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 (µM)
Literature/Reference (µM)
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. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
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 5µL of this stock solution 500µL of octanol and 500µL 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
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
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 Ф0 (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
Tolbutamide
1.02491478
0.37
Indomethacin
1.61177733
1
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
1
2
3
4
5
-2
-3
HPLC Log D
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
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
2.66287464
2.59
Ketoconazole
3.101795496
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
1.5
2
2.5
3
3.5
-1
Measured HPLC Log D
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
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
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
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
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
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 drug’s 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 37°C. 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.
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
Permeability Screening Using Caco-2 Monolayer
Purpose
Intestinal epithelium permeability is a critical characteristic that determines the rate and extent of human
absorption and ultimately affects the bioavailability of drug candidates. The caco-2 permeability assay
allows rapid assessment of membrane permeability to rank-order compounds as high, medium and low
permeability. Interaction with efflux pump such as P-glycoprotein could also be identified using Caco-2
model.
Assay protocol
Caco-2 cells are grown on transwell polycarbonate membrane inserts for 21 days. Growth and
differentiation is monitored by the measurement of transepithelial electrical resistance during culture. The
test compound is added in triplicate in the donar compartment. Samples are collected from the receiver
compartment at specified time points. To assess the influence of P-glycoprotein on the transport process of
a compound, the apical-to-basolateral (A-B) vs baolateral-to-apical (B-A) transport is compared. For
quality control and for classification of test compounds, reference compounds with high and low
transepithelial flux are included in each assay. After each test run the integrity of the monolayer is checked
using Lucifer Yellow. Compound concentrations are measured in both compartment using UPLC-UV or by
LC/MS to calculate the apparent permeability coefficient of the compound. The A-B permeability allows to
rank-ordering of compounds with potential low bioavailability. Paap <1 x 10-6 cm/s (low permeability); Paap
between 1 x 10-6cm/s and 10 x 10-6 cm/s (medium permeability) and Paap > 10 x 10-6 cm/s (high
permeability)
Drugs
Caco-2 Cell
Permeability
(cm/s)
%Fraction
Absorbed in
Humans
Amiloride
Atenolol
Cimetidine
Furosamide
Metformin
Norfloxacin
Sulpiride
Terbutaline
Alprenolol
Desipramine
Ketoconazole
Ketoprofen
Hydralazine
Phenytoin
Propranolol
Verapamil
4.62E-07
2.17E-07
4.69E-07
1.08E-07
2.85E-07
5.15E-07
4.44E-07
1.43E-07
5.35E-06
6.76E-06
6.34E-06
9.73E-06
2.00E-05
1.49E-05
9.06E-06
8.65E-06
50
79
66
52
35
35
50
93
95
76
90
90
90
90
95
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
Reproducble Papp of propranolol over a 14 week period
A-B Papp (10-6 cm/s)
10
1
Propranolol
0.1
0
2
4
6
8
10
12
14
16
Week
P-glycoprotein inhibition
P-glycoprotein is expressed not only in the intestine but also in other tissues such as the blood-brain barrier, liver and
kidney, where it limit brain penetration, involved in biliary secretion and renal tubular secretion. Compound that are
effluxed in the gut show poor bioavailability as well as compounds that interact with P-gp as inhibitors could display
significant drug-drug interactions. Therefore, identifying compounds that potentially interact with P-gp is of particular
importance during drug discovery and lead optimization.
% Inhibition of Taxol transport in presence of Pgp Substrate
A toB
Papp (cm/s)
9.34E-07
Papp(B-A) - Papp (A-B)
% inhibition
B to A
2.44E-06
1.51E-06
Digoxin
2.37E-06
1.44E-06
4.65
Vinblastine
2.66E-06
1.73E-06
-14.61
Hoechst 33342
1.77E-06
8.36E-07
44.49
Rhodamine 123
1.80E-06
8.66E-07
42.50
Reserpine
1.28E-06
3.46E-07
77.03
Verapamil
1.26E-06
3.26E-07
78.35
Quinidine
1.06E-06
1.26E-07
91.63
All substrate concentration is 50µM except Verapamil & Quinidine
which is 200µM
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
Metabolic Stability in microsomes
Purpose
The assessment of metabolic stability of new chemical entities is essential in the lead optimization process
to determine the liability of the structure towards certain isozymes of P450 system. By using liver
microsomes of different animals species and human, potential species related differences in the rate of
metabolism can be detected
Assay protocol
Incubation solutions are prepared containing microsomes in phosphate buffer and a NADP-generating
system. The reaction is initiated by addition of the NADP-generating system after pre incubation at 37°C
with test compounds. The reaction is stopped by addition of acetonitrile at 0, 15 30, 45 & 60 min and
samples are prepared to determine the loss of parent compound by LC/MS/MS. % parent remaining is a 2
point screen and 5 point testing is done T1/2 determination.
Metabolism of testosterone in Rat microsmes, Human Microsomes and
recombinant CYP 3A4
1.4e+5
rCYP450 3A4
Rat Microsomes
Human Microsomes
1.2e+5
Peak Area
1.0e+5
8.0e+4
6.0e+4
Human t1/2 = 27 min
4.0e+4
2.0e+4
0.0
Rat t1/2 = 5.25 min
0
5
10
15
20
25
30
35
Time (min)
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
Concentration 6-OH testosterone after incubation of testesterone in different
concentration of human microsomal protein
40
Concentration (µg/mL)
30
20
10
Protein Conc 1mg/mL
Protein Conc 0.5mg/mL
Protein Conc 0.25mg/mL
0
0
5
10
15
20
25
30
35
Time (min)
Metabolism of testosterone in human microsomes
1.4e+5
1.2e+5
Testosterone
6-OH Testosterone
Peak Area
1.0e+5
8.0e+4
6.0e+4
4.0e+4
2.0e+4
0.0
0
5
10
15
20
25
30
Time (min)
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
In Vitro Evaluation of CYP-P450 Inhibition
Purpose
Cytochrome P450 are the principle xenobiotic metabolizing enzymes in the liver. Many drug-drug
interaction involve CYP inhibition. By testing portential drug candidates at an early stage on CYPinhibition, new chemical entities leading to adverse side effects could be identified early on.
Assay protocol
Mixture of KH2PO4, compounds and NADPH are preincubated at 37°C for 5 minutes in a shaking water
bath. 25µL of human liver microsomes (HLM) standing on ice is added to each sample and vortexed. For 0
time 200µL of this mixture is immediately transferred into tube containing 50µL of 1M formic acid to stop
the reaction. These sets of tubes is centrifuged at 14000 pm for 6 minutes. 100 µL of supernatant is
transferred into injection vials and analyzed for the metabolite concentration of each substrate using an
LC/MS/MS method developed at NAEJA Pharmaceutical Inc. For CYP 2D6 and CYP 3A4 30 minute
samples are collected. Metabolites of the substrate reaction: 6ß hydroxyl testosterone for CYP3A4 &
Dextrorphan for CYP2D6. Test on other isozymes are also available
Inhibition of CYP 450 with NAEJA azoles and comparison with Itraconazole
at 1µg/mL
CYP 450
Itraconazole
NPI-2903
NPI-2921
NPI-2836
NPI-2869
2C9
41.00
85.69
84.28
69.48
74.49
3A4
91.30
97.52
89.90
89.56
80.10
2D6
0.30
40.66
45.51
30.94
58.40
2A6
0.00
10.82
11.05
0.00
0.00
2C19
27.90
76.91
47.21
91.95
89.19
2E1
0.00
0.00
0.00
0.00
0.00
CYP 2C9:
Tolbutamide (150µM) [4-hydroxy
tolbutamide]
In vitro inhibition
of Cytochrome P450 enzymes by azoles
CYP 3A4:
Testosterone (100µM) [4-Androsten-6B,17B-Diol-3-one]
CYP 2D6:
Dextromethorphan (20µM) [Dextrorphan]
100.00
Coumarin (5µM) [7-hydroxycoumarin]
90.00
S-Mephenytoin (50µM) [4-hydroxy mephenytoin]
80.00
Chlorzoxazone (40µM) [6-hydroxy chlorzoxazone]
70.00
CYP 2A6:
CYP 2C19
CYP 2E1:
60.00
% Inhibition
50.00
40.00
30.00
NPI-2869
20.00
NPI-2903
10.00
NPI-2921
0.00
NPI-2836
Itraconazole
2C9
3A4
2D6
2A6
2C19
2E1
CYP 450 Isoforms
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
Intrinsic clearance
Vmax/Km evaluation
Matrices: Recombinant CYP isoforms, Liver microsomes and S9
Michaelis-Menten data fitting using parent compound disappearance rate
Determination of Intrinsic clearance of testosterone in human liver microsomes
Michaelis-Menten
5
Rate (nmol//min)
4
3
2
1
0
0
20
40
60
80
100
120
140
[Substrate] (µM)
Vmax = 5.6
Km = 44.5
Hanes-Woolf
[Substrate] (µM)/Rate (nmol//min)
30
-60
-40
25
20
15
10
-20
5
0
20
40
60
80
100
120
140
[Substrate] (µM)
Vmax = 5.6
Km = 44.5
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
In Vitro Evaluation of CYP-P450 Induction in Cryopreserved rat
hepatocytes
Assay procedure: Hepatocytes : Female Sprague Dawley Rat - Celsis #F00005-P. Hepatocytes were seeded on 48-well
collagen I coated plate @ conc 11x104cells/well, volume 200µl/well and Hepatocytes were seeded on 96-well collagen I
coated plate @ conc 3.5x104cells/well, volume 100µl/well
Media: InVitro GRO CP media containing of Torpedo Antibiotic @ conc 1ml antibiotic/45ml media. The cells were
incubated overnight at 37C 5%CO2. At 24hours old in vitro GRO CP media was replaced with the fresh one. At 48hours
in vitro GRO CP media was placed with in vitro GRO HI containing Torpedo Antibiotic (1ml/45ml media) and inducers
were added at different concentrations. At 72 hrs the procedure followed at 48hrs is repeated. At 96 hours- Metabolism of
Testosterone (CYP3A) and Ethoxyresorufin (CYP1A) assays are performed as follows:
CYP3A: The media was removed and the hepatocytes were washed twice with InVitroGRO KHB. 100µl of 100µM
Testosterone in KHB was added to each well and placed in the incubator for 1hour. Then 100µl of incubating mixture
from each well was transferred to 100µl acetonitrile on 96-well plate. Samples were filterd through Whatman GF/B Filter
and analysed for 6β-hydroxytestosterone amount by LC/MS/MS.
CYP1A: The media was removed and the hepatocytes were washed twice with InVitroGRO KHB. 100µl of 3mM
Salicylamide solution in KHB was added to the hepatocytes and placed in the incubator for 10 min. The Salicylamide
solution was aspirated and replaced with 100µl of 10µM Ethoxyresorufin (ERR). The plate was placed in the incubator for
1hour. Then 100µl of incubating mixture from each well was transferred to 100µl acetonitrile on 96-well dark plate. The
amount of Resorufin is determined by fluorescent counter at excitation 530 nm and emission 580 nm.
Induction of CYP1A in Cryopreserved Female Rat
Hepatocytes (48-well plate)
Inducers
Naïve
DMSO 0.8%
DMSO 0.1%
Omeprazole
Dexamethasone
Rifampicin
3Methycholanthrene
350
300
250
200
150
100
50
0
BetaNaphthoflavone
Naïve
DMSO 0.8%
DMSO 0.1%
Omeprazole
Dexamethasone
Rifampicin
3Methycholanthrene
%VC
350
300
250
200
150
100
50
0
BetaNaphthoflavone
%VC
Induction of CYP3A in Cryopreserved Female Rat
Hepatocytes (48-well plate)
Inducers
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
In Vivo Pharmacokinetics in Catheterised Rats and Mice
Purpose
Obtaining early stage pharmacokinetic data in the evaluation of new chemical entities is crucial
for successful designing of animal pharmacology and toxicology studies. The pharmacokinetic
profiles of compounds are provided following dosing by the intended route and by the
determination of relevant pharmacokinetic parameters such as clearance, volume of distribution
and bioavailability. NAEJA provides animal pharmacokinetic data in rats and mice.
Experimental Protocol
In vivo rat PK (Serial sampling)
Male rats are cannulated with a permanent catheter in the jugular vein for blood sampling. The
compound can be dosed by the selected route of administration and repeated blood sampling is
performed from each animal. Typically 8-9 samples are taken during 24 hr post dosing and
replenishing with equal volume of saline each time.
Typical protocol is a single dose intravenous and oral administration with blood collection from
jugular vein catheter with UPLC-MS/MS detection method for plasma. Deliverables include
Cmax, Tmax, AUClast, AUCINF, Vd, T1/2, Cl and F
In vivo mouse PK (Parallel sampling)
Typical protocol is a single dose intravenous and oral administration with blood collection from
each animal by cardiac puncture (n=3 mice per time point) with UPLC-MS/MS detection method
for plasma. Deliverables include Cmax, Tmax, AUClast, AUCINF, Vd, T1/2, Cl and F
The plasma samples generated after proper sample preparation method are measured by
LC/MS/MS and pharmacokinetics parameters are generated using Winnonln program (Pharsight
Corporation)
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com
Plasma concentration of cysteine protease inhibitors in rats after oral administration
Concentration (µg/mL)
10
NPI-8176
NPI-3481
NPI-3474
NPI-3469
1
0.1
0.01
0
2
4
6
8
10
Time (hr)
Plasma concentration of NPI-3469 in rats after oral administration different formulation
Concentration (µg/mL)
10
1
0.1
0.01
0.001
NPI-3469- Formulation-I, Microemulsion
NPI-3469- Formulation -II; Cosolvent
NPI-3469-Formulation-III; HPßCD
0.0001
0
2
4
6
8
10
Time (hr)
Department of Biopharmaceutics & Pharmacokinetics, NAEJA Pharmaceutical Inc. 4290-91A Street,
Edmonton, Canada, T6E5V2; Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com;
www.naeja.com