4 June 2009
SOLVO introducing PSC 833 the specific P-gp inhibitor and new drug transporter services (mouse Bcrp1, OAT3, OCT2-metformin)
We are pleased to inform you that SOLVO Biotechnology introduces the specific P-gp inhibitor PSC 833 (valspodar, Amray) and three new PrediScreen services
- PSC 833 (valspodar, Amray)
- mouse Bcrp1 vesicular transport assay
- OAT3 uptake transporter assay
- OCT2 uptake transporter assay using metformin as substrate
PSC 833, a non-immunosuppressive cyclosporine D analog, is a 2nd generation ABCB1/P-gp/MDR1 specific reversal agent. We have confirmed that PSC 833 is a specific P-gp inhibitor by measuring the inhibitory potential of PSC 833 on a group of apically localized efflux transporters (Fig. 1). In the vesicular transport assay, the IC50 for P-gp mediated N-methyl-quinidine (NMQ) transport inhibition was ~10nM, about 2 logs below the IC50 for ABCG2/BCRP/MXR mediated estrone-3-sulfate (E3S) inhibition and more than 2 logs below the IC50 for inhibition of dehydroepiandrosterone-sulfate (DHEAS) transport by ABCC4/MRP4. Dose-dependent inhibition of estradiol-17β-glucuronide (E217βG) transport mediated by MRP2 was not clearly detected.
In addition, PSC 833 was found to be very effective in the monolayer efflux assays. In MDCKII-MDR1 cells the digoxin efflux ratio was reduced from 11.2 to 1.22 (Fig.2).
The fact that a broad set of preclinical and clinical data is available for this compound (reviewed in Tai 2000 Curr Opin Mol Ther 2:459) makes PSC 833 especially valuable for in vitro and in vivo ADME studies.
Figure 1. Specificity of PSC 833 to inhibit major apical efflux transporters in the vesicular transport assay. NMQ, E3S, DHEAS and E217βG transport was measured individually in the presence of various concentrations of PSC 833 using radioactive probe substrates. Relative transport (vesicular uptake) values were calculated.
Figure 2. Bidirectional permeability of labelled digoxin in MDCKII-MDR1 cells in the presence and absence of PSC 833. Digoxin (10 µM) apparent permeability (Papp) was calculated.
For more details please click here.
Mouse Bcrp1 Vesicular Transport Assay
The BCRP transporter (ABCG2/MXR) belongs to the family of ABC transporters. Besides its role in cancer resistance, BCRP can influence the ADME/Tox properties of drugs and nutrients. BCRP is localized on the apical membrane of polarized cells and can be found at the major barriers in the body, including the intestine, the BBB and the liver. BCRP has wide substrate specificity transporting both hydrophilic and hydrophobic compounds.
SOLVO's mouse Bcrp1 vesicular transport assay uses isolated membrane vesicles from MDCKII cells expressing mouse Bcrp1 and operates with 3H-estrone-sulfate (E3S) as the probe substrate. Mouse Bcrp1 transports E3S efficiently with negligible background transport in control vesicles (Figure 3).
Figure 3. E3S transport into mouseBcrp1 and control vesicles.
IC50 values were determined for a set of known BCRP interactors for both the human and mouse transporter using E3S as the probe substrate. Table 1 compares these IC50 values revealing good correlation between the two species.
Table 1. Comparison of IC50 values generated in the mouseBCRP1 and human BCRP vesicular transporter inhibition assay
| BCRP interactors | IC50(µM) mouseBcrp1 | BCRP |
| Leflunomide | 1.56 | 3.86 |
| Chlorothiazide | 134 | 62 |
| Prazosine | 2.14 | 1.80 |
| Dantrolene | 0.54 | 0.25 |
| Mitoxantrone | 62.3 | 104 |
| Ko134 | 0.056 | 0.04 |
| Sulfasalazine | 1.64 | 0.31 |
| DHEAS | 39.2 | 43.1 |
For more details please click here.
OAT3 uptake transporter assay
OAT3 is an integral membrane protein and appears to be localized at the basolateral membrane of the kidney. OAT3 is a multispecific transporter accepting a variety of structurally unrelated compounds as substrates including clinically important drugs. OAT3 accepts amphipathic and hydrophilic organic anions and some organic cations. OAT3 mainly accounts for the uptake of benzylpenicillin (PCG), dehydroepiandrosterone-sulfate (DHEAS), uremic toxins (indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanopropionate), in part for the uptake of estrone-sulfate (E3S), but has only a limited contribution to the uptake of hydrophilic and small organic anions.
A simple method for measuring OAT3 mediated transport is the cellular uptake assay. SOLVO generated a CHO-K1 cell line stably expressing OAT3 and developed an inhibition assay for the determination of the interaction of test drugs with this transporter. The interaction is detected as inhibition of the initial rate of 3H-estrone-sulfate transport by human OAT3 into cells expressing the OAT3 transporter.
Figure 4. Inhibition of OAT3 mediated uptake of 3H-estrone-sulfate by a set of compounds.
OCT2 uptake transporter assay using metformin as substrate
Organic cation transporters in the kidney play essential physiologic and pharmacological roles in the uptake of cationic drugs and endogenous organic ions. Moreover, human organic cation transporter 2 (hOCT2) is the most abundant organic cation transporter in the basolateral membranes of renal proximal tubules. OCT2 is also expressed in the small intestine and in the brain, where it may play a role in dopamine transport and be a target for anti-Parkinsonian drugs. OCT2 seems to play predominant role in secretion of low molecular weight organic cations (generally monovalent and hydrophilic).
The cellular uptake assay is a simple method to measure OCT2 mediated transport. SOLVO has developed a CHO-K1 cell line stably expressing OCT2 and a high-throughput assay for the determination of the interaction of test drugs with this transporter. The assay measures the inhibition of the initial rate of human OCT2 mediated 14C-metformin uptake.
Metformin was suggested as a superior substrate for renal OCT2 over liver OCT1, since OCT2 plays a dominant role in metformin pharmacokinetics. This explains why metformin is eliminated through renal excretion.
Nine compounds were tested for inhibition of metformin uptake into OCT2 transfected CHO cells (Fig.5.). As expected, ASP, verapamil, cimetidine, prazosin, quinidine, quinine, alprenolol and metoprolol inhibited the OCT2 transporter, whereas atenolol didn't. Dose dependence of OCT2 mediated 14C-metformin uptake inhibition with increasing verapamil and quinidine concentration is shown in Figure 6.
Figure 5. Inhibition of OCT2 mediated uptake of 14C-metformin by a set of compounds.
Figure 6. Dose dependent inhibition of OCT2 mediated uptake of 14C-metformin by verapamil and quinidine
For more details please click here.
Should you have any further questions, please contact us at sales@solvo.com.
With our best regards,
SOLVO Biotechnology
Business Development Team
CONTACT INFORMATION:
SOLVO Biotechnology
2 Gyár St. H-2040 Budaörs, HUNGARY
E-mail: sales@solvo.com
web: www.solvo.com
Phone: +36-23/503-940
Fax: +36-23/503-941
SOLVO distributors:
in US and Canada: XenoTech LLC www.xenotechllc.com,
in Europe: SOLVO and Tebu-bio www.tebu-bio.com,
in Japan: Nosan www.nosan.co.jp,
in India: Krishgen www.krishgen.com
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