Transcript A Short Course in Pharmacokinetics

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Pharmacodynamics
Chris Town
Research Pharmacokinetics
March 22, 2005
Christopher Town, Ph.D.
Definitions
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Pharmacodynamics:
the study of the biochemical and physiological effects
of drugs and the mechanisms of their actions,
including the correlation of action and effects of drugs
with their chemical structure; also, the relationship
between drug concentration and effect.
March 22, 2005
Christopher Town, Ph.D.
Click toPharmacodynamics
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• Principle: The Activity of all compounds is caused
by interaction with receptors in the body
– Receptors are biomolecules in the body
• Enzymes
• Cell Surface Receptors
• Nuclear receptors
• Etc.
March 22, 2005
Christopher Town, Ph.D.
Drug Responses from Different Types of Interactions
with Receptors
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• Direct reversible effects
– Ca Channel Blockers
– Beta-Blocker
• Indirect reversible effects
– Nuclear Hormone Receptors
– Anti-coagulants
• Irreversible effects
– Anti-Cancer drugs
– Antibiotics
March 22, 2005
Christopher Town, Ph.D.
Click toPharmacodynamics
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Processes driven by:
Cmax (Maximum Concentration in Plasma)
AUC (Overall Exposure)
Caverage (Concentration during study)
Examples of different processes in Antibiotics:
Beta-lactams (time dependent, no persistance)
Tetracyclines (time-dependent, with persistance)
Flouroquinloline (Concentration dependent, with
persistance
Examples in organs and tissues:
Nuclear Hormone Receptors
Cell Surface Receptors
Enzyme inhibitors
March 22, 2005
Christopher Town, Ph.D.
Linear to
Pharmacodynamic
Response
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Response (% of Maximum)
Linear Response to Drug Concentration
100
80
60
40
20
0
0.0001 0.001
0.01
0.1
1
10
100
Drug Concentration (g/l)
Receptor in Central Compartment
March 22, 2005
Christopher Town, Ph.D.
PK Model with Effect Compartment
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Drug in
Central
Compartment
k12
Drug in
Tissue
k21
k1e
k10
Effect
Compartment
March 22, 2005
ke0
Christopher Town, Ph.D.
Hysteresis
in Plot
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Response (% of Maximum)
Response to Drug Concentration
100
80
60
40
20
0
0.0001 0.001
0.01
0.1
1
10
100
Drug Concentration (g/l)
Receptor Not in Central Compartment
March 22, 2005
Christopher Town, Ph.D.
ClickPharmacodynamic
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• The interaction between different drugs and
individual receptors may show different affinities and
different potency and efficacy of response
• The reaction between drugs and receptors may have
different response times
– Different between tmax and Maximum response
• Modeling requires accurate bioanalytics and
reasonable measurement of response with time
March 22, 2005
Christopher Town, Ph.D.
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Exposure Screening
March 22, 2005
Christopher Town, Ph.D.
Typical
Screening
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toDiscovery
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titleCascade
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In Vitro Enzyme Assay
Cell Based Assay
Selectivity
CYP Inhibition screening
Microsomal stability (high CL)
Exposure screening
Caco-2 permeability
IV/PO PK studies
solubility
Screening
PK
Acute in vivo efficacy model
Expanded PK (definitive, TK,
Formulations, etc…)
Chronic Efficacy model
DP1 Candidate
March 22, 2005
Christopher Town, Ph.D.
Defining
PK/DM
Issuestitle
in a Project
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How do we help define the issues?
• Conduct a series of investigative studies on the Initial lead
– IV/Oral PK study (in efficacy species)
– Microsomal stability (efficacy & other species)
• Metabolite ID work for very unstable compounds
– Caco-2 permeability study (+/- Pgp inhibitor)
– CYP inhibition (5 major isoforms)
– Protein binding
• (especially for compounds with major disparity between
intrinsic and cell-based activity)
– Solubility and Pharmaceutical Technology assessment
(+solution/suspension comparison, as needed)
– P450 induction
– Target level analysis (e.g. tumor, brain, etc..)
– In vitro plasma stability
– Multi-cannulated rat model (as needed)
March 22, 2005
Christopher Town, Ph.D.
Screening
to aid title
in Chemical
ClickoftoInitial
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Master
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Design Screen to address Compound’s shortcomings
– Metabolically Unstable (Microsomal Stability)
– Poor Absorption (CaCo-2 Cell Permeability)
– Rapid Elimination (IV administration in rats)
– CYP 450 Inhibitor (P450 Inhibition)
• Perform full test with random compounds to
determine if problems with the chemical series have
changed.
March 22, 2005
Christopher Town, Ph.D.
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The Screening
Cascade title
for HSL
HSL inhibition
<100 nM IC50
Cellular activity assay
<100 nM EC50
Selectivity
50x vs. ACPH, LPL, HL, PL, AChE
Fasted mouse model
Toxicity Screening
Microsome / blood stability
March 22, 2005
Efficacy at 4h at 10 mg/kg p.o.
100 / 300 mpk in rat
Mouse, rat, dog, human
PK plasma levels
Supports q.d. dosing
Diabetic rodent
> 50 mg/dL glucose decrease
Christopher Town, Ph.D.
Discovery
Project
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Lead Structure determined from HTS
•
•
•
•
Active in vitro activity
Low exposure in vivo
Active in vivo
PK issues may play a role
O
O
N
O
March 22, 2005
N
Christopher Town, Ph.D.
Analysis
fromtitle
In vitro
Studies
ClickoftoCompounds
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• Concentrations determined using LC/MS/MS with a
simple assay
– No Internal Standard
• Calculate Concentration from change in instrument
response (Peak Area)
• Compare remaining drug concentration in presence
and absence of NADPH (a necessary Cofactor for
enzymatic breakdown) over time.
March 22, 2005
Christopher Town, Ph.D.
Compound
Stability
in Liver
Microsomes
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% of InitialConcentration of Compound 8
100
80
60
Human
40
Mouse
20
Beagle
Rat
0
0
10
20
30
40
Time (min)
Compound 8 most unstable in rat and dog liver microsomes
more stable in human and mouse
March 22, 2005
Christopher Town, Ph.D.
Stability
in RatMaster
Blood and
Plasma
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Disappearance of Compound 8 from rat blood and plasma
Compound 8 (g/l)
100
10
Blood
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0
2
4
6
8
10
Plasma isolated
from blood
Plasma
Time (min.)
Compound 8 appears to break down in plasma and whole
blood and apparently concentrates in Red Blood Cells
March 22, 2005
Christopher Town, Ph.D.
Stability
Human
Blood title
and Plasma
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Stability of Compound 8 in Human Blood and Plasma
Compound 8 (mg/l)
100
Plasma
Blood
Plasma isolated
from blood
10
1
0
10
20
30
40
50
60
Time (h)
Compound 8 is quite stable in human blood and plasma
(similar findings with dog blood and plasma)
March 22, 2005
Christopher Town, Ph.D.
Analysis
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In vivo
Studies
ClickoftoCompounds
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• Concentrations determined using LC/MS/MS
• Need Internal Standard
• Calibration curve made from 8 standards (5 – 5000 g/l
Calculate Concentration from change in instrument response
(Peak Area)
• Quality Control samples added to sample run in duplicate or
triplicate
• QC samples spread throughout sequence to make certain
instrument is not changing over time
• Need a simple sample preparation procedure that works with
most drugs
• Rugged LC column that can handles hundreds of samples
• LIMS system helps keep track of data.
March 22, 2005
Christopher Town, Ph.D.
HPLC System
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March 22, 2005
Christopher Town, Ph.D.
Rugged
for LC
System
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Leap Technologies CTS Pal Autoinjector
March 22, 2005
Christopher Town, Ph.D.
Versatile
LC/MS/MS
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Applied Biosystems API 4000
March 22, 2005
Christopher Town, Ph.D.
Micestyle
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Concentration of Compound
8o
in
mouse
blood
after
oral dosing
C
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BAY5-9241(ug/l)
I
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Compound is measurable in whole blood when rapidly collected and
inactivated. Efficacy appears to be Cmax driven.
March 22, 2005
Christopher Town, Ph.D.
Compound
RatsMaster
after Oraltitle
Administration
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Rat Blood Concentrations after 100 mg/kg, PO
1000
O
N
O
N
Compound 8 (g/l)
O
100
IC50
10
1
0
1
2
3
4
Time (h)
Compound 8 does not reach high concentrations in rats after high doses
March 22, 2005
Christopher Town, Ph.D.
Compound
8 inMaster
rat blood title
and plasma
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 Compound 8 breaks down in whole rat blood and
plasma.
 Following isolation of plasma after spiking whole
blood with Compound 8, the compound is found to
concentrate more in red blood cells than in plasma.
 Compound 8 has about a 50-fold lower concentration
than expected in serum when the compound has
been spiked into whole blood prior to isolation of the
serum.
 Analysis by LC/MS/MS allows the compound to be
measured in whole blood or red blood cells without
interference from the matrix.
March 22, 2005
Christopher Town, Ph.D.
8 in Rat title
Bloodstyle
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1000
Compound 8 (g/l)
iv - 5 mg/kg
po systemic - 20 mg/kg
po portal - 20 mg/kg
100
10
1
0.1
0
1
2
3
4
5
6
7
Time (hr)
Comparison of portal vein concentrations and systemic
concentrations suggests high first past
March 22, 2005
Christopher Town, Ph.D.
Microsomal
Several Lead
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title Compounds
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% Compound Remaining at 10 Minutes
Mouse Mouse Rat
Rat Rat (STZ) Dog Human
Compound (CD1) (Balb/c) (SD) (Wistar) (Wistar) (Beagle) (pool)
Compound 8 68.7
43.5
39.7
58.9
61.2
55.5
80.2
Compound 30
31.7
18.0
43.4
34.4
82.2
Compound 57
52.9
51.9
61.3
63.4
81.8
Compound 65 17
69.7
Compound 72
44.8
78.0
77.1
81.6
72.2
Compound 76
51.8
72.2
74.8
91.7
83.1
Compound 79
29.4
59.4
62.5
90.6
77.9
The stability of all compounds in rodent is low
The extent of in vitro instability is not predictive of in vivo exposure
March 22, 2005
Christopher Town, Ph.D.
Lead Structures
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F
O
O
MeO
O
O
N
N
N
N
O
O
Compound 72
Compound 76
O
O
N
N
O
Compound 57
March 22, 2005
Christopher Town, Ph.D.
Exposure of 3 lead HSL Compounds in Rats
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Rat Blood Concentrations of HSL inhibitors
After Oral Administration of 10 mg/kg
100
Concentration (M)
10
1
Compound 76
Compound 57
0.1
Compound 72
0.01
0.001
0
1
2
3
4
Time (h)
Compound 57 shows highest concentration at 1 h
March 22, 2005
Christopher Town, Ph.D.
Efficacyto
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Compound
57 in Fasted Mice
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BAY
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Plasma FFA (% of Initial Level)
120
mg/kg, p.o.
0
1
3
10
110
100
90
80
70
60
0
1
2
3
4
5
6
Hours
Compound 57 shows dose-dependent efficacy in mice
March 22, 2005
Christopher Town, Ph.D.
PK after Intravenous Administration
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Concentrations of Compound 57 in blood after
intravenous administration
10
Dog - 2 mg/kg
Rat - 5 mg/kg
Compound 57 (uM)
1
0.1
0.01
0.001
0
2
4
6
Time (hours)
Species
Dog
Rat
Dose AUC0-tn
C0
mg/kg (mg*h)/l mg/l
2
235
846
5
161
2160
t1/2
h
1.14
0.70
Cl
l/h/kg
4.25
6.19
Vdss
l/kg
4.64
4.91
High clearance and volume of distribution in rat and dog
March 22, 2005
Christopher Town, Ph.D.
Identifying
best compound
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HSL inhibition
> 800 (> 450 synthesized)
Cellular lipolysis assay
> 350 compounds
Selectivity
> 300 compounds
Fasted mouse model
50 compounds
Toxicity Screening
29 compounds
2-Week Rat Tox
4 compounds
Dog Tox
(1 compound)
DP1
Pharmacokinetics did not play a major role in final compound choice
March 22, 2005
Christopher Town, Ph.D.
Compound
in vivo Properties
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•
shows good efficacy but low systemic exposure
•
shows low stability in liver microsomes from all
species tested, and rat blood and plasma
•
high volume of distribution in both rat and dog
•
showed toxicity in gut wall and testes
•
might be concentrating in the tissues where it was
causing toxicity
•
radiolabeled material was made and a whole body
autoradiography in rats was undertaken to check for
accumulation
March 22, 2005
Christopher Town, Ph.D.
of HSL Project
ClickConclusion
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• Could not move forward into development because of issues of
toxicity and inadequate PK
• Chemistry could not change core of molecule without losing
activity
• The program has not moved forward into clinical studies
March 22, 2005
Christopher Town, Ph.D.
Structure
HSL style
Inhibitors
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toThree
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Master title
1. Novo Nordisk
2. Aventis
3. Aventis
4. Bayer
Ebdrup, S.; Sorensen, L. G.; Olsen, O. H.; Jacobsen, P. Synthesis and
Structure-Activity Relationship for a Novel Class of Potent and
Selective Carbamoyl-Triazole Based Inhibitors of Hormone Sensitive
Lipase. J. Med. Chem. 2004; 47, (2), 400-410.
March 22, 2005
Christopher Town, Ph.D.
Click toHSL
editKnockout
Mastermice
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• HSL-deficient male mice are infertile
• Testes of male HSL-deficient mice are abnormal
• HSL appears to play a major role in spermatogenesis
Infertility and testicular defects in hormone-sensitive lipase-deficient
mice. Chung S, Wang SP, Pan L, Mitchell G, Trasler J, Hermo L.
Endocrinology. 2001 Oct;142(10):4272-81.
March 22, 2005
Christopher Town, Ph.D.
Lessons
Learned
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• It is very difficult to advance to human studies with efficacious
compounds that have bad PK
• There is little that you can do with a chemical series if the core
is metabolically unstable
• It is normally best to start a project with more than one chemical
series
• Even the most promising methods of treatment can’t produce a
product unless a compound with good PK and tox profiles can
be found
March 22, 2005
Christopher Town, Ph.D.
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Contributors
Metabolics
Research Technologies
Mike Burns
Tom Claus
Kevin Clairmont
Mary Guinness
Christine Keiper
Yaxin Li
Michelle Daly
Arthur Salhanick
Vicki Trouern-Trend
Terri Witman
Ling Yang
Bob Dreyer
Tim Garrison
Gregg Hirschfeld
Pharmaceutical
Technology
Marian Carthy
Ed Povilaitis
Alma Tarampi
Jim Williams
Chemistry
Furahi Achebe
Jon Brice
Bill Bullock
AnnMarie Campbell
Jim Cook
Lamont Cranston
Rob Dally
David Dickson
Sookhee Ha
Lee Huang
Zhenqiu Hong
Harold Kluender
Qingjie Liu
Derek Lowe
Steve Magnuson
Ingo Mugge
David Miller
Laszlo Musza
Tony Paiva
Brent Podlogar
Ning Qi
Mareli Rodriguez
Ming Wang
Wai Wong
Xiaoquing Yin
Preclinical
Paul Adams
Vipin Agarwal
Tanja AlebicKolbah
Joan Bienvenue
Sue Jenkins
Changfu Cheng
Ron Mays
March 22, 2005
Tim Nicholas
Matthew Prevost
Jose Rivera
Wolfgang Rossberg
Kenny Seaver
Chris Town
Yaodong Xu
Debbie Young
Core Team
Kevin Clairmont Wolfgang Rossberg
Tom Claus
Chris Town
Derek Lowe
Jim Williams
Nick Livingston
Christopher Town, Ph.D.