Our first Science letter: Efflux transporters: boon or blight for big pharma?

August, 2005

Dear Colleague,

I am Bernadette Kovács - Business Development Associate of Solvo Biotechnology. It is my pleasure to welcome you as a Reader of our new Solvo Science letter. We are launching this letter to inform you - our valued Customers - about new and exciting developments in the world concerning Membrane Transporters. I hope that you will find these bulletins informative and stimulating. The first Science letter's author is my colleague, Attila Nemeth M.D. Ph.D. M.B.A. OecD., Associate Professor of Medicine & Genetics at the National Institute for Psychiatry & Neurology of Hungary, who is the Business Development & Strategy Consultant of Solvo Biotechnology.

Efflux transporters: boon or blight for big pharma?

Drug transporters are expressed in many tissues such as the intestine, liver, kidney, brain, placenta and Sertoli cells. They play key roles in drug absorption, distribution, transport, metabolism and excretion (ADTME). The use of transporter function offers the possibility of delivering a drug to the target organ, avoiding distribution to other organs (thereby reducing the chance of toxic side effects); controlling the elimination process; and/or improving oral bioavailability. A significant number of drug candidates entering clinical development are dropped at some stage due to unacceptable pharmacokinetic and toxicodynamic properties. As a consequence, many pharmaceutical companies now carry out rational high-throughput (HT) drug metabolism and pharmacokinetic (DMPK) screening systematically (fail fast - fail cheap). The high-throughput screening (HTS) for absorption using PAMPA, PAMPORE, Caco-2 monolayers and the screening for metabolic stability and metabolic enzyme inhibition via cytochrome P450 (CYP450) recombinant microsomes or human liver microsomes in combination with various drug transporters (transportocytes) have become extremely popular. Transporters have been classified as primary, secondary, or tertiary active transporters. Secondary or tertiary active transporters, mainly uptake transporters (328 transporters in 43 families) such as OAT, OATP, NTCP, OCT, OCTN, and PEPT, are driven by an exchange or cotransport of intracellular and/or extracellular ions. The driving force for primary active transporters like ATP-binding cassette (ABC) transporters (48 transporters in 7 families), such as MDR, MRP, and BCRP, is ATP hydrolysis.

Drug pumps are pimps for drug interactions?

Drug-drug interactions (DDIs) due to uptake and/or efflux transporters can be enrolled into two categories: (i) competition for the substrate binding sites, (ii) change in the expres sion level of the transporters. Pgp, MRP1, MRP4, BCRP are responsible for the cellular extrusion of many kinds of drugs in the central nervous system (CNS). Drug-drug interactions via brain Pgp between loperamide, a substrate for Pgp, and quinidine, an inhibitor of Pgp, have been reported. Although the antidiarrheal agent loperamide is a potent opiate, it does not produce opioid CNS effects at usual doses in pa tients. However, respira tory depression occurred when loperamide (16 mg) was given with quinidine at a dose of 600 mg. These changes were not explained by increased plasma loperamide con centrations. Thus, inhibition of Pgp by quinidine increases the entry of loperamide into the CNS with re sulting opiate-induced respiratory depression. Pgp, MRP2, BCRP are expressed on the brush-border membrane of enterocytes and excrete their substrates into the lumen, resulting in a potential limi tation of net absorption and oral drug bioavailabil ity. The intestinal Pgp content correlates with the AUC after oral admin istration of digoxin, a Pgp substrate, in humans. Pgp inhibitors, such as quinidine, valspodar, and verapamil, are known to increase plasma concentrations of digoxin because they block its biliary and/or urinary excretion via Pgp. Since the therapeutic window of digoxin is very narrow, changes in its plasma concen tration are potentially deleterious. Transporter induction represents a new type of DDIs. The AUC value of oral digoxin is significantly lower during rifampin treatment but the effect is less pronounced after i.v. administra tion of digoxin. The renal clearance and half-life of digoxin are unaltered by rifampin. However, rifampin treatment increases the intestinal Pgp content 3.5-fold, which correlates with the AUC value after oral but not intravenous adminis tration of digoxin. Since rifampin also induces intestinal MRP2, coadministration of rifampin is expected to in crease the secretion into the lumen of MRP2 substrates, such as glutathione or glucuronide conjugates. St. John’s wort is one of the most commonly used over-the-counter (OTC) herbal medicines in the US and is widely used in the treatment of mild de pression. Coadministration of St. John’s wort and the HIV pro tease inhibitor indinavir reduced the latter’s exposure by 57% in healthy volunteers due to induction of intestinal Pgp. In addition, the administration of St. John’s wort resulted in an 18% reduction in digoxin exposure after a single dose of digoxin, and a 1.4-and 1.5-fold increase in the expression of duodenal Pgp and CYP3A4, respectively. Although St. John’s wort induces both Pgp and CYP3A4, like rifampin, the reduction in the oral bio availability of digoxin may be caused by the induction of intestinal Pgp due to a lack of significant metabolism via CYP3A4. It has been shown that treatment with water-soluble vitamin E [TPGS]) enhances the absorption of cyclosporine in healthy volunteers or liver transplant recipients. Another report has demonstrated that TPGS also increased the solubility of amprenavir, an HIV protease inhibitor, and inhibited the efflux transport systems and enhanced the perme ability of amprenavir through Caco-2 cell monolayers. Surfactants, such as Cremo phor EL or Tween 80, have been found to be potent inhibitors of Pgp. Both are used as formulation vehicles (pharmaceutical excipients) for a vari ety of poorly water-soluble drugs, including the antican cer agents paclitaxel and docetaxel. The use of these surfactants may increase the intestinal absorption of some drugs through Pgp inhibition and, thus, improve the drug bioavailability of Pgp substrates. In the liver, xenobi otics are metabolized by the so-called phase I and II en zymes, which are mainly CYPP450 and conjugat ing enzymes, respectively. After these enzymatic reactions, the conjugated metabolites produced are pumped out from hepatocytes into the bile. These mainly canalicular efflux transporters (MDR1, MDR3, MRP2, BSEP, BCRP) play a physiologically important role as the “phase III” xenobiotic detoxification system. BSEP, located on the canalicular membrane, mediates the transport of bile acids such as taurocholic acid. Cholestasis induced by some drugs is mediated, at least in part, by inhibition of BSEP, resulting in intracellular accumulation of cyto toxic bile salts. The immunosuppressant, cyclosporine, has been shown to produce cis-inhibition of BSEP-mediated bile salt transport. A similar mechanism has been postulated for rifampin and glib enclamide. In contrast, the choles tatic estrogen metabolite, E2-17βG, causes trans-inhibition of BSEP-mediated bile salt transport and, therefore, exerts its cholestatic action only after its excretion by MRP2 into the canalicular lumen.

Mammalian pumps take after human ones?

There is a species difference in the transport of organic anions via MRP2 across the bile canalicular membrane. The ATP-dependent transport activity (Vmax/Km) of the MRP2 substrate DNP-SG in mouse, rat, guinea pig, rabbit, dog, and human canalicular membrane vesicules (CMVs) is widely variable. Another report has shown that the transport activity of glutathione conjugates and unconjugated anions (prav astatin, BQ-123, and methotrexate) in human CMVs was ~3-to 76-fold lower than that in rat CMVs, whereas the transport activity of glucuronides was similar in the two species. At Solvo Biotechnology there are several HTS techniques available whereby species differences between various transporters could be evaluated. For instance, according to the latest comparative analysis of human MRP2 vs. rat Mrp2 and human MDR1 vs. rat mdr1 via ATP-ase as well as IO Sf9 vesicular transport system, several important observations could be demonstrated (see attached data; H. Glavinas’ poster and O. von Richter’s presentation). In the utilized membranes the expression levels of the two transporter proteins were the same. However, according to the ATP-ase assay results the rat Mrp2 EC50 values were significantly higher for the tested drugs mirroring the higher Km values of these moieties. Moreover, ATP-ase activities could not been observed with NEM-GS, sulfinpyrazone and furosemide on rat Mrp2 expressed Sf9 membranes. Looking at the vesicular transport-based assays, one of the important endogenous MRP2 substrate, E2-17β-G showed different kinetic profile vs. rat Mrp2. Human MRP2 exhibited higher affinity to E2-17β-G and gave the sigmoid Hill-plot curve. This substantiated two binding sites for E2-17β-G in human vs. only one in rat. Furthermore, MK-571 – an important MRP2 modulator – stimulated only the human MRP2 dependent E2-17β-G transport.

Tools for toying with pumps

Monolayer assays: two major drawbacks of using selected cell lines are usually noted: (i) transporter expression pattern changes with time, and (ii) the cell line overexpresses other transporters with overlapping substrate specificities. After growing and seeding the transfected cell-lines, viability must be assessed by transendothelial elec trical resistance. Markers for paracellular, transcellular, and Pgp-dependent efflux must be included in each experiment. Efflux transporter substrates could be analyzed by cassette liquid chromatogra phy or HPLC with tandem mass spectrometry (LC/MS/MS) along with the test compounds. The apparent permeability (Papp) should be calculated. Data are usually presented as the average Papp (nm/s) ± SD. Involvement of a Pgp-mediated efflux mechanism is indicated if the B→A/A→B ratio is > 2.0. The expressed transporter protein must be evaluated qualitatively and quantitatively in cell lysates by Western blot analysis utilizing special transporter specific antibodies. In order to determine the robustness and reliability of cell lines, transporter expression and activity must be monitored over passage numbers and time. Usually transporter functional activity is more consistent between passages than protein levels detected by Western blotting. Vesicular transport (VT): This is a widely used technique that can be applied in different HT assays. Utilization of inside-out (IO) vesicles provides major advantages, such as (i) the test compounds and ATP have direct access to the cytoplasmic domains of the transporter, and (ii) since the direction of the transport is occurring from outside to inside, the transport can be assayed by measuring the amount of substrate trapped inside the vesicle. Although membrane vesicles can be made from practically any kind of cells that express the transporter, the most frequently used membrane preparations utilize baculovirus-infected Sf9, Sf21, and High Five insect cells. The advantage of this expression system is the particularly high transporter protein expression level (around 5-10 % of the total membrane protein is the expressed transporter). In addition, the heterologously expressed transporter is the only mammalian protein in this system. Data and commercial availability of different vesicular membrane preparations can be attained at www.solvo.com. Dye inhibition assays: calcein AM and its analogues are ferquently used due to their favorable technical properties. Intracellular calcein AM is hydrolyzed by endogenous esterases, yielding a fluorescent product, calcein. Accumulation of the fluorescent dye can be detected without the need for washing steps, making this assay suitable for HT settings. The presence of Pgp in the cell membrane strongly reduces calcein accumulation, and inhibitors of Pgp produce an increased rate of accumulation. Calcein has advantageous spectral properties, such as bright fluorescence, that is insensitive to pH or to Ca2+ - Mg2+ concentrations, and does not show spectral changes upon accumulation or binding to intracellular compartments. Both calcein AM and free calcein are transported by MRP1, thus the patented calcein assay by Solvo Biotechnology is applicable for the quantitation for MDR1 and MRP1 activities. A suitable MDQ kit also exists for the specific diagnostic detection of MDR1 and MRP1 activities in clinical tumor samples. A similar dye transport-based assay, utilizing Hoechst 33342 has been developed to measure the activity of MXR/BCRP, the third MDR-ABC transporter, with broad substrate specificity. BCRP besides its role in MDR, takes integral part in the secretion of clinically important drugs such as topotecan, mitoxantrone, flavopiridol, methotrexate, fluorescent dyes, different toxic compounds in normal food (PhIP) or a chlorophyll derivative pheophorbide A. Solvo Biotechnology is the only membrane transporter company which possesses all four main in vitro techniques (ATPase, vesicular transport, dye transport, nucleotide trapping assays) to look into the BCRP mediated efflux transport in a more thorough fashion (see attached data; C. Xia’s presentation). ATPase activity assay: The ATPase assay is one of the most widely used assays to search for compounds that interact with different ABC transporters. The assay requires relatively high levels of transporter protein expression, thus mostly membrane vesicles prepared from recombinant baculovirus infected insect cells (e.g. Sf9 cells) are used for this purpose. These membrane preparations show a vanadate-sensitive ATPase activity that is modulated by interacting compounds. The rate of ATP hydrolysis is determined by measuring the liberation of inorganic phosphate. In the presence of transported substrates the ATPase activity of the transporter increases (activation protocol). Inhibitors that act in a non-competitive fashion, or some slowly transported compounds inhibit the ATPase activity of the stimulated transporter (inhibition protocol). The main advantage of the ATPase assay is in its simplicity and reproducibility. HT version of the assay has been developed. Nucleotide trapping: In ABC transporters the catalytic reaction involves the formation of a transition state complex that contains an occluded nucleotide. An occluded form of ADP can be stabilized by vanadate, which provides the basis of the screening method, called vanadate-dependent nucleotide trapping. Such a nucleotide trapping is concentration dependently modulated by the transported substrates. Experimentally the isolated cell membranes are incubated with α-32P labeled azido-ATP, and after washings in the presence of cold ATP, the trapped, labeled nucleotides are covalently bound to the transporter by UV photo-cross linking. Nucleotide trapping is a low throughput assay, however some subtle interactions of drugs with transporters can only be shown by this assay.

Screening strategy

Depending on the overall aims of the test and the transporter assayed, different methods and screening strategies should be applied. In order to screen a large library of compounds, fluorescence-based whole cell assays and the ATPase assay seem to be the methods of choice. This gives a combination of direct and indirect assays, however, even these large-scale screening methods should be used in a complementary fashion, as false positive or false negative results could be obtained if only one of these methods are applied. Screening for transported substrates for a given ABC transporter, indirect methods used alone may be misleading, because both substrates and inhibitors might give you positive results. On the contrary, indirect assays, utilizing reporter substrates, are the methods of choice to screen for reverting agents. The Papp values (PAMPA) and the ATPase assay results of the investigational compounds can provide reliable guidance in the selection of result-driven further assays (VT and/or dye inhibition). Good correlations could be observed in the measured parameters with PAMPA vs. monolayer (MDKC II/Caco-2) as well as ATPase activation/inhibition vs. calcein dye inhibition assays. Drugs which are in the high solubility range, ATPase and dye inhibition assays can be more reliable vs. low or medium-range solubility molecules where vesicular transport (VT) and monolayer assays are prone to be more advantageous. Moreover, if we look at the Biopharmaceutical Classification System / Biopharmaceutical Drug Dispoition Classification System (BCS/BDDCS) grids, low solubility/high permeability Class 2 drugs (e.g. carbamazepine, cyclosporine, digoxin, ketoconazole, tacrolimus) metabolize predominantly via efflux transporters; whereas high solubility/low permeability Class 3 molecules (e.g. cimetidine, ranitidine) are carried by uptake transporters (see attached data; O. von Richter’s presentation).