Metabolite Kinetics II
Arthur G. Roberts
Question
• Is there a drug that you need to reduce the
dosage after subsequent dosing?
– Answer: Yes, if the drug exhibits product
inhibition.
Outline
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In vitro cell systems
In vitro plasma binding
In vitro methods
Separation and metabolite quantification
In vitro Systems
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Purified Recombinant proteins
Baculovirus insect cell expressed proteins
Liver microsomes
Liver cytosol
Liver S9
Liver Cancer Cell Lines
Transfected Cancer Cell Lines
Hepatocytes
Liver slices
Isolated perfused liver (in situ)
Purified Recombinant Proteins
Clone Gene
Insert Gene into Cells
Grow Cells
E. coli
E. coli, Yeast, Sf9 insect cells
Crack Cells
Isolate by
Centrifugation
Column
Chromatography
Microsomes/Supernant
e.g. Ni2+ affinity resin
Baculovirus insect cell expressed
proteins
Liver Cancer Cell Lines
• Hep G2
– Hepatocellular carcinoma
• C3A
– Hepatoblastoma
• PLC/PRF/5
– Hepatoma
Transfected Cell Lines
• Overexpression of Phase I and Phase II
enzymes into Cells
• Cell Lines
– V79 Chinese hamster
– Hep G2
– MLC-5 human lymphoblast
Hepatocytes
trypan blue dye exclusion method
Liver Slices
Isolated Perfused Liver
In vitro Methods
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Practical aspects
Plasma protein binding
Metabolite Formation Kinetics
Inhibition
Enzyme Mapping/Reaction Phenotyping
Metabolic Stability
Metabolite Profiling
Computational Methods
Cross species ADME
Practical Aspects
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Incubation vessel
Assay buffer
Cofactor sources
Initiating incubations
Maintaining incubations
Stopping incubations
Storage
Other Considerations
Other Considerations
• Many enzymes are inhibited or stimulated by
organic solvent.
• Flavin-containing monooxygenase (FMO)
nortorious sensitive freezing.
• UGT enzymes in tissue fractions demonstrate
“latency”
– detergents or pore forming agents alamethicin
• Industry institutes automation.
In vitro plasma binding
Drug
Drug
Metabolite Formation Kinetics
• Purpose: Determine kinetic parameters
– Km, Vmax, estimate Clint=Vmax/Km
• Metabolite formation linear with [E]
• low [E], below Km
• Beware of substrate depletion because
velocities are measured from initial slopes
Metabolite Formation Kinetics
phenacetin (CYP1A2)
tolbutamide (CYP2C9)
Hyperbolic
alprazolam (CYP3A4)
midazolam (CYP3A4)
Atypical
Sigmoidal
Product Inhibition
(Donato, 2010)
Inhibition
• Inhibition major cause of drug-drug interactions (DDIs)
– CYPs
• Reversible Inhibition
– Competitive, noncompetitive, uncompetitive, mixed-type
• Suicide/Mechanism-based (Reactive Intermediate)
– Covalently binds to enzyme (kills enzyme)
– Idiosyncratic reactions
• toxic/fatal immunogenic response
– Potency increases with time
• Auto-inhibition (non-linear PK)
– Rational Drug Design
• Specific
• Few side effects.
Enzyme Mapping/Reaction
Phenotyping
• Estimation of the relative contributions of
specific enzymes to the metabolism of a test
compound
• Approaches
– Recombinant Proteins (Specific)
– Selective Inhibition (Microsomes, Pooled
Specimens)
• Beware of inhibitor depletion (partial inhibition)
– Correlation (Multiple Specimens)
(Chapter 15 of Drug Metabolism in Drug Design and Development)
Example #1: Selective Inhibition with
Dextromethorphan in microsomes
Uninhibited
ketoconazole
(CYP3A4)
quinidine
(CYP3A4)
hexamethoxyf
lavone
(HMF)
(UGT1A1)
Method #1
Dextromethorphan
(Cough Suppressant)
X
X
M1
20%
X
X
20%
X
M2
20% M3
20%
M4
20%
M5
X
Lutz, 2008
Example #2: Correlation with
midazolam, CYP3A4 and CYP3A5 in
microsomes
[CYP3A4]
[CYP3A5]
Midazolam 1’-OH
Hydroxylation
0.02 mg/mL
0.01 mg/mL
1.2 nmol/mg*min
0.01 mg/mL
0.01 mg/mL
1.0 nmol/mg*min
Rate
etc.
CYP3A4 rate
O
[CYP3A5]/[CYP3A4]
Which one has the higher activity (CYP3A4 or CYP3A5)? How to determine the activity of CYP3A5?
Reaction Phenotyping: Drug
Development
• Discovery
– High Throughput/Major CYPs
• Early Development
– Metabolites and Polymorphic CYPs
• Full Development
– Detailed kinetics
– HLM and recombinant protein
Metabolic Stability
• Required for Lead Compounds
– Must be stable in blood  target
• Drug metabolism major clearance pathway
• Desirable pharmacokinetic properties
(Masimirembwa, 2003)
Metabolic Stability
Which one has the higher metabolic stability?
Estimating Clint from Metabolic
Stability
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Low [Enzyme]
Low [S] << Km
Measure parent at different times (Substrate Depletion)
Estimate the elimination rate constant (K) or the t1/2
Calculate the Clint, in vitro and Clint, in vivo
Metabolite Profile
• identify possible active, toxic and inactive
metabolites
• recombinant protein to liver hepatocytes
• assists in the interpretation of in vivo animal
experiments.
Computational: Identification of Hot
and Soft Spots
• Hot spots
– Reactive metabolic site on a drug.
• Soft spots
– Reactive metabolic site on a drug that leads to
rapid metabolism (i.e. metabolic instability)
(Cross, 2010 Drug Discovery Today)
Computational: Metabolism Prediction
Software
• Proprietary
– Metasite
– ADMET Predictor
• Free
– SMARTCyp (http://www.farma.ku.dk/smartcyp/)
– MetaPrint2D (http://wwwmetaprint2d.ch.cam.ac.uk/metaprint2d/)
Cross Species ADME
• Appropriate animal selection
– animals with “human livers” (Strom, 2010)
• In-vivo metabolic profile
– pharmacological and biological activity
– monitoring (bioanalytical method)
– human toxicity
• Reaction Phenotyping (Animal vs. Human)
(p. 212, Drug Metabolism in Drug Design and Development)
Separation and Identification
• Separation
– Liquid Chromatography
• Metabolite Identification
– Radio flow detection/scintillation counting
– Mass Spectrometry
– NMR
– Fluorescence spectroscopy
– UV-visible spectroscopy
Example
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0.05 mg/L of Ro15-4513
0.001 mg/mL liver microsomes
Ro15-4513 was depleted at a rate of 0.02 hr-1
In vitro plasma binding, 90%
In vivo human Clh = 56 to 70 L/hr
Clh in vitroin vivo, in vitroExtraction Ratio,
in vitro in vivo correlation?