Transporter Substrate Identification
As a leading CRO, BOC Sciences provides in vitro technology-based services for the transporter substrate identification, an essential part of the optimization of pharmacokinetic properties of lead compounds.
What is Transporter Substrate Identification?
The pharmacokinetic properties of a drug are influenced not only by the physicochemical properties of the drug itself, but also by the metabolic enzymes and transporters in the human body. Membrane transporters influence the distribution of drugs in the body by selective transport of some drugs. In general, the more lipid-soluble drugs are transported across membranes by passive diffusion in the body, so they are distributed more uniformly in all organs and tissues. Hydrophilic drugs are generally less permeable to membranes, and some of them require the participation of membrane transporters for transmembrane transport, which results in the absorption and transport of drugs being affected by the type of transporters and tissue distribution, thus causing large differences in the concentration levels of drugs in various organs and tissues.
The transporter substrate identification refers to in vitro methods to evaluate whether a drug under investigation is a substrate for transporters, including MDR1 (P-gp), BCRP, OAT1, OAT3, OCT2, MATE1, MATE2K, OATP1B1 and OATP1B3. If a new chemical entity (NCE) is an inhibitor of a transporter, the pharmacokinetic properties of the drug are altered, leading to a drug-drug interaction (DDI), which requires a substrate test for the corresponding transporter.
Fig.1 Model of the transporter and substrate.
Transporters and Substrates
| Transporters | Specific Substrates | Specific Inhibitors |
| P-gp/MDR1 | Digoxin, loperamide, irinotecan, doxorubicin, vincristine, paclitaxel, fexofenadine | Cyclosporine, quinidine, tariquidar, verapamil |
| BCRP/MXR | Mitoxantrone, amethopterin, topotecan, imatinib, irinotecan, statins, sulfate conjugates, porphyrins | Estrone, 17beta-estradiol, fumitremorgin C |
| OATP1B1 | Bromosulphophthalein, oestrone-3-sulphate, oestradiol-17β-glucuronide, statins, repaglinide, valsartan, olmesartan, bilirubin glucuronide, bilirubin, bile acid | Saquinavir, ritonavir, lopinavir, rifampin, cyclosporine |
| OATP1B3 | Bromosulphophthalein, cholecystokinin 8, statins, digoxin, fexofenadine, telmisartan, valsartan, olmesartan, oestradiol-17β-glucuronide, bile acid | Rifampin, cyclosporine, ritonavir, lopinavir |
| Organic Anion Transporter 1 (OAT1) | para-aminohippurate, adefovir, cidofovir, zidovudine, lamivudine, zalcitabine, aciclovir, tenofovir, ciprofloxacin, methotrexate | Probenecid, neomycin |
| Organic Anion Transporter 3 (OAT3) | Oestrone-3-sulphate, nonsteroidal anti-inflammatory drug, cefaclor, ceftizoxime, bumetanide | Probenecid, neomycin |
| Organic Cation Transporter 2 (OCT2) | N-Nethylpyridinium, tetraethylamine, dimethylpyridinium, procainamide, ranitidine, amantadine, amiloride, oxaliplatin, varenicline | Cimetidine, cetirizine, testosterone, quinidine |
Table. 1 Major transporters and their substrates, inhibitors
Our Methods
In Vitro System
The evaluation system of the transporter substrate identification includes Inside-out membrane vesicles, Caco-2 cells and transfected cell lines (e.g. MDCK, LLC-PK1, etc.). When performing substrate evaluation of efflux transporters the appropriate test system needs to be selected based on the nature of the compound. For example, considering that compounds with high lipid solubility (e.g. LogP/LogD >3) tend to bind non-specifically to membrane vesicles and filtration devices and thus hide their ATP-based transport, the Caco-2 system is used for substrate evaluation. For compounds with high water solubility, the vesicle system is used.
Concentration Design
The transporter substrate identification is a qualitative test, and generally 2-3 concentrations are designed, which should cover the relevant concentration range such as the plasma concentration at the effective dose, combined with the maximum solubility and other information. It should be noted that if the compound is found to have an inhibitory effect on the transporter in the early stage, the substrate concentration design should be as much as possible below the value of the half-inhibitory concentration (IC50) or the inhibition constant.
Evaluation Criteria
When the efflux ratio (ER) or uptake ratio (UR) is ≥ 2 and the activity is inhibited by 50% or more by a known inhibitor, the substance to be tested is considered as a substrate for this transporter. If the in vitro system expresses multiple transporters, the results should be verified using two or more known inhibitors. Once the in vitro assay confirms that the drug under investigation is a substrate for one or some of the transporters, a DDI assessment needs to be considered.