Solution Properties NAEJA

Determination of Aqueous solubility by shake Flask Method
Purpose
Approximately more than 30% of lead molecules fail early in development due to unfavorable
physicochemical profiles. Poor solubility is the reason for many of these pharmacokinetic
failures. Solubility is a thermodynamic parameter and which is related to the kinetic process of
dissolution followed by absorption. pH plays an important role for ionizable and zwetterionic
molecules. A standard technique to determine the thermodynamic aqueous compound solubility is
the shake flask method.
Assay protocol
Solubility measurement is performed under equilibrium conditions at pH 7.4. Approximately 1mg
of powder of the test compound is dissolved in phosphate buffer at pH 7.4. This solution is
shaken for 24 hr until equilibrium is reached. After separation of the solid by filtration, the
concentration of the compound in the filtrate is determined by LC-UV or LC/MS using one point
standard in DMSO solution.
Compound
NAEJA (M)
Literature/Reference (M)
Albendazole
1.94
1.87
Amiodarone
<0.003
<0.003
Bifonazole
0.12
0.11
Tamoxifen
0.27
Cinnarizine
0.02
0.05
Nimodipine
2.65
2.0
Glyburide
12.73
14.66
Griseofulvin
15.43
22.01
Ketoconazole
6.46
4.95
Nifedipine
16.50
24.48
Flutamide
71.52
88.99
Phenytoin
73.06
91.33
Chlorzoxazone
1041
1934
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. # 2 4290 91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: jkhan@naeja.com; Tel:
Direct: 1-780-989-9827;www.naeja.com
Measurement of Octanol/Water Partition (Log P) and Distribution

Coefficient (Log D)
Purpose
Lipophilicity is an important physicochemical parameter that influences the pharmacokinetic and
pharmacodynamic behaviour of compounds and plays major role in their absorption ,disposition and
plasma protein binding. The octanol/water partition coefficient is a widely used parameter to measure
lipophilicity. The Log P value is the ratio of the concentrations of neutral substance in two immiscible
phases water and octanol. Log D is the log partition at a particular pH. The properties of n-octanol are
thought to resemble those of lipid bilayer membranes and therefore distribution into n-octanol simulates to
an extent their ability to passively diffuse across biological membranes.
Assay protocol
Assay is based on shake-flask procedure. Buffer at pH 7.4 is generally used as the aqueous phase. The
compounds are dissolved in DMSO. To 5L of this stock solution 500L of octanol and 500L of buffer is
added and shaken for a period of 4 hrs. The amount of compound in the buffer and n-octanol is determined
by LC-UV or LCMS
Compound
NAEJA
Literature/Reference
Albendazole
3.47
3.47
Amiodarone
3.53
>3.5
Griseofulvin
2.40
2.32
Ketoconazole
3.69
3.58
Nifedipine
3.28
3.25
Tamoxifen
4.16
4.9
Warfarin
0.94
0.84
Atenolol
-1.80
-2.02
Caffeine
-0.09
-0.02
Cyclobenzaprine
2.60
2.82
Tolbutamide
0.40
0.37
Fluconazole
0.70
0.50
Indomethacin
1.26
1.0
Lidocaine
1.64
1.78
Omeprazole
2.26
2.27
Measurement of Lipohilicity using Reversed phase High

Performance Liquid Chromatography (HPLC)
Partitioning from aqueous/organic mobile phases into standard (usually C-18) statinary phases can be used
as a direct measurement of lipophilicity. However, these phases do not have properties identical to octanol,
therefore to cover a wide range of lipophilicity , various concentrations of the organic solvent in the mobile
phase must be used. To compare retention using different organic-phase concentrations, they are normally
extrapolated to a zero organic solvent concentration. This gradient HPLC system is calibrated by a few
compounds by plotting the gradient retention times values in the function of the isocratically determined 0 (Organic
solvent concentration) values. The gradient retention times can be converted to volume percentage organic-phase
concentrations called the Chromatographic Hydrophobicity Index (CHI). The conditions used cover a 6-logP unit range
o lipophilicity and simple data processing can be used to covert the gradient retention time to CHI values. The CHI
values can also be projected to a logarithmic scale that is more appropriate for free energy-related comparisons with the
usual logP and logD parameters using the following equation:
CHILogD=0.054 CHI-1.467
Compounds
HPLC log D7.4
Octanol/Water
pH 7.4
Pyridoxine
-0.59926698
-0.59
Atenolol
-0.45017217
-0.52
Acetazolamide
-0.35183304
-2.02
Ampicilline
-0.0409545
-1.72
Triamterene
0.49515237
1.16
Pentoxifylline
0.55542474
0.32
0.37
Tolbutamide
1.02491478
Indomethacin
1.61177733
Glyburide
2.19863988
2.28
Cyclobenzaprine
2.51269065
2.82
Lidocaine
2.71254114
1.78
Reserpine
3.83868279
4.16
BifonazoleM
3.99094983
4.45
Correlation of CHI Log D7.4 and measured octanol/water pH

7.4 Log D
y = 1.2001x - 0.5778
R2 = 0.8846
Octanol/Water Log D
5
4
3
2
1
-1
0
-1 0
-2
-3
HPLC Log D
Compounds
Measured
HPLC Log
D7.4
Literature
HPLC Log D7.4
Terbutaline
-0.434832984
-0.64
Cinoxacin
-0.425359872
-0.65
Amiloride
-0.422202168
-0.49
Theophylline
-0.387467424
-0.51
Ciprofloxacin
-0.122220288
-0.08
Cimetidine
-0.027489168
-0.09
Naproxen
0.733517496
0.66
Ketoprofen
0.812460096
0.53
Warfarin
0.888244992
0.93
Tolbutamide
1.014553152
0.95
Quinidine
1.106126568
0.98
Propranolol
1.434527784
1.27
Phenytoin
1.800821448
1.72
Testosterone
2.574458928
2.86
Nifedipine
Ketoconazole
2.66287464
3.101795496
2.59
2.67
Correlation of Measured HPLC Log D7.4 and Literature HPLC

Log D 7.4
y = 1.0032x - 0.1035
R2 = 0.9826
Literature HPLC Log D
3.5
3
2.5
2
1.5
1
0.5
-1
0
-0.5 -0.5 0
0.5
1.5
2.5
3.5
-1
Measured HPLC Log D
Measurement of Plasma Protein Binding using Reversed phase

High Performance Liquid Chromatography (HPLC)
A fast gradient HPLC method will be utilized to determine Human Serum Albumin (HSA) binding of
discovery compounds using chemically bonded protein stationary phases. The HSA binding values will be
derived from gradient retention times that will be converted to the logarithm of the equilibrium constants
(log K HSA). Three types of assay will be done on each compound in RSA504 column (Rat Serum
binding); MSA504 column (Mouse serum binding) and CT-291054 column (Human Serum binding). All
the compounds will be analyzed by all three columns to give us HSA binding to rank order compound for
potential protein binding in vivo.
Compound
NaAcetate(tm)-HSA
Ampicillin-HSA
Chlorpromazine-HSA
Cimetidine-HSA
Cinoxacin-HSA
Ciproflaoxacin-HSA
Indomethacin-HSA
Ketoconazole-HSA
Ketoprofen-HSA
Nifedipine-HSA
Phenytoin-HSA
Propranolol-HSA
Quinidine-HSA
Terbutaline-HSA
Tolbutamide-HSA
Verapamil-HSA
Warfarin-HSA
Lot #
tr
0.275
0.484
2.555
0.597
1.077
1.544
4.014
2.294
4.025
1.853
1.478
1.297
1.256
0.507
2.946
1.304
3.367
k'=(tr-tm)/tm
P=100(k'/k'+1)
Published
% Protein Bound
HSA %
Linear Gradiant
0.76
43.18
21.2
8.29
89.24
94.7
1.17
53.94
21.2
2.92
74.47
60.1
4.61
82.19
45.0
13.60
93.15
99.5
7.34
88.01
93.0
13.64
93.17
97.3
5.74
85.16
69.5
4.37
81.39
75.5
3.72
78.80
62.0
3.57
78.11
61.7
0.84
45.76
28.8
9.71
90.67
96.0
3.74
78.91
74.4
11.24
91.83
97.9
Protein Binding using Chiral HSA column (Human Serum Albumin)

Compound
NaAcetate(tm)-HSA
Chlorpromazine-HSA
Cimetidine-HSA
Cinoxacin-HSA
Ciproflaoxacin-HSA
Ketoconazole-HSA
Nifedipine-HSA
Phenytoin-HSA
Propranolol-HSA
Quinidine-HSA
Terbutaline-HSA
Tolbutamide-HSA
Verapamil-HSA
Lot #
tr
0.955
16.324
1.551
2.067
6.786
12.628
5.397
3.496
3.705
3.317
1.599
18.099
3.031
k'=(tr-tm)/tm
P=100(k'/k'+1)
Published
% Protein Bound
HSA %
Isocratic Gradiant
16.09
94.15
94.7
0.62
38.43
21.2
1.16
53.80
60.1
6.11
85.93
45.0
12.22
92.44
93.0
4.65
82.30
69.5
2.66
72.68
75.5
2.88
74.22
62.0
2.47
71.21
61.7
0.67
40.28
28.8
17.95
94.72
96.0
2.17
68.49
74.4
Chemical Stability in Aqueous solution/plasma

Purpose
In aqueous solutions and plasma drug can be subjected to some form of chemical degradation. Chemical
degradation may also give rise to harmful degradation products. However, the most common consequence
of drugs instability is a loss of activity. Therefore it is important to determine chemical stability of a
compound early in the drug discovery.
Assay protocol
Chemical stability is performed at a certain pH level. The compound is added to standard buffer solution or
other matrix of interest and incubated at 37C. Aliquots are removed at defined timepoints. For each time
point the pH of the solution and peak area of parent compound is measured by UPLC-UV-ELSD.
140
120
100
80
0.1M B uffer
60
0.1N HCl
40
20
0
0
0.25
0.5
24
T im e ( hr)
120
100
80
Rat P lasma
60
Human P lasma
40
20
0
0
24
T im e ( hr)

Solution Properties NAEJA

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Solution Properties NAEJA

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Determination of Aqueous solubility by shake Flask Method

Measurement of Octanol/Water Partition (Log P) and Distribution

Measurement of Lipohilicity using Reversed phase High

HPLC log D7.4

Correlation of CHI Log D7.4 and measured octanol/water pH

Correlation of Measured HPLC Log D7.4 and Literature HPLC

Literature HPLC Log D

Measurement of Plasma Protein Binding using Reversed phase

Protein Binding using Chiral HSA column (Human Serum Albumin)

Chemical Stability in Aqueous solution/plasma

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