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

• In vitro cell systems• In vitro plasma binding• In vitro methods• Separation and metabolite quantification

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)

Purified Recombinant Proteins

Grow Cells

Crack Cells

Isolate by Centrifugation

Column Chromatography

E. coli, Yeast, Sf9 insect cells

Microsomes/Supernant

e.g. Ni2+ affinity resin

Clone Gene

Insert Gene into Cells E. coli

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

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

Practical Aspects

• 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 Kineticsphenacetin (CYP1A2) tolbutamide (CYP2C9)

alprazolam (CYP3A4)midazolam (CYP3A4)

Atypical

SigmoidalProduct Inhibition

(Donato, 2010)

Hyperbolic

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)

hexamethoxyflavone(HMF)(UGT1A1)

Lutz, 2008

20%

20%

20%

20%

20%

Dextromethorphan(Cough Suppressant) X

XM1

M2

M3

M4

M5

X X

X X

Method #1

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

etc.

Rate

[CYP3A5]/[CYP3A4]O

CYP3A4 rate

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

• 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://www-

metaprint2d.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

• 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 vitroin vivo, in vitroExtraction Ratio, in vitro in vivo correlation?