1. Metabolite Kinetics II
Arthur G. Roberts

2. Question
Is there a drug that you need to reduce the dosage after subsequent dosing?
Answer: Yes, if the drug exhibits product inhibition.

3. Outline In vitro cell systems In vitro plasma binding In vitro methods
Separation and metabolite quantification

4. In vitro Systems 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)

5. Purified Recombinant Proteins
Clone Gene
Insert Gene into Cells
E. coli
Grow Cells
E. coli, Yeast, Sf9 insect cells
Crack Cells
Isolate by Centrifugation
Microsomes/Supernant
Column Chromatography
e.g. Ni2+ affinity resin

6. Baculovirus insect cell expressed proteins

8. Liver Cancer Cell Lines
Hep G2
Hepatocellular carcinoma
C3A
Hepatoblastoma
PLC/PRF/5
Hepatoma

9. Transfected Cell Lines
Overexpression of Phase I and Phase II enzymes into Cells
Cell Lines
V79 Chinese hamster
Hep G2
MLC-5 human lymphoblast

10. Hepatocytes trypan blue dye exclusion method
They often use the tryptan blue method to assess viability. selectively colour dead tissues and cells blue.
trypan blue dye exclusion method

11. Liver Slices

12. Isolated Perfused Liver

13. In vitro Methods Practical aspects Plasma protein binding
Metabolite Formation Kinetics
Inhibition
Enzyme Mapping/Reaction Phenotyping
Metabolic Stability
Metabolite Profiling
Computational Methods
Cross species ADME

14. Practical Aspects Incubation vessel Assay buffer Cofactor sources
Initiating incubations
Maintaining incubations
Stopping incubations
Storage
Other Considerations

15. 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.

16. In vitro plasma binding
Drug
Drug

17. 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

18. Metabolite Formation Kinetics
phenacetin (CYP1A2)
tolbutamide (CYP2C9)
Hyperbolic
alprazolam (CYP3A4)
midazolam (CYP3A4)
Atypical
Sigmoidal
Product Inhibition
(Donato, 2010)

19. 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.

20. 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)

21. Example #1: Selective Inhibition with Dextromethorphan in microsomes

22. Uninhibited ketoconazole (CYP3A4) quinidine (CYP3A4)
shorthand:
P = CH3-O-R-N-CH3
M1= CH3-O-R-NH
M2= HO-R-N-CH3
M3= HO-R-NH
M4= GluO-R-N-CH3
M5= GluO-R-NH
hexamethoxyflavone
(HMF)
(UGT1A1)

23. X X X Method #1 Dextromethorphan (Cough Suppressant) 20% M3 M1 M4 M2
Specific CYP3A4 inhibitor ketoconazole X M2 M5
Lutz, 2008

24. Example #2: Correlation with midazolam, CYP3A4 and CYP3A5 in microsomes

25. Midazolam 1’-OH Hydroxylation 0.02 mg/mL 0.01 mg/mL 1.2 nmol/mg*min
[CYP3A4]
[CYP3A5]
Midazolam 1’-OH Hydroxylation
0.02 mg/mL
0.01 mg/mL
1.2 nmol/mg*min
1.0 nmol/mg*min
etc.
Rate
[CYP3A5]/[CYP3A4] O
CYP3A4 rate
Which one has the higher activity (CYP3A4 or CYP3A5)? How to determine the activity of CYP3A5?

26. Reaction Phenotyping: Drug Development
Discovery
High Throughput/Major CYPs
Early Development
Metabolites and Polymorphic CYPs
Full Development
Detailed kinetics
HLM and recombinant protein

27. Metabolic Stability Required for Lead Compounds
Must be stable in blood  target
Drug metabolism major clearance pathway
Desirable pharmacokinetic properties
(Masimirembwa, 2003)

28. Metabolic Stability
Which one has the higher metabolic stability?

29. Estimating Clint from Metabolic Stability
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

30. Metabolite Profile
identify possible active, toxic and inactive metabolites
recombinant protein to liver hepatocytes
assists in the interpretation of in vivo animal experiments.

31. 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)

32. Computational: Metabolism Prediction Software
Proprietary
Metasite
ADMET Predictor
Free
SMARTCyp (
MetaPrint2D (

33. 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)

34. Separation and Identification
Liquid Chromatography
Metabolite Identification
Radio flow detection/scintillation counting
Mass Spectrometry
NMR
Fluorescence spectroscopy
UV-visible spectroscopy
detects radiolavels

35. Example 0.05 mg/L of Ro15-4513 0.001 mg/mL liver microsomes
Ro 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?