Drug-transporter interaction testing in drug discovery and development

The human body consists of several physiological barriers that express a number of membrane transporters. For an orally absorbed drug the intestinal, hepatic, renal and blood-brain barriers are of the greatest importance. The ATP-binding cassette(ABC) transporters that mediate cellular efflux and the solute carrier transporters that mostly mediate cellular uptake are the two superfamilies responsible for membrane transport of vast majority of drugs and drug metabolites. The total number of human transporters in the two superfamilies exceeds 400, and about 40-50 transporters have been characterized for drug transport. The latest Food and Drug Administration guidance focuses on P-glycoprotein, breast cancer resistance protein, organic anion transporting polypeptide 1B1(OATP1B1), OATP1B3, organic cation transporter 2(OCT2), and organic anion transporters 1(OAT1) and OAT3. The European Medicines Agency's shortlist additionally contains the bile salt export pump, OCT1, and the multidrug and toxin extrusion transporters, multidrug and toxin ex-trusion protein 1(MATE1) and MATE2/MATE2 K. A variety of transporter assays are available to test drugtransporter interactions, transporter-mediated drugdrug interactions, and transporter-mediated toxicity. The drug binding site of ABC transporters is accessible from the cytoplasm or the inner leaflet of the plasma membrane. Therefore, vesicular transport assays utilizing inside-out vesicles are commonly used assays, where the directionality of transport results in drugs being transported into the vesicle. Monolayer assays utilizing polarized cells expressing efflux transporters are the test systems suggested by regulatory agencies. However, in some monolayers, uptake transporters must be coexpressed with efflux transporters to assure detectable transport of low passive permeability drugs. For uptake transporters mediating cellular drug uptake, utilization of stable transfectants have been suggested. In vivo animal models complete the testing battery. Some issues, such as in vivo relevance, gender difference, age and ontogeny issues can only be addressed using in vivo models. Transporter specificity is provided by using knock-out or mutant models. Alternatively, chemical knock-outs can be employed. Compensatory changes are less likely when using chemical knockouts. On the other hand, specific inhibitors for some uptake transporters are not available, limiting the options to genetic knock-outs.

SPECIFIC UPTAKE OF MONOCLONAL ANTIBODY-CONJUGATED METHOTREXATE BY HUMAN LYMPHOCYTIC LEUKEMIC B CELLS

Objective: To analysis the uptake of free MTX and MTX conjugated to tumor specific monoclonal antibody by target and nontarget cells. Methods: The folate antagonist methotrexate (MTX) was conjugated to two monoclonal antibodies (Mab) directed against human chronic lymphocytic leukemia (CLL), Dal B01 and Dal B02, by an active ester method. Both conjugates were more cytotoxic toward the target tumor cell line D101 than to the nontarget cell line MOLT3, and Dal B02MTX conjugate was more inhibitory to D101 cells than free MTX in a 6 h pulse exposure assay. Results: Drug uptake studies revealed that D101 cells took up much more Dal B01 and Dal B02conjugated MTX than free MTX. The amounts of drug taken up by D101 cells incubated with Dal B01 and Dal B02conjugated MTX were always 3 to 5fold higher than that taken up by MOLT3 cells, although the latter took up more drug when incubated with free MTX. Furthermore, tumor cells incubated with Dal B01 or Dal B02conjugated MTX retained much larger amounts of drug for a prolonged period of time than those incubated with free MTX. Conclusion: The enhanced specific cytotoxicity of Dal B01 and Dal B02MTX conjugates toward target tumor cells is therefore likely due to (I) delivery of larger amounts of MTX to target cells when the drug is conjugated to Mab; (ii) longer retention of Mabconjugated MTX by target cells; and (iii) slow, prolonged release of MTX from the surfacebound or endocytosed conjugates, rendering them into a sustained release dosage form.

Effect of chirality on conformation and cellular uptake of poly(S-(o-nitrobenzyl)-L,D-cysteine) polypeptides

A series of poly（S-（o-nitrobenzyl）-L,D-cysteine） polypeptides with different chirality was synthesized and their molecular structures,secondary conformations,drug release and biological properties were thoroughly investigated.The chirality of the polypeptides had effect on secondary conformations and the cellular uptake behavior of the related nanoparticles.