Solubility-pH profile of drugs.
Experiences and surprises in logS
measurements
Krisztina Takács-Novák
Semmelweis University, Budapest
PhysChem FORUM 9 September 17, 2010. Barcelona
Overview of presentation
¾ new protocol of saturation shake-flask method
¾ revisit of Henderson-Hasselbalch (HH) relationship
¾ surprising experiences in solubility determination
Development of physico-chemical profiling
1971 lipophilicity, ionization
Leo, Hansch, Elkins: Chem. Rev. 71, 525-616 (1971)
1995 solubility, permeability (introduction of BCS)
Amidon, Lennernas, Shah, Crison: Pharm. Res. 12, 413-420 (1995)
2002 pharmaceutical profiling
Kerns, Di: Curr. Top. Med. Chem. 2, 87-98 (2002)
• solubility
• ionization
• lipophilicity
• permeability
+
• integrity
• stability
• metabolism
• protein binding
• CYP-450 inhibition
The importance of solubility in drug research
● important molecular property that influences the intestinal
absorption → determines bioavailability
● useful during lead selection and optimization and serves as a
screening parameter
● required for biopharmaceutical classification (BCS)
● necessary for salt selection and optimization of formulation
Boom in solubility research from 90-ies
• new solubility determination methods
• demand for standardization
• claim for HT approaches
• claim for compound-sparing assays
• affords for in silico prediction
¾ but the largest need is a better understanding of
solubility reactions, particularly in case of ionizable
molecules in order to give correct interpretation of
experimental data
Physico-chemical profiling at Semmelweis Univ.
¾ We started with logP measurement (1977)
• basic research: lipophilicity of amphoteric drugs
ion-pair partition
• method development in collaboration with Sirius
(1992-)
• we measured logP values for pharm. companies
logP, pKa, S, Pe
2003
1994
2003
How we involved in solubility measurement?
¾ We got the first request for solubility determination
in 1990
sample: deprenyl (Jumex®) (Chinoin)
method: saturation shake-flask (SF)
¾ In the last decade we measured more logS than logP
more samples → more experiences → more surprises
CH3
CH3
N
N
N
O
N
OH
HN
Cl
N
COOH
O
CH3
O
H 2N
N
O
CH3
S
N
F
O
O
CH3
N
N
N CH3
CH3O
O
N
Cl
O
O
N
N
H
H
N
O
O
O
O
F
H3 C
OH
OH
CH3
O
N
F
O
OH
CH3
CH3
Why it is difficult to estimate the solubility?
Cl
O
H
N
O
H2N S
COOH
N
O
CH3
CH3
logP: 2.56
logP: 5.04
logS: -4.39
logS: -4.39
¾ Solubility is affected by:
• particle size
• crystallinity
• polymorphism
• aggregation, micelle formation
• adsorption on the solid excess, etc.
We use these solubility definitions:
¾ Equilibrium (= thermodynamic) solubility (S)
the concentration of compound in a saturated solution when solid is
present and solution and solid are at equilibrium
¾ Intrinsic solubility (So)
the equilibrium solubility of the free acid or base form of an ionizable
compound at a pH where it is fully unionized
¾ Apparent solubility (SpH)
the equilibrium solubility of an ionizable compound at a pH where
ionized form is also present
¾ Solubility of salt (Ssalt)
Ssalt = K sp
We use these methods:
¾ Saturation shake-flask (SF)
temperature: controlled (25.0 or 37.0 °C)
medium: Britton-Robinson or Sörensen buffer
phase-separation: sedimentation
conc. measurement: UV spectroscopy
¾ Chasing Equilibrium Solubility Method (CheqSol)
potentiometric approach
GLpKa (Sirius Anal. Instr. UK)
Stuart, Box: Anal Chem. 77, 983-990 (2003)
Box, Völgyi, Baka, Stuart, Takács-Novák, Comer: J. Pharm. Sci. 95. 12981307 (2006).
New protocol of SF method
Baka, Comer, Takács-Novák: J. Pharm. Biomed. Anal. 46, 335-341 (2008)
Aim of the work:
¾ to investigate the critical experimental conditions of SF
method
¾ to reveal their influence on intrinsic solubility
¾ to create a standardized protocol
Factors studied:
• composition of buffer
• amount of solid excess
• temperature
• equilibration time
• technique of phase-separation
Model compound:
H
N
Cl
O
H2N S
O
S
O
NH
O
• ionizable
• sparingly soluble
• stable
• UV active
Results
So: 556 ± 13.2 µg/ml n=18
pH: 6.0
pKa1: 8.75 pKa2: 9.88
Effect of buffer solution
1. Britton-Robinson
(I=0.089)
2. Sörensen-phosphate (I)
(I=0.076)
3. Sörensen-citrate (II)
(I=0.318)
Solubility (ug/ml )
¾ effect of buffer solution
1000
800
600
779
556
565
400
200
0
Britton-Rob
Sörensen I
Sörensen II
Buffe rs
¾ the So is influenced by the ionic strength of the buffer, it is related
to the changes in the activity coefficient (Streng et al. J. Pharm. Sci. 1984)
¾ effect of solid excess
Amount of solid excess
700
Intrinsic solubility (ug/ml)
the amount of solid
weighted was from 4 mg
to 20 mg by 4 mg steps
650
600
550
500
450
400
0
2
4
6
8
10
12
14
16
18
20
22
24
26
Solid (mg/5ml)
¾ the So is not influenced by the solid excess
we recommend using small but sufficient excess (~ 5-10 mg/5ml)
to avoid the difficulties in sampling
¾ effect of equilibration time
• stirring (agitation)
• sedimentation (phase-separation)
Impact of stirring-time
Impact of sedimentation-time
(when sedimentation: 24 hours)
(when stirring: 48 hours)
640
600
620
580
556
600
S (ug/ml)
S (ug/ml)
560
540
520
500
580
556
560
540
520
480
500
460
0,5
1
2
6
12
24
48
Time (h)
¾ equilibration time: 6 + 18 = 24 hours
1
2
4
6
8
Time (h)
12
18
24
New protocol of SF method:
• buffer: B-R (pH 2.5 – 11.5) or Sörensen-phosphate (pH 3-7)
• temperature: 25°C or 37°C (± 0.1)
• solid excess: small excess (~ 5-10 mg/5ml)
• incubation time: 24 hours (6 + 18)
• phase-separation: sedimentation
• concentration measurement: UV spectroscopy
¾ it allows to determine equilibrium solubility faster, less than
36 hours
¾ it was applied in the last 3 years at more than 50 compounds
and only one necessitated longer equilibration time
Revisit of HH relationship
Völgyi, Baka, Box, Comer, Takács-Novák: Anal. Chim. Acta 673, 40-46 (2010)
¾ the pH-dependence of equilibrium solubility of
ionizable molecules is described by HH equation
(
+ log(1 + 10
HA
log SpH = log S 0 + log 1 + 10 pH − pK a
B
log SpH = log S 0
pK a − pH
)
)
¾ the HH relationship can be used to predict the solubility
at physiologically relevant pHs ( 1.5, 5.5, 6.8, 7.4) if pKa
and logSo are known
¾ validity of HH equation has been widely investigated
• investigated 25 amines
• commented limited
applicability
• computational basis
• re-analyzed literature
data
¾ we performed a systematic study using structurally
diverse compounds
4 monoprotic bases
1 diprotic base
1 ampholyte
¾ we measured
• pKa values
• logSo values by two methods (SF, CheqSol)
¾ we determined the logSpH values over a wide pH range
(0-12) by SF method
Papaverine hydrochloride
H3CO
N
HCl
H3CO
5,5
pHmax
OCH3
5,0
OCH3
4,5
Exp. conditions:
logS
4,0
method: SF new protocol
3,5
tempr.: 25.0 °C
3,0
2,5
Results:
2,0
1,5
pKa: 6.36
0
2
4
6
8
pH
10
12
So: 17 µg/ml (SF)
17 µg/ml (CheqSol)
¾ HH valid
¾ pH< 2 common-ion effect
logSo [µM]: 1.70
Bergström (2004)
Exp. conditions:
method: small-scale SF
medium: phosphate buffer
tempr.: 22.5±1°C
Results:
pKa:
9.1
logSo: 0.3
Our
7
pHmax
Exp. conditions:
6
method: SF new protocol
medium: BR buffer
tempr.: 25.0±0.1°C
logS
5
4
Results:
pKa:
8.71
logSo: 1.98
3
2
1
0
2
4
6
pH
8
10
12
¾ HH valid
¾ pH< 2 common-ion effect
Our
Bergström (2004)
6
pKa:
pHmax
8.8
logS
logSo: 0.8
5
pKa:
9.32
4
logSo: 1.24
3
2
1
0
2
4
6
8
10
12
pH
¾ HH relationship is valid if accurate pKa and logSo values are
used
¾ aggregation may alter the curve but before proposing
aggregation/micelle formation etc., the experimental data
should be scrutinized and tested rigorously
Quetiapine hydrogen fumarate
O
OH
N
N
N
HOOC
1/2
COOH
[BH22+2Cl-]
S
6
diprotic base:
BH22+
5
- H+
BH+
logS
3.56
- H+
B
6.83
fumaric acid:
4
- H+
[BH+FumH-]
FumH2
FumH
2.8
3
0
2
4
6
8
10
-
- H+
Fum 2-
4.0
12
pH
blue line: theoretical HH curve
¾ HH valid up to pH 6
¾ at pH 2 dichloride salt is ppt
¾ pH< 2 common-ion effect
green line: model includes salt
solubility
What we have learnt from this study?
¾ the experiments must be carefully designed and performed
¾ the most critical point is the precise pH control during the
measurement (before and after the incubation), otherwise false
conclusions can be drawn
¾ the common-ion effect below pH 2 is significant even in case
of non-hydrochloride salts
¾ deviation from HH relationship often due to the inaccuracy of
the applied method
¾ before supposing special molecular interactions and applying
complicated equations for calculation the experiments must be
checked
Surprising experiences
Example 1.
2 samples: free base
pKa: 5.66
hydrochloride salt
SpH at pHs: 1.2, 4.5, 6.8 at 37 °C
6
5
SpH [µg/ml]
1.2
4.5
6.8
8.0
base
28 650
36
10
13
HCl salt
63 300
110
45
30
pH
logS
4
3
2
1
0
1,2
4,5
6,8
8
pH
Different solubility data!
pH: 1.2 (0.1M HCl)
¾ different crystal structures
pH: 4.5 – 8.0 (Sörensen)
Example 2.
2 polymorphs: A form
pKa: 9.63
B form
at pHs: 1.2 - 6.8 SpH > 50%
SpH [µg/ml]
8.9
9.2
12
A form
1 800
1280
460
B form
2 080
1290
460
pH
No significant difference in solubility data!
¾ A and B forms convert to the base with identical crystal structure
Example 3.
2 samples: hydrochloride salt
mesylate salt
SpH at pHs: 1.2, 3.1, 4.5, 6.8 at 37 °C
difficulties:
at pH 1.2 stable supersaturated solution formed
at pH 6.8 opalescent colloid is appeared
SpH [µg/ml]
1.2
3.1
4.5
6.8
HCl salt
48 000
± 1000
370
± 10
13
±1
~0.5
CH3SO3H salt
47 000
± 1000
360
± 10
62
±5
~0.5
pH
Identical solubility
of the salts!
¾ complicated and unrevealed acid-base chemistry
¾ analysis of solid is necessary
Example 4.
OH
OH
requested solubility data:
at pHs: 1.2, 4.5, 6.8
F
O
N
F
at 37 °C
pKa: 9.7
monovalent week acid
SpH [µg/ml]
pH: 1.2
pH: 4.5
pH: 6.8
dest.
water
1.2
0.8
1.0
2.1
below pH 7: unionized
SpH = So
Management insisted on the measurement!
¾ understanding of ionization equilibria is useful before the
solubility is ordered to measure
Summarizing
¾ solubility measurement requires careful experimental design
and perfect understanding of solubility reactions
¾ care must be taken to avoid the interactions with buffer
components
¾ the pH must be controlled before and after the incubation
period
¾ attention has to be paid to reach the equilibrium state
¾ analysis of the solid at the end of the incubation can reveal
molecular transformation (like polymorph transition, new salt
formation)
¾ the interpretation of results sometimes more challenging than
we realize
„Solubility still remains deceptively easy to
untrained eye and quite difficult to those
interested in precise data and clear
interpretation of results.”
Alex Avdeef (2007)
Acknowledgements
¾ thanks to colleagues at Semmelweis University
Gergely Völgyi
Edit Baka
¾ thanks also to collaborators, especially
at Sirius Anal. Instr. (UK)
John Comer
Karl Box
at pION (USA)
Alex Avdeef
El Camino