Drug Absorption and Transport
Essential studies in drug discovery for predicting how a compound will behave in the human gastrointestinal (GI) tract. These studies help assess a drug’s bioavailability, permeability, and interaction with transporters, which are critical for oral drug development.
Permeability evaluation
Parallel Artificial Membrane Permeability Assay (PAMPA)
| Protocol: Permeability at four different pH’s (4.0, 5.0, 6.5 and 7.4) | |
|---|---|
| Assay Format | Stirwell PAMPA Sandwich |
| Buffer System | Phosphate buffer |
| pH values used | 4.0/7.4, 5.0/7.4, 6.5/7.4 and 7.4/7.4, |
| Assay Controls | Furosemide, Antipyrine, Piroxicam, Propranolol |
| Assay concentration | 10 µM |
| Incubation time | 4 hours |
| Incubation Temperature | Room temp. |
| Reaction volume | 200 µL |
| Replicates | n=3 |
| % DMSO content | <1% |
| Analysis | LC-MS/MS |
| Deliverables | Permeability (Pe, x 10-6cm/sec) |
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
- Unidirection: Apical to Basal direction (A to B)
- Bidirection: Apical to Basal / Basal to Apical direction (A to B / B to A)
- 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 wild type (WT)
| Protocol | |
|---|---|
| Transport direction | As per client request |
| Cell Line | MDCKII-WT (NKI) |
| 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 |
| 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
- Unidirection: Apical to Basal direction (A to B)
- Bidirection: Apical to Basal / Basal to Apical direction (A to B / B to A)
- 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
- Unidirection: Apical to Basal direction (A to B)
- Bidirection: Apical to Basal / Basal to Apical direction (A to B / B to A)
- 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
- Unidirection: Apical to Basal direction (A to B)
- Bidirection: Apical to Basal / Basal to Apical direction (A to B / B to A)
- 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
Vesicle transport assays
- BSEP / NTCP
Vesicle transport assays
- BSEP / NTCP
Uptake Transporters (Substrate evaluation)
Hepatocyte Uptake Assay
Oil spin method
| Protocol | |
|---|---|
| Species | As per the client request |
| Hepatocytes | Transporter qualified |
| Time points | 0.5, 1.5, 3.0 and 5.0 min |
| Test compound Incubation concentration | 1 µM |
| Inhibitor | Rifamycin SV (0.5 mM) |
| Cell density | 1*106 cells/mL |
| Assay Control | Rosuvastatin |
| Replicates | n=3 |
| Incubation time | 5 min at 37 °C |
| Analysis | LC-MS/MS |
| Final DMSO concentration | 0.1% |
| Test compound requirement | ~1mg of powder or 10 µL of 10 mM DMSO stock |
Hepatocytes
- Mouse hepatocytes
- Rat Hepatocytes
- Dog hepatocytes
- Monkey hepatocytes
- Human hepatocytes
Media Loss method
| Protocol | |
|---|---|
| Species | As per the client request |
| Hepatocytes | Transporter qualified |
| Time points | 0.5, 1.5, 3.0 and 5.0 min |
| Test compound Incubation concentration | 1 µM |
| Inhibitor | Rifamycin SV (0.5 mM) |
| Cell density | 1*106 cells/mL |
| Assay Control | Rosuvastatin |
| Replicates | n=3 |
| Incubation time | 5 min at 37 °C |
| Analysis | LC-MS/MS |
| Final DMSO concentration | 0.1% |
| Test compound requirement | ~1mg of powder or 10 µL of 10 mM DMSO stock |
Hepatocytes
- Mouse hepatocytes
- Rat Hepatocytes
- Dog hepatocytes
- Monkey hepatocytes
- Human hepatocytes
