analytical.com

/413© 2007

Sirius and ACD

We have a copy of ACD ChemSketch v 4.55, May 2000We use it for drawing chemical structuresDatabase is useful for structures of drugs, and referencesWe use it for predicting pKa, logP and solubility, before designing experiments to measure them– How many pKas?– Which are acids, which are bases?– Approximate values?– Predicted logP often correlates with solubility, so helps us decide

whether to use cosolvent for pKa measurement

We published a paper citing our use of ACD software [1]Sirius RefinementPro software produces a file of measured data ready to be imported into ACD software

[1] Box, K. Bevan, C. Comer, J. Hill, A. Allen, R. Reynolds, D. High-throughput measurement of pKa values in a mixed-buffer linear pH gradient system. Anal. Chem. 2003 (75(4)) pp 883-892

/414© 2007

Themes covered by this talk

Variations in published solubility valuesSolubility definitionsReasons for incorrectly reported solubility valuesKinetic vs. equilibrium solubilityLoperamide – an example of an incorrect resultLogP vs. logS0, and predicting BCS classSolubility vs. pH, and the validity of Henderson-Hasselbalch

From next Monday, you will be able to download these slides from

http://www.sirius-analytical.com/downloads/Downloads/downloadsacd.htm

/415© 2007

Acknowledgements

Colleagues at Sirius– Karl Box, Tom Gravestock, Jon Mole, Ruth Allen (Chemistry)– Martin Stuart (Software)

Academic collaborators– Krisztina Takács-Novák, Edit Baka (Semmelweis University, Budapest)– Farah Huque (University of Cardiff)– Rebeca Ruiz (University of Barcelona)– Slavica Eric (University of Belgrade)

Not forgetting…– Tony Williams (ACD)

From next Monday, you will be able to download these slides from


/416© 2007

How to measure solubility by shake-flask

Weigh sample, add buffer, stir for a long time (e.g. 24 hours or more) to create saturated solution

Sample must be present in excess,

i.e. precipitate always there

Separate solution and

solid, by

Sedimentation, Centrifugation or

Filtration

Check crystalline form

(polymorph) of solid

Analyse concentration in solution

Concentration = equilibrium solubility

Don’t forget to check pH of solution!

This information provides solubility at a

measured pH

/417© 2007

Variation of aqueous solubility in literature (25ºC)

8 A. Avdeef, C. M. Berger, Pharm. Res. 17 (2000), pp. 85-89.11 S. H. Yalkowsky, Y .He, Handbook of Aqueous Solubility Results , CRC Press, Boca Raton, (2003)12 T. Higuchi, F. M. L. Shih, T. Kimura, J. H. Rytting, J. Pharm. Sci . 68 (1979), pp. 1267-1272 13 A. Fini, G. Fazio, G. Feroci, Int. J. Pharm . 126 (1995), pp. 95-10214 S. Pinsuwan, A. Li, S. Yalkowsky, J. Chem. Eng. Data 40 (1995), pp.623-62615 S. Pinsuwan, P. B. Myrdal, Y. C. Lee, S. H. Yalkowsky, Chemosphere 35(11) (1997), pp. 2503-251316 C. A. S. Bergström, K. Luthman, P. Artursson, Eur. J. Pharm. Sci. 22 (2004), pp387-39817 R. H. Levy, M. Rowland, J. Pharm. Sci. 60 (1971), pp. 1155-115918 P. Ruelle, U. W. Kesserling, J. Pharm. Sci . 87(8) (1998), pp. 998-101419 M. F. Powell, In Analytical Profiles of Drug Substances, K. Florey, Ed. Academic Press: San Diego, 1986, Vol. 29 K. Kawakami, K. Miyoshi, Y. Ida, Pharm. Res . 22 (2005), pp. 1537-154330 D. K. Madan, D. E. Cadwallader, J. Pharm. Sci. 62 (1973) pp.1567

Table from: Baka, E. Comer, J E A. Takács-Novák, K. Study of equilibrium solubility measurement by saturation shake-flask method using hydrochlorothiazide as model compound, J. Pharm. Biomed. Anal., 2007 (submitted)

CompoundSolubility range (mg/ml)

Solubility range (µg/ml)

Cholesterol [12, 30 ] 0.025, 2600Dexamethasone [11, 29] 89.1, 121.0Diclofenac [8. 12. 13] 0.6 - 2.4Digoxin [11,29] 28.0, 97.9Estradiol [11,29] 0.16, 5.00Hydrocortisone [11, 29] 280, 359Ibuprofen [8, 13, 14, 15] 20 - 80Indomethacin [11,19] 4.00, 14.0Lidocaine [16, 17, 18, 19] 2.30 - 4.60Progesterone [11, 29] 7.90, 200Riboflavine [11, 29] 66.0, 99.9

/418© 2007

Uncertainty of experimental data

Anthracene0.86 log units [2] 17 laboratories

NOH

OH

CH3

CH3 CH3

TerfenadinelogS = -6.17[6], -4.67[5,8], -7.74[7]

[1] Jorgensen, W L. Duffy, E M. Adv. Drug Deliv. Rev. 2002, 54, 355-366[2] Kishi, H. Hashimoto, Y. Chemosphere 18 (1989) 1749-1759][3] Pogliani, L. J. Chem. Inf. Comput. Sci. 1996, 36, 1082-1091[4] Dannenfelser, R M. Yalkowsky, S H. Sci. Total. Environ. 1991, 109-110, 625-628 [5] Analytical Profiles of Drug Substances[6] Bergström, C.A. Wassvik, U.N. Luthman, K. Artursson, P. J. Chem. Inf. Comput. Sci. 44 (2004) 1477-1488 [7] Sirius Analytical Ltd. – measured in-house[8] Rytting, E. Lentz, K. A. Chen, X –Q. Qian, F. Venkatesh, S. The AAPS Journal 7 (2005) E78-E105[9] Dollery, C. Therapeutic Drugs, 2nd edition 1998, Churchill Livingstone, London

N

NHN

NH

O

NH2

GuaninelogS = -3.58 [3,8], -1.86[4]

“The average uncertainty in experimental logS measurements for reasonably complex organic molecules is likely no better than 0.6 log unit” [1]

N

O

NO

Cl

LoperamidelogS= -7.13 [7], -4.38 [9]

/419© 2007

Software for prediction of solubility

% Compounds predicted within

0.42 0.4062.338.5AlogP98

0.61 0.6072.937.7Cerius2ADME0.51 0.4967.241.0WSKOWWIN0.42 0.39 66.434.4ADMEWORKS Pred.

0.53 0.5174.644.3Absolv 2

0.73 0.7273.142.9SPARC

0.35 0.3445.723.3CHEMICALC

0.57 0.5773.847.6QikProp

0.44 0.4277.962.3MOLPRO0.60 0.5978.754.9ESOL0.48 0.4681.146.7PredictionBase0.67 0.6681.151.6AlogS0.73 0.7285.259.0ACDLabs0.67 0.6586.059.8ChemSilico0.74 0.7386.959.0ADME Boxes0.76 0.7486.972.1Admensa0.82 0.8291.064.8SimulationPlus

R2 q2±1.0 log unit±0.5 log unitModel

Predictive abilities of some software for aqueous solubility prediction

Dearden’s study [1]Test set: 122 compounds [2]

“Prediction of aqueous solubility can be almost as good as

experimental measurements for most of the compounds”

[1] Dearden JC Expert Opin. Drug Discov. (2006) 31

[2] Rytting, E. Lentz, K. A. Chen, X –Q. Qian, F. Venkatesh, S. The AAPS Journal 7 (2005) E78

/4110© 2007

Rytting’s published solubility values

Rytting et al published two tables of measured solubilityTable 1 lists experimental logS of 321 drugs from the literatureTable 2 lists 122 logS values measured in Rytting’s lab. All samples are also in table 1, with slightly different resultsSearching through solubility results measured at Sirius, we found we had measured 25 of the Rytting compounds, using CheqSol methodology

Compound Sirius LogS

Rytting 321

Rytting 122

Amitriptyline -4.39 -4.46Chlorzoxazone -2.61 -2.83 -2.87Diclofenac -5.45 -5.10Flufenamic Acid -5.35 -4.62 -4.41Flurbiprofen -4.11 -3.74 -3.87Folic Acid -5.31 -5.44 -4.89Haloperidol -5.47 -4.43 -4.98Hydrochlorothiazide -2.68 -2.69 -2.78Ibuprofen -3.61 -3.42 -3.63Lidocaine -1.85 -1.77Mefanamic Acid -6.34 -3.77 -5.18Metoclopramide -3.59 -3.18Nadolol -1.57 -1.01 -1.01Naproxen -4.14 -4.16 -3.79Nitrofurantoin -3.33 -3.48 -3.49Norfloxacin -2.75 -3.06 -2.76Phenobarbital -2.28 -2.37Phenylbutazone -4.39 -2.64 -4.13Prochlorperazine -4.87 -4.40Propranolol -3.50 -0.71Quinine -2.81 -2.79 -2.43Sulfamerazine -3.10 -1.22 -3.11Sulfathiazole -2.70 -2.81 -2.18Sulindac -4.52 -5.00Terfenadine -7.74 -4.67 -4.87

/4111© 2007

y = 0.99x + 0.01R2 = 0.94

-8

-6

-4

-2

0

-8 -6 -4 -2 0LogS Sirius

LogS

Ryt

ting

Rytting 321 compounds

Linear (Rytting 321 compounds)

18 of Rytting’s Table 1 results compared with Sirius

Sirius results compare really well with 18 results from Rytting table 1, which were all values taken from the literature

/4112© 2007

18 Rytting Table 1 results plus 7 outliers

These outliers from Rytting Table 1 are all much higher than Sirius measured values

y = 0.99x + 0.01R2 = 0.94

-8

-6

-4

-2

0


LogS

Ryt

ting


Rytting 321 outliers


/4113© 2007

17 of Rytting’s Table 2 results compared with Sirius

These 17 results were measured by Rytting’s group using at least 24 hours shaking followed by 15 minutes centrifugation at 14,000 rpmSupernatants analysed by HPLC with UV detectionSome outliers high compared with Sirius measured values

y = 0.95x - 0.05R2 = 0.93

-8

-6

-4

-2

0


LogS

Ryt

ting

Rytting 122


Linear (Rytting 122)

/4114© 2007

Rytting tables 1 and 2 vs. Sirius

Who’s right – Sirius or the published values?Before trying to answer that question, let’s look at some definitions

y = 0.99x + 0.01R2 = 0.94

-8

-6

-4

-2

0


LogS

Ryt

ting





Terfenadine

Mefan-amic acid

PhenylbutazonePropranolol

Sulfamerazine

Haloperidol

Flufenamic acid

/4115© 2007

What does the word “solubility” mean?Thousands of references in PubMed, June 2007

To be sure of what we are measuring, we drafted our own definitions

Solubility 63,949Biological solubility 8,135Native solubility 1,813Water solubility 1,795Natural solubility 1,736Aqueous solubility 1,177Buffered solubility 397Equilibrium solubility 105Intrinsic solubility 62Turbidimetric solubility 35Thermodynamic solubility 30Unbuffered solubility 21Kinetic solubility 9

Dissolve excess sample in water, no buffer. Sample controls the solution pH.

How many people actually measure this pH?

/4116© 2007

Solubility definitionsKinetic (or Turbidimetric) Solubility is the concentration of a compound in solution at the time when an induced precipitate first appears

– This measurement is popular at early stage assessment– The result obtained is often significantly higher than the equilibrium solubility

Equilibrium (or Thermodynamic) Solubility is the concentration of compound in a saturated solution when excess solid is present, and solution and solid are at equilibrium– This is the true solubility of a molecule– It is traditionally measured by shake-flask, which is accurate but slow

Nowadays, high throughput shake-flask measurements are made in parallel– Other novel techniques have been developed e.g. Sirius CheqSol

Intrinsic Solubility S0 is the equilibrium solubility of the free acid or base form of an ionizable compound at a pH where it is fully un-ionized

Intrinsic solubility must be used for training software to predict solubility of ionisable drugs

/4117© 2007

Reasons for incorrectly reported intrinsic solubility

Shake-flask problemsResult reported was not measured at pH where sample was unionisedPrecipitate was present, but it was a poorly soluble salt, not the unionised sampleIncorrect separation, e.g. sample passed through filterNot waiting long enough for equilibrium

Other problemsNatural solubility result was reportedKinetic solubility result was reportedResult measured in a solvent was reportedSolubility reported for a different polymorph

/4118© 2007

Kinetic solubility and Equilibrium solubilitySome compounds can’t form super-saturated solutions– When the pH is right, they fall out of solution immediately– Their kinetic and equilibrium solubilities are identical

Other compounds form super-saturated solutions– Their kinetic solubilities are higher than their equilibrium solubilities

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0 2000 4000 6000 8000

Equilibrium solubility, µg/mL

Kin

etic

sol

ubili

ty, µ

g/m

L

ChasersNon-chasersLinear (Non-chasers)

0

200

400

600

800

1000

1200

1400

1600

0 200 400 600 800 1000 1200 1400 1600

Chasers

Non-chasers

/4119© 2007

Salt solubility reported, not intrinsic solubility

Monoprotic basepKa values used– 8.90 (measured at Sirius)

N

O

NO

Cl

Loperamide has been reported in the literature to have higher solubility than we found– Reported: LogS0 = -4.38 [1], equivalent to 41.7µM– Measured at Sirius: LogS0 = -7.13, equivalent to 0.073µM

[1] Dollery, C. Therapeutic Drugs, 2nd edition 1998, Churchill Livingstone, London

Loperamide

/4120© 2007

Loperamide pKa measurement

R2 = 1.00

pH-metric measurementSix titrations in water-methanol (14-55%)Result 8.90 by YS extrapolationpsKa values at methanol percentages used in CheqSol experiments are interpolated from YS graph

/4121© 2007

Loperamide solubility exampleSolubility measured in water-methanol mixtures (6 titrations, 20-55% methanol)

This example shows experiment done in 34.1% methanol

Interpolated psKa of 8.46

Top graph – chasing equilibrium

Bottom graph – Bjerrum plot, showing Precipitation Point and CheqPoint.

Sample is a Chaser, because its kinetic solubility (calculated from the precipitation point) is higher than its intrinsic solubility (calculated from the CheqPoint)

/4122© 2007

Loperamide solubility extrapolation

Intrinsic solubility– Extrapolated LogS = -7.13– Equivalent to 0.073µM

Natural solubility and pH– 0.83µM, pH 7.67,

determined by mass balance calculation

Solubility at pH 7.4– 2.39µM, interpolated from

solubility-pH profile

Salt solubility – see next slide

R2 = 0.99

Intrinsic solubility, S0

Solubility at pH 7.4

Natural solubility

Salt solubility

/4123© 2007

Loperamide verdict

Loperamide is extremely insoluble in free base formIt’s also poorly soluble in salt formWe measured solubility of several loperamide salts

Solubility Profile of Loperam ide with Various Counter-Ions

-8

-7

-6

-5

-4

-3

-2

-1

0

2 4 6 8 10 12

pH

LogS

0.1M mesylate

0.15M mesylate

0.025M HCl

0.075M HCl and0.025M Besylate0.15M HCl

0.05M Besylate

0.07M Benzoate

0.15M Besylate

0.15M Benzoate

0.15M Tosylate

Loperamide solubility reported in the literature – LogS of -4.38, equivalent to 41.7µM

We believe that the reported solubility is for a salt form of loperamide

/4124© 2007

What can we learn from logP vs. logS0?We measured PAMPA permeability and logP of 39 drugsAll marketed compoundsResults show that PAMPA permeability is proportional to logP [1]– Diverse set of 39 drug-like

compounds– LogP alkane-water measured

pH-metrically using Sirius GLpKa

[1] K. Box, J. Comer, F. Huque, Correlations between PAMPA permeability and log P. In Pharmacokinetic profiling in drug research: biological, physicochemical and computational strategies. Wunderli, H. Kraemer, S. Folkers, G. Testa, B., editors, Verlag Helvetica Chimica Acta, 2006, pp 243-257

/4125© 2007

-10

-8

-6

-4

-2

0

2

-2 0 2 4 6 8 10

Log P

Log

S 0

Melting point above 140ºC. Compounds can all form supersaturated solutions

Melting point below 140ºC. Can supersaturate

Melting point below 140ºC. Cannot supersaturate. Most have CNS activity

For points between grey lines: LogS0 = -1.03 LogP – 0.53

High solubility region

Low solubility region

Measured LogP vs. measured logS0 for 84 drugs

Pramoxine

/4126© 2007

-10

-8

-6

-4

-2

0

2

-2 0 2 4 6 8 10

Log P

Log

S

12

34

CLASS 1 CLASS 3

CLASS 2 CLASS 4

More permeable (higher logP)

Mor

e so

lubl

e

Comparing results with Biopharmaceutics Classification (BCS)

No BCS data for 5 compounds marked yellow.

/4127© 2007

-10

-8

-6

-4

-2

0

2

-2 0 2 4 6 8 10LogP

LogS

Measured values from previous slides

/4128© 2007

-10

-8

-6

-4

-2

0

2

-2 0 2 4 6 8 10LogP

LogS

Predicted values calculated using a popular program (not ACD!)

Tendency for solubility values to be calculated too high

Terfenadine

/4129© 2007

-10

-8

-6

-4

-2

0

2

-2 0 2 4 6 8 10LogP

LogS

Folic acid

Loperamide

Am

anta

dine

Dilti

azem

Piroxicam

Glipizide Meclofenamic acid

Mefenamic acidWar

farin

Probe

necid

Predicted properties can differ significantly from measured values

Biggest errors are in predicted solubility

Errors lead to mis-prediction of BCS class

MeasuredPredicted

/4130© 2007

-10

-8

-6

-4

-2

0

2

-2 0 2 4 6 8 10LogP

LogS

Amantadine

Diltiazem

1

2

111333222444

High permeability

Hig

h so

lubi

lity

Measured class 1 – predicted class 2

Measured■ Predicted

/4131© 2007

Measured 2 and 4 – predicted 1 and 3

-10

-8

-6

-4

-2

0

2

-2 0 2 4 6 8 10LogP

LogS

Folic acid

Loperamide

Piroxicam

GlipizideMeclofenamic acid

Mefenamic acidW

arfa

rinPr

oben

ecid

1

2

3

4 Measured■ Predicted

111333222444

High permeability

Hig

h so

lubi

lity

/4132© 2007

Solubility vs. pH for promethazine, a weak base with one pKa– weak acid would be mirror-image

LogS = the concentration of unionized sample in solution in equilibrium with excess undissolved sampleGraph based on Henderson-Hasselbalch principles, drawn using pKa and intrinsic solubility (S0)

[H][H])(KSS a0 +

=

logS

pH (Concentration scale)

-4

-3

-2

-1

0

1

4 6 8 10 12

Intrinsic solubility

pKa

Solubility and pH for ionisable molecules

a

a0

K[H])(KSS +

=

acid base

If the sample has more than one pKa, or it forms an insoluble salt, the equations are more complicated

N

S

NCH3CH3

CH3

/4133© 2007

Henderson Hasselbalch vs. Shake-flask

Green lines are calculated from pKaand CheqSol S0 values measured at SiriusBlue points are shake-flask data [1]Agreement is good where samples are unionizedWhy does diclofenac solubility deviate from the green line at higher solubilities?

Propranolol

Diclofenac

[1] Shake flask data in the next four slides is from Avdeef, A. Berger, C M. Brownell, C. Pharm. Res. 2000, 17(1), 85-89

BasepKa = 9.54S0 = 81µg/mL (314µM)

O

NH

OH

CH3

CH3

NH

OH

O Cl

Cl

AcidpKa = 3.99

S0 = 0.9µg/mL (4.1µM)

/4134© 2007

Deviations from solubility-pH profile

The graph may deviate at higher logS values for two possible reasons:– Either the ionized sample may

precipitate as a salt with a counter-ion

– Or all the compound used in the experiment may dissolve (“Solubility Limit”)

logS

pH

-5

-4

-3

-2

-1

0 2 4 6 8

Either: poorly soluble salt

or:all sample dissolved

/4135© 2007

Reaching the solubility limit

9.4 g/litre

29.6 g/litre

296 g/litre

Although it was not confirmed in the publication, it is likely that the solubility limit was reached in this example The concentration at the red points is shownIt is impractical to do shake-flask solubility experiments at very high concentrations, especially for new chemical entities available only in small quantities

Diclofenac

0.00075 g/litre

/4136© 2007

Solubility vs. pH for four drugsKetoprofen(acid, one pKa)

O

O

OH

CH3

800 g/litre

Hydrochlorothiazide (acid, two pKas)

94.2 g/litre

SSNH

O O

NH

O

O

NH2

Cl

146 g/litre

Ibuprofen(acid, one pKa)

O

OH

CH3

CH3

CH3

Famotidine (ampholyte, two pKas)

7.4 g/litre

/4137© 2007

Chlorprothixene

0.0000511M

Promethazine

0.0000499M

Pramoxine

0.0000485M

Does Henderson-Hasselbalch always draw true solubility-pH profiles?

A recent publication [1] suggested that Henderson-Hasselbalch did not always predict solubility-pH profiles accurately.

We re-measured properties of some molecules and found that the published profiles may have been based on questionable values for pKa or solubility

The next slides show results for chlorprothixene, promethazine and pramoxine

Shake flask solubilities were measured by HPLC with UV. Note the low UV signal for pramoxine

[1] Bergström, C A S. Luthman, K. Artursson, P. Accuracy of calculated pH-dependent aqueous drug solubility, Eur. J. Pharm. Sci., 2004, 22, 387-398

/4138© 2007

New profile (Sirius 2007): pKa = 9.52, LogS = -0.76

Published profile:pKa = 8.4 [1], logS = 0.2

Solubility of benzenesulfonate salt, (Sirius, 2007)LogS = 2.26

pH 12

No. of buffers: 10

[1] Encyclopedia of Therapeutic Drugs, Churchill Livingstone, New York, 1991

-1

0

1

2

3

4

0.0 2.5 5.0 7.5 10.0 12.5pH

LogS

(

S =

[B] +

[BH

+]).

Verdict: Sirius red profile fits all shake-flask points except at pH 12. It’s a coincidence that the published profile fits the mid-pH points, as the pKa and solubility are both questionable

Published black line = “best fit”

/4139© 2007

0

1

2

3

4

5

0.0 2.5 5.0 7.5 10.0 12.5pH

LogS

(

S =

[B] +

[BH

+]).

New profile (Sirius 2007) pKa = 8.99, LogS = 1.81


pH 12.07

No. of buffers: 11

Verdict: shake-flask data fits Sirius profile much better than published profile in mid-pH region

[1] Encyclopedia of Therapeutic Drugs, Churchill Livingstone, New York, 1991

/4140© 2007

2

3

4

5

6

7

0.0 2.5 5.0 7.5 10.0 12.5pH

LogS

(

S =

[B] +

[BH

+]).

New profile (Sirius 2007) pKa = 7.12, LogS = 2.98


Verdict: Nothing fits well. Different pKas used. Pramoxine is a liquid at high pH. UV absorbance is low. Could these properties make it hard to measure by shake-flask?

pH 12.08

No. of buffers: 9

[1] Casanovas, A M. Labat, C. Courriere, P. Oustrin, J. 1982, Eur. J. Med. Chem. 17, 333-337

/4141© 2007

Conclusions

There may have been shortcomings in experimental techniques used in the past to measure PhysChem propertiesIt can be risky to use published values without checking themIt’s useful when customers can improve predictions by making use of good quality results to train softwarePeople must work harder to get good experimental resultsThe danger: Too much automation, Not enough thinkingGood experimental design is critical for getting the right resultACD software helps us to design good experiments

Sirius – Instruments and PhysChem Analytical Services

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