Drug-Drug Interaction
These are a critical component of drug discovery and development. They help predict how a new drug might interact with other medications, potentially altering its safety or efficacy. These studies are especially important given the prevalence of polypharmacy—patients taking multiple drugs simultaneously.
CYP P450 reaction phenotyping
| Protocol | |
|---|---|
| Buffer pH | 100 mM potassium phosphate buffer, pH 7.4 |
| Incubation matrix | Bactosomes (Cypex) |
| NADPH Concentration | 1 mM |
| Incubation time | 60 min at 37 °C |
| Test compound incubation concentration | 1 µM |
| Probe substrate incubation concentration | 5 µM |
| Recombinant CYP450 enzymes | CYP1A2; CYP2B6; CYP2C8; CYP2C9; CYP2C19; CYP2D6 and CYP3A4/5 |
| Replicates | n=3 |
| Analysis | LC-MS/MS |
| Test compound Requirement | ~1 mg of powder compound or 20 µL of 10 mM DMSO stock |
Flavin Monooxygenases
Monoamine oxidases
Aldehyde dehydrogenases
Enzyme inhibition
Reversible
Single concentration
| Protocol | |
|---|---|
| Incubation matrix | Pooled Human Liver microsomes |
| CYP Enzymes | As per client request |
| Buffer | 100 mM Potassium Phosphate buffer, pH 7.4 |
| Reaction volume | 200 µL |
| Replicates | n=2 |
| Final NADPH concentration | 1 mM |
| Preincubation time | 10 min @ 37 °C (To ensure all reagents reaches 37 °C) |
| Test compound concentration | 10 µM or as per client requirement |
| Analysis | LC-MS/MS |
| Test compound requirement | ~1 mg of powder compound or 30 µL of 20 mM DMSO stock |
CYP P450 enzymes
- CYP1A2
- CYP2B6
- CYP 2C9
- CYP2C19
- CYP2D6
- CYP3A4/5
Determination of IC50
| Protocol | |
|---|---|
| Incubation matrix | Human Liver microsomes |
| Buffer | 100 mM Potassium Phosphate buffer, pH 7.4 |
| CYP Enzymes | As per client request |
| Reaction volume | 200 µL |
| Replicates | n=2 |
| Final NADPH concentration | 1 mM |
| Preincubation time | 10 min @ 37 °C (To ensure all reagents reaches 37 °C) |
| Test compound concentration | 7-point IC50 curve from 50 µM by 3.33-fold serial dilution or as per suggested concentration by client |
| Analysis | LC-MS/MS |
| Test compound requirement | ~3 mg of powder compound or 100 µL of 20 mM DMSO stock |
CYP P450 enzymes
- CYP1A2
- CYP2B6
- CYP 2C9
- CYP2C19
- CYP2D6
- CYP3A4/5
Determination of Ki
| Protocol | |
|---|---|
| Incubation matrix | Human Liver microsomes |
| CYP Enzymes | As per client request |
| Buffer | 100 mM Potassium Phosphate buffer, pH 7.4 |
| Reaction volume | 200 µL |
| Replicates | n=3 |
| Final NADPH concentration | 1 mM |
| Preincubation time | 10 min @ 37 °C (To ensure all reagents reaches 37 °C) |
| Substrate concentration | 0.5 Km to 5 Km |
| Test compound concentration | 6-point curve from 50 µM by 3-fold serial dilution or as per suggested concentration by client |
| Analysis | LC-MS/MS |
| Test compound requirement | ~4 mg of powder compound or 150 µL of 20 mM DMSO stock |
CYP P450 enzymes
- CYP1A2
- CYP2B6
- CYP2C8
- CYP 2C9
- CYP2C19
- CYP2D6
- CYP3A4/5
Determination of Kinact/Ki
| Protocol | |
|---|---|
| Incubation matrix | Human Liver microsomes |
| CYP Enzymes | As per client request |
| Buffer | 100 mM Potassium Phosphate buffer, pH 7.4 |
| Reaction volume | 200 µL |
| Replicates | n=3 |
| Final NADPH concentration | 1 mM |
| Preincubation time | 15 min (37 °C at 70 rpm) in water bath (To ensure all reagents reaches 37 °C) |
| Primary incubation time | 1, 5, 10, 15, 20, 25 and 30 min at 37 °C with NADPH |
| Test compound concentration | 7-point IC50 curve from 50 µM by 3.33-fold serial dilution or as per suggested concentration by client |
| Analysis | LC-MS/MS |
| Test compound requirement | ~3 mg of powder compound or 70 µL of 20 mM DMSO stock per CYP450 isoform |
CYP P450 enzymes
- CYP1A2
- CYP2B6
- CYP2C8
- CYP 2C9
- CYP2C19
- CYP2D6
- CYP3A4/5
Irreversible (CYP P450) : Time Dependent Inhibition (TDI)
| Protocol | |
|---|---|
| Incubation matrix | Human Liver microsomes |
| CYP Enzymes | As per client request |
| Buffer | 100 mM Potassium Phosphate buffer, pH 7.4 |
| Reaction volume | 200 µL |
| Replicates | n=2 |
| Final NADPH concentration | 1 mM |
| Preincubation time | 10 min @ 37 °C (To ensure all reagents reaches 37 °C) |
| Primary incubation time | 30 min with/without NADPH |
| Test compound concentration | 50 µM or as per suggested concentration by client |
| Analysis | LC-MS/MS |
| Test compound requirement | ~1 mg of powder compound or 30 µL of 20 mM DMSO stock |
CYP P450 enzymes
- CYP1A2
- CYP2B6
- CYP2C8
- CYP 2C9
- CYP2C19
- CYP2D6
- CYP3A4/5
IC50 shift
Non-Dilution method
| Protocol | |
|---|---|
| Incubation matrix | Human Liver microsomes |
| CYP Enzymes | As per client request |
| Buffer | 100 mM Potassium Phosphate buffer, pH 7.4 |
| Reaction volume | 200 µL |
| Replicates | n=2 |
| Final NADPH concentration | 1 mM |
| Preincubation time | 10 min @ 37 °C (To ensure all reagents reaches 37 °C) |
| Primary incubation time | 30 min with/without NADPH |
| Test compound concentration | 7-point IC50 curve from 50 µM by 3.33-fold serial dilution or as per suggested concentration by client |
| Analysis | LC-MS/MS |
| Test compound requirement | ~3-4 mg of powder compound or 150 µL of 20 mM DMSO stock |
CYP P450 enzymes
- CYP1A2
- CYP2B6
- CYP2C8
- CYP 2C9
- CYP2C19
- CYP2D6
- CYP3A4/5
Dilution method
| Protocol | |
|---|---|
| Incubation matrix | Human Liver microsomes |
| CYP Enzymes | As per client request |
| Buffer | 100 mM Potassium Phosphate buffer, pH 7.4 |
| Reaction volume | 200 µL |
| Replicates | n=2 |
| Final NADPH concentration | 1 mM |
| Preincubation time | 10 min @ 37 °C (To ensure all reagents reaches 37 °C) |
| Primary incubation time | 30 min with/without NADPH |
| Test compound concentration | 7-point IC50 curve from 50 µM by 3.33-fold serial dilution or as per suggested concentration by client |
| Analysis | LC-MS/MS |
| Test compound requirement | ~3-4 mg of powder compound or 150 µL of 20 mM DMSO stock |
CYP P450 enzymes
- CYP1A2
- CYP2B6
- CYP2C8
- CYP 2C9
- CYP2C19
- CYP2D6
- CYP3A4/5
CYP P450 Induction (human hepatocytes)
CYP activity measurement
| Protocol | |
|---|---|
| Test system | Hepatocytes seeded in a collagen-coated 96-well plate and overlaid with matrigel |
| CYP isoforms | CYP1A, CYP2B, CYP3A |
| Positive controls | As per the species |
| Vehicle controls | 0.1% DMSO |
| Test compound concentration | 1 Conc. OR 4 Concs. (As per client's suggestion) |
| Inducer treatment duration | 72 h |
| Enzyme substrates | Phenacetin (100 µM), Bupropion (500 µM), Midazolam (20 µM) |
| Substrate incubation time | 30 min |
| Incubation conditions | 37 °C, 5% CO2, 95% RH |
| Replicates | 3 |
| Test compound requirement | ~1mg of powder or 100 µL of 10 mM DMSO stock |
| Analysis | UV spectrophotometer (Cytotoxicity) |
| LC MS/MS (Metabolites: Acetaminophen, Hydroxy-bupropion, Hydroxy-midazolam) | |
Species
- Rat
- Dog
- Human
m-RNA expression
| Protocol | |
|---|---|
| Test system | Hepatocytes seeded in a collagen-coated 96-well plate and overlaid with matrigel |
| CYP isoforms | CYP1A, CYP2B, CYP3A |
| Positive controls | As per the species |
| Vehicle controls | 0.1% DMSO |
| Test compound concentration | 1 Conc. OR 4 Concs. (As per client's suggestion) |
| Inducer treatment duration | 72 h |
| Enzyme substrates | Phenacetin (100 µM), Bupropion (500 µM), Midazolam (20 µM) |
| Substrate incubation time | 30 min |
| Incubation conditions | 37 °C, 5% CO2, 95% RH |
| Replicates | 3 |
| Test compound requirement | ~1mg of powder or 100 µL of 10 mM DMSO stock |
| Analysis | UV spectrophotometer (Cytotoxicity) |
| RT-qPCR (Cyp gene expression) | |
Species
- Rat
- Dog
- Human
Transporters (Inhibitor evaluation)
Caco-2
| Protocol | |
|---|---|
| Transport direction | As per client request |
| Cell Line | Caco-2 (Evotec & ATCC) |
| Test system | Millicell and Corning 96 well plates (0.4 μm pore size, 0.11/ 0.143 cm2 surface area), characterized by TEER >200 ohms*cm2 |
| Incubation conditions | 5% CO2, humidified cell culture incubator maintained at 37 °C |
| Incubation time | 0 and 120 min (OR) 0,30, 60, 90 and 120 min |
| Buffer pH conditions | Donor: HBSS or FaSSIF pH 7.4 or pH 6.5 Receiver: HBSS pH 7.4 with or without 1% BSA |
| Test compound incubation concentration | 2 µM or soluble concentration |
| Replicates | n=2 |
| Analysis | LC-MS/MS |
| Assay controls | Digoxin, Atenolol, Propranolol and Rosuvastatin |
| Final DMSO concentration | 0.1% |
| Assay controls concentration | 10 µM |
| Test compound requirement | ~1mg of powder or 10 µL of 10 mM DMSO stock |
Transport directions
- Bidirection with inhibitor: Apical to Basal / Basal to Apical direction (A to B / B to A) with inhibitor
- Determination of Km, Vmax, Ki and IC50
MDCK II - MDR1
| Protocol | |
|---|---|
| Transport direction | As per client request |
| Cell Line | MDCKII-MDR1 (NKI)-SOLVO |
| Test system | Millicell and Corning 96 well plates (0.4 μm pore size, 0.11/ 0.143 cm2 surface area), characterized by TEER >40 ohms*cm2 |
| Incubation conditions | 5% CO2, humidified cell culture incubator maintained at 37 °C |
| Incubation time | 0 and 120 min |
| Buffer pH conditions | Donor: HBSS pH 7.4 Receiver: HBSS pH 7.4 with 1% BSA |
| Test compound incubation concentration | 2 µM or soluble concentration |
| Inhibitor concentration | 10 µM (Elacridar or Zosuquidar) |
| Replicates | n=2 |
| Analysis | LC-MS/MS |
| Assay controls | Digoxin, Atenolol and Propranolol |
| Final DMSO concentration | 0.2% |
| Assay controls concentration | 10 µM |
| Test compound requirement | ~1mg of powder or 10 µL of 10 mM DMSO stock |
Transport directions
- Bidirection with inhibitor: Apical to Basal / Basal to Apical direction (A to B / B to A) with inhibitor
- Determination of Km, Vmax, Ki and IC50
MDCK II – BCRP
| Protocol | |
|---|---|
| Transport direction | As per client request |
| Cell Line | MDCKII-BCRP (SOLVO) |
| Test system | MDCKII-BCRP monolayer grown on Millicell and Corning 96 well plates (0.4 μm pore size, 0.11/ 0.143 cm2 surface area) for 5 days, characterized by TEER >70 ohms*cm2 |
| Incubation conditions | 5% CO2, humidified cell culture incubator maintained at 37 °C |
| Incubation time | 120 min |
| Buffer pH conditions | Donor: HBSS pH 7.4 Receiver: HBSS pH 7.4 with 1% BSA |
| Test compound incubation concentration | 2 µM or soluble concentration |
| Inhibitor concentration | 10 µM (Ko143) |
| Replicates | n=2 |
| Assay controls | Prazosin, Atenolol & Propranolol |
| Final DMSO concentration | 0.2% |
| Assay controls concentration | 10 µM |
| Test compound requirement | ~1mg of powder or 10 µL of 10 mM DMSO stock |
Transport directions
- Bidirection with inhibitor: Apical to Basal / Basal to Apical direction (A to B / B to A) with inhibitor
- Determination of Km, Vmax, Ki and IC50
Transfected HEK-293
| Protocol | |
|---|---|
| Cell line | Transfected HEK-293 |
| Transporter | As per client request |
| Incubation Condition | 37 °C, uptake of test compound at 1 min and 5 min |
| Incubation volume | 75 uL |
| Test compound and assay control concentration | 2 µM |
| Assay controls | As per the transporter |
| Replicates | n=3 |
| Analysis | LC-MS/MS |
| Inhibitor | As per the transporter |
| Test compound requirement | ~1mg of powder or 10 µL of 10 mM DMSO stock |
Transporters
- BSEP
- NTCP
- OATP1B1/1B3
- OAT1/OAT3
- MATE1 and 2
- PEPT1 and 2
- Determination of Km, Vmax, Ki and IC50
