CypExpress™ – A Human Cytochrome P450 Catalytic System

  • Each CypExpress product is comprised of a specific, unmodified recombinant human CYP, P450 oxidoreductase cofactors, and antioxidants encapsulated in a semipermeable shell – allowing rapid diffusion of substrates and products.
  • These enzymes are expressed in a eukaryotic system providing an optimized environment for efficient catalysis.
  • CypExpress is a very stable dry powder that is ready to use. Just mix with substrate, incubate and then separate by centrifugation. CypExpress can then be reused for a second reaction.

CypExpress Applications

  • Broad Metabolic Screens (e.g. liver slices &/or microsomes)
  • P450 Phenotyping
       P450 Inhibition
  • Metabolite Identification
  • Structural Characterization of Metabolites
       Drug-­Drug Interactions
       Trapping/Isolation of Reactive Intermediates
  • Synthesis of Key Metabolites
       NOTE: CypExpress protocols for these applications are in development

CypExpress Advantages

CypExpress offers numerous advantages over existing methods of NCE screening and metabolite catalysis.

  • These unmodified human P450 enzymes produce the same metabolite isomers as the human liver
  • Cleaner HPLC results
  • Better metabolite yields
  • Compatible with multiple buffers and higher solvent concentrations
  • Stable dry powder is ready to use
  • Easy to separate using just low speed centrifugation
  • Multiple reaction cycles
  • Significant savings over current methods

P450 Metabolite Identification

CypExpress metabolites are clearly identified by HPLC

  • Cleaner baselines than other systems


  • A lot more metabolite per dollar
  • Cleaner baselines than other system


Scale Up Metabolite Synthesis

  • High percentage conversion of NCEs to metabolites
  • Multiple cycles, if needed, can be run in one day
CypExpress Isoform Cycles Substrate Reaction Product
2C9 1 x 1.5 hr Diclofenac 59.2 mg in 400 mL 4'-hydroxydiclofenac 53.6 mg (90.5%)
2D6 2 x 3 hr Dextromethorphan 27 mg in 200 mL Dextrorphan 7.5 mg (27.8%)
3A4 2 x 3 hr Testosterone 14.4 mg in 200 mL 6β-Testosterone 7.01 mg (48.7%)

Typical Metabolite Yields from Microtiter Plate* Reaction with Different CypExpress

Substrate, 200 µM CypExpress Metabolite Micro Plate µM of Metabolite ± SD*
2D6 Dextrorphan 24‐Well 92.15 ± 0.95
Dextromethorphan 2D6 Dextrorphan 96‐Well 58.17 ± 3.84
Testosterone 3A4 6-­‐β-­‐OH-­‐Testosterone 24-Well 52.37 ± 0.76
Testosterone 3A4 6-­‐β-­‐OH-­‐Testosterone 96‐Well 46.92 ± 7.70
Diclofenac 2C9 4’-­OH-­Diclofenac 24-­Well 80.55 ± 8.32
Diclofenac 2C9 4’-­OH-­Diclofenac 96‐Well 184.89 ± 5.03
S-­Mephenytoin 2C19 S-­4’-­OH-­Mephenytoin 24‐Well 28.73 ± 4.25
S-­Mephenytoin 2C19 S-­4’-­OH-­Mephenytoin 96‐Well 18.07 ± 1.51

*Suitable for rapid P450 phenotyping and metabolite identification

P450 Metabolite Identification

  • More metabolite per dollar

CypExpress = A Better System

System Isoform Needs G6PDH Recommended Reaction Conditions Cost Unwanted Byproducts Ease of Use Metabolite Yield/$
Temp Buffer Time
Microsomes Multiple YES 37 °C Phosphate 30 min $$
Many +/- ++
Bacterial Specific YES 37 °C Phosphate 30 min $$ Few +/- +
Baculoviral Specific YES 37 °C Phosphate 30 min $$$$ Few - +/-
CypExpress Specific NO 20 °C to
37 °C
Phosphate, Tris or Other Min to days $ Few +++ ++++

P450 Phenotyping

At present, the following CypExpress systems are available:

  • 2C9, 2C19, 2D6, 3A4, 1A2 and a vector control
  • These isoforms account for >80% of hepatic P450 metabolism
  • CypExpress systems for additional P450s will be available soon

CypExpress is very stable in both dry form, and in buffers during long incubations

  • Just mix with NCEs to be tested (no other components needed)
  • Incubate with aeration, centrifuge (or filter) and analyze

Phenotyping & Metabolite ID in Microplates

  • CypExpress can be readily used in 96- or 24- or 48-well formats
  • The suspension is easily dispensed by multi-channel pipeting or automated pipeting methods
  • Substrate can be used at 100-200 µM for 10-45+% metabolite yield
  • Adequate aeration is important
  • Recommend ≤1/2 full wells
  • Metabolites are easily identified by HPLC

CypExpress 2D6 Catalyzed synthesis of Dextromethorphan (DOM) from Dextrorphan (DOH) in a microplate


Scale-up Synthesis using CypExpress

Example: CYP2C19-Catalyzed Conversion of Mephenytoin (MP) to 4-Hydroxymephenytoin (HMP)

Cycle Reaction Sample μM of MP Reaction Hours μM of HMP
1 Suspension 500 4 36.07
1 Supernatant 500 4 31.77
2 Suspension Not added 4 94.75
2 Supernatant Not added 4 94.75

Substrate inhibition is observed for high [MP], but with CypExpress a lot more HMP is produced in a second reaction cycle

The Bottom Line

CypExpress offers numerous advantages over previous P450 systems for:

  • P450 Profiling
  • Metabolite identification
  • Metabolite catalysis
  • Unmodified human P450 & NADPH oxidoreductase
  • Expressed in a eukaryotic system
  • Includes G6PDH, NADP+, Mg++ and antioxidant systems
  • Cleaner HPLC results and better metabolite yields
  • Compatible with multiple buffers and higher solvent concentrations
  • Stable, easy to use dry powder
  • Easy to separate using just low speed centrifugation
  • Multiple reaction cycles
  • Significant savings over current methods