Cyclosporins: structure-activity relationships for the inhibition of the human FPR1 formylpeptide receptor

The human formylpeptide receptor (FPR) is a seven-transmembranous G-protein-coupled receptor (7TM-GPCR) for chemotactic peptides of bacterial origins, possibly involved in the recruitment and activation of neutrophils in various inflammatory diseases of mucosal epithelia. Mutational analyses suggest that interactions of formylated peptides with FPR occur on the outer exoplasmic leaflet/domains of the plasma membrane. The immunosuppressive and antifungal antibiotic cyclic undecapeptide cyclosporin A (CsA; cyclo-[MeBmt(1)-Abu(2)-MeGly(3)-MeLeu(4)-Val(5)-MeLeu(6)-Ala(7)-D-Ala(8)-MeLeu(9)-MeLeu(10)-MeVal(11)]) and some tested analogues such as [Ala(2)]-CsA, [Thr(2)]-CsA, [Val(2)]-CsA, and [Nva(2)]-CsA were able of inhibiting the binding of formylpeptides to the FPR, with [D-MeVal(11)]-CsA (CsH) being much more active than the other analogues. CsH is devoid of immunosuppressive and antifungal activities, and its large potency for human FPR inhibition is of inverse agonism origin. Formylpeptide binding to FPR-expressing cells does not only induce chemotaxis; it also causes a rapid release of granule enzymes in the extracellular medium, allowing the easy monitoring of any inhibition of FPR function "in vivo" (with intact live cells). With such an assay, CsH was confirmed to be the most potent FPR inhibitory cyclosporin, although a far related immunosuppressive cyclosporin analogue, FR901459 ([Thr(2), Leu(5), Leu(10)]-CsA), was found to display a high FPR inhibitory activity (FPR-InhA). To establish structure-activity relationships (SAR) for FPR function inhibition, 59 cyclosporins were now studied by this standardized assay (with differentiated human leukemic cell line HL-60 as FPR-expressing cells and with N-acetyl-beta-D-glucosaminidase release as read-out). These SAR confirmed the low FPR-InhA of classical cyclosporins, where such activity was only seldom found: the most active ones ([Thr(2), Ile(5)]-CsA, [aMeIle(11)]-CsA, and [MeAla(11)]-CsA) remained 3-10-fold less potent than CsH. In contrast, the SAR disclosed that N(10)-desmethylated cyclosporins were particularly prone to display a large FPR-InhA: their most potent one was a [Thr(2), Gly(3), Leu(5), D-Hiv(8), Leu(10)]-CsA, found to be only 2-4-fold less active than [D-MeVal(11)]-CsA (CsH), with which it shows six differences out of 11 residues. Because the free conformations of both CsH and N(10)-desmethylated cyclosporins differ from those of "classical" (N(10)-methylated, [L-MeVal(11)]-using) cyclosporins, these potent FPR inhibitory cyclosporins probably bind to FPR pharmacophores for which classical cyclosporins show little affinity. Moreover, because the conformations of the N(10)-desmethylated cyclosporins widely differ from the CsH one, they probably bind to different pharmacophores on the FPR molecules.     

Aureobasidins: structure-activity relationships for the inhibition of the human MDR1 P-glycoprotein ABC-transporter

Cyclic depsipeptide cyclo-[D-Hmp(1)-L-MeVal(2)-L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle+ ++(6)-L-MeVal(7)-L-Leu(8)-L-betaHOMeVal(9)], the antifungal antibiotic aureobasidin A (AbA), was reported to interfere with ATP-binding cassette (ABC) transporters in yeast and mammalian cells, particularly the MDR1 P-glycoprotein (Pgp), a transmembrane phospholipid flippase or "hydrophobic vacuum cleaner" that mediates multidrug resistance (MDR) of cancer cells. In a standardized assay that measures Pgp function by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL(100)) cells as highly resistant Pgp-expressing cells and the cyclic undecapeptide cyclosporin A (CsA) as a reference MDR-reversing agent (IC(50) of 3.4 microM), AbA was found to be a more active Pgp inhibitor (IC(50) of 2.3 microM). Out of seven natural analogues and 18 chemical derivatives of AbA, several were shown to display even more potent Pgp-inhibitory activity. The Pgp-inhibitory activity was increased about 2-fold by some minor modifications such as those found in the naturally occurring aureobasidins AbB ([D-Hiv(1)]-AbA), AbC ([Val(6)]-AbA), and AbD [gammaHOMeVal(9)]-AbA). The replacement of the [Phe(3)-MePhe(4)-Pro(5)] tripeptide by an 8-aminocaprylic acid or the N(7)()-desmethylation of MeVal(7) led to only a 3.3-fold decreased capacity to inhibit Pgp function, suggesting that the Pgp inhibitory potential of aureobasidins, though favored by the establishment of an antiparallel beta-sheet between the [D-Hmp(1)-L-MeVal(2)-L-Phe(3)] and [L-aIle(6)-L-MeVal(7)-L-Leu(8)-] tripeptides, does not critically depend on the occurrence of the [L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle(6)] type II' beta-turn secondary structure. In contrast, the most potent Pgp inhibitors were found among AbA analogues with [betaHO-MeVal(9)] residue alterations, with some data suggesting a negative impact of the [L-Leu(8)-L-betaHOMeVal(9)-D-Hmp(1)] gamma-turn secondary structure on Pgp inhibitory potential. The [2,3-dehydro-MeVal(9)]-AbA was the most potent Pgp inhibitory aureobasidin, being 13-fold more potent than AbA and 19-fold more potent (on a molar basis) than CsA. Finally, there was no correlation between the SAR for the human MDR1 Pgp inhibition and the SAR for Saccharomyces cerevisiae antifungal activity, which is mediated by an inositol phosphoceramide synthase activity.     

Competitive and allosteric interactions in ligand binding to P-glycoprotein as observed on an immobilized P-glycoprotein liquid chromatographic stationary phase

A liquid chromatographic stationary phase containing immobilized P-glycoprotein (Pgp) was synthesized using cell membranes obtained from Pgp-expressing cells. The resulting Pgp-stationary phase was used in frontal and zonal chromatographic studies to investigate the binding of vinblastine (VBL), doxorubicin (DOX), verapamil (VER), and cyclosporin A (CsA) to the immobilized Pgp. The compounds were added individually to the chromatographic system with or without ATP in the running buffer. Using this approach, dissociation constants were calculated for VBL (23.5 +/- 7.8 nM), DOX (15.0 +/- 3.2 microM), VER (54.2 +/- 4.7 microM), and CsA [97.9 +/- 19.4 nM (without ATP) and 62.5 +/- 4.6 nM (with ATP)]. The compounds were also added in pairs using standard competitive chromatography procedures. The results of the study demonstrate that competitive interactions occurred between VBL and DOX, cooperative allosteric interactions occurred between VBL and CsA and ATP and CsA, and anticooperative allosteric interactions occurred between ATP and VBL and VER. The chromatographic studies indicate that the immobilized Pgp was modified by ligand and cofactor binding and that the stationary phase can be used to study drug-drug binding interactions on the Pgp molecule.     

SDZ PSC-833--a novel potent in vitro chemosensitizer in multiple myeloma.

Multiple myeloma cell lines and patient tumor samples with and without the expression of the classical multidrug resistance (MDR) phenotype were investigated in vitro for drug induced cytotoxicity and modulation of drug resistance. Overall there was a good correlation in the cell lines between MDR expression, as measured by immunocytochemistry with monoclonal antibodies against P-glycoprotein 170 (Pgp), and in vitro resistance to doxorubicin (dox) and vincristin (vcr). Drug resistance in the cell line RPMI 8226 dox 40, expressing a high level of Pgp, was almost completely reversed by the novel non-immunosuppressive cyclosporin A (CsA) analog SDZ PSC-833 (PSC), while the chemosensitizers verapamil, CsA and quinine, in clinically achievable concentrations, were much less effective. In cell lines with low Pgp expression, PSC and the other chemosensitizers seem equally effective. The patient tumor samples were selected to represent different combinations of Pgp expression, drug resistance and effects of chemosensitizers. PSC and CsA appeared equally potent and resistance modulation was detected not only in Pgp positive, but also in Pgp negative tumor samples. Furthermore, in one case of a Pgp expression myeloma, chemosensitizers were without effect. These findings indicate the need to incorporate in vitro chemosensitivity assays with Pgp determination when the effects of MDR modulating chemosensitizers are to be studied in the clinic.     

The potent immunosuppressive cyclosporin FR901459 inhibits the human P-glycoprotein and formyl peptide receptor functions

By sequestering cytosolic calcineurin into a molecular complex with cyclophilin and its consequent T-cell dysfunction, some cyclosporins, such as CsA and FR901459 ([Thr2-Leu5-Leu10]-CsA), display potent immunosuppressive activity. Independently on this property, cyclosporins may display one or more other biological activities mediated by interaction with cell surface glycoproteins. Several cyclosporins inhibit the function of human MDRI-encoded P-glycoprotein (Pgp), a flippase known to cause cancer multidrug resistance, but also expressed by some normal immunocompetent cells and by normal epithelial cells which control drug bioavailability in vivo. CsA is known to be a potent Pgp inhibitor with a 3.2 microM IC50 in an assay where the most potent derivative SDZ PSC 833 gives a 0.49 microM IC50. FR901459 is now shown to be a good Pgp inhibitor, being 2-fold weaker only (IC50 of 6 microM) than CsA. Some cyclosporins may also inhibit the function of the human FPR1-encoded formyl peptide receptor (FPR), a chemotactic receptor whose absence is known to impair antibacterial immunity. Yet this inhibition is very weak for all, but one of them, CsH, whose 0.15 micro/M IC50 makes it a much more potent FPR inhibitor than CsA (IC50 >10 microM in the same assay). FR901459 is now shown to be a very potent inhibitor of FPR function (IC50 of 0.6 microM). Since CsH shows little Pgp-inhibitory activity and has no known immunosuppressive activity, FR901459 displays a unique pharmacological profile: like CsA, it inhibits T-cell function; less than CsA, it can inhibit Pgp function on selected leukocyte subsets and on epithelial barriers known to control drug bioavailability; however, much more efficiently than CsA, it can inhibit the FPR function, a receptor involved in some leukocytic inflammatory responses to chemotactic peptides.     

Conferone from Ferula schtschurowskiana enhances vinblastine cytotoxicity in MDCK-MDR1 cells by competitively inhibiting P-glycoprotein transport

Overexpression of the protein transporter P-glycoprotein (Pgp, MDR1) at the cell surface is a major cause of multidrug resistance (MDR) and poor response to treatment in cancer chemotherapy and therapy for leishmaniasis. The present study shows that conferone, a sesquiterpene coumarin ether isolated for the first time from Ferula schtschurowskiana, endemic in Uzbekistan, enhances the cell toxicity of vinblastine (VBL) in MDR1-transfected Madin-Darby canine kidney (MDCK-MDR1) cells. Conferone presents the advantage to mediate this effect at safe concentrations. At 10 microM, it efficiently competes with the photoactivatable cyclosporin A analogue (SDZ 212 - 122) for the binding to Pgp and accumulates [3H]-VBL to a higher extent than cyclosporin A or cnidiadin. [3H]-VBL accumulation is dose-dependent and correlates with the inhibition of Pgp photolabeling affinity, supporting the hypothesis that conferone sensitizes MDCK-MDR1 cells to VBL by competitively inhibiting drug efflux. In MDCK-MDR1 cells, [3H]-VBL accumulation appears to be almost completely dependent on inhibition of Pgp transport. However, the strict specificity of conferone to this efflux pump has to be demonstrated in cell lines expressing other protein transporters. Collectively, our findings identify conferone as a powerful modulator of Pgp transport and a promising molecule for the treatment of MDR malignancies and leishmaniasis. Complementary in vitro and in vivo studies are, however, needed to assess the value of conferone as a reversal drug in human therapy. Considering its high affinity for Pgp, conferone may have an additional usefulness as a tool for the design or the (hemi)synthesis of agents probing Pgp. To our knowledge, this is the first report identifying sesquiterpene coumarins from Ferula as possible drug candidates for the reversion of MDR encoded by the MDR1 gene or the synthesis of agents probing Pgp.     

SDZ PSC 833 and SDZ 280-446 are the most active of various resistance-modifying agents in restoring rhodamine-123 retention within multidrug resistant P388 cells.

Multidrug resistance (MDR) of tumor cells may result from overexpression of P-glycoprotein (Pgp) but may be down-modulated by resistance-modifying agents (RMAs). The cyclosporin SDZ PSC 833 and the cyclopeptolide SDZ 280-446 were found to be the strongest RMAs known to date for restoring the sensitivity of MDR cells to anticancer drugs, as well as for restoring their retention of daunomycin, a fluorescent anthracycline. Using rhodamine-123 (Rhod-123), another fluorescent probe of Pgp function which also differentiates sensitive and MDR cells, several RMAs were compared for their capacity to inhibit Pgp function. At variance with the data obtained with the daunomycin probe, a series of RMAs did not detectably restore Rhod-123 retention by the MDR cells. With the remaining RMAs, achieving the same levels of Rhod-123 retention required 3 times lower RMA concentrations when the RMA was added to the MDR cells for both the initial uptake and the efflux of Rhod-123 rather than for its uptake only. Nevertheless, the data emphasized the large superiority of SDZ PSC 833 and SDZ 280-446 over all other RMAs.     

Valspodar: current status and perspectives

Valspodar (Amdray, SDZ PSC 833) is derived from cyclosporin, but lacks the immunosuppressive and most of the collateral activities of cyclosporin A (CsA, Sandimmune, Neoral); it exhibits an enhanced capacity to chemosensitise tumour cells showing the classical type multiple drug-resistance (MDR) associated with MDR1 P-glycoprotein (Pgp) overexpression. This valspodar-mediated chemosensitisation of MDR tumour cells is reviewed with regard to its mechanism of inhibition on Pgp flippase function, and its potential inhibition of anticancer drug (ACD) metabolisation by CYP3A enzymes is discussed. Potent inhibition of the membranous and cytoplasmic detoxification mechanisms expressed by cells at the absorption and clearance borders in the body by valspodar results in the many pharmacokinetic interactions with other drugs that are substrates of either, or both, Pgp and CYP classes of detoxifying enzyme. In view of the present ability to restrict oral bioavailability of valspodar within a narrow range, and to adapt adequately the chemotherapeutic dosages to achieve their equivalent exposure in the presence or absence of valspodar, current clinical data on its efficacy and safety permit optimism for ongoing Phase III trials. The potential of valspodar to increase exposure or to modulate the biodistribution of other chemotherapeutics, such as HIV protease inhibitors to the brain, is further evoked, as this might become another application of the new drug. This evaluation of valspodar compared to CsA attempts to interpret its mechanisms of action, rather than to serve as a complete and comparative repertoire of all published preclinical and clinical data.     

Cytotoxic activity of cyclosporin A and [3-keto-Bmt1]-[Val2]-cyclosporin (SDZ PSC 833) on tumour cells from patients with haematological malignancies

Objective and method: The fluorometric microculture cytotoxic assay was employed for characterisation of the cytotoxic effect of cyclosporin A (CsA) and its non-immunosuppressive analogue SDZ PSC 833, [3-keto-Bmt¹]-[Val²]-cyclosporin (PSC) in tumour cells from patients with haematological or solid tumours. Results: Tumour cells from patients with chronic lymphocytic leukaemia (CLL) or non-Hodgkin's lymphoma (NHL) were found to be more sensitive to both drugs than those of tumour cells from patients with acute lymphocytic leukaemia (ALL), acute myoblastic leukaemia (AML) and various solid tumours. There was a close correlation between the effects of the two drugs (correlation coefficient 0.71), but CsA was slightly more active than PSC in most diagnoses. No tumour cell sample showed sensitivity to PSC without also being sensitive to CsA. There was a moderate level of correlation between the activity pattern of CsA and doxorubicin (correlation coefficient 0.66), whereas the correlations with other cytostatics, such as vincristine, cytarabine and melphalan, were low (correlation coefficient −0.11 to 0.33). Conclusion: The results indicate that PSC shares the direct cytotoxic properties of CsA, but is slightly less potent. Clinical testing of the cytotoxic effect of these agents in haematological malignancies seems warranted and the apparent non-cross-resistance with standard agents makes cyclosporins a potentially useful adjunct to chemotherapy in those diagnoses. Received: 12 August 1996 / Accepted in revised form: 10 December 1996     

Reversal of multidrug resistance in mouse lymphoma cells by phenothiazines

Various compounds were tested with regard to their reversal of multidrug resistance (MDR) in mouse tumor cells transfected with the human MDR1 gene. Phenothiazines containing aromatic moieties were bound through stacking interaction involving the polarization of the aromatic aminoacid substituents at the target site of p-glycoprotein (Pgp) 170, as a consequence of their large dipoles (as in the binding of phenothiazine to calmodulin-like structures). Acting as a calcium channel blocker, verapamil may induce conformational changes in the calcium channel-like structures of the transmembrane regions of Pgp. Most probably the tyrosine moieties of Pgp are involved in the action of verapamil and phenothiazines. Tomato lectin specifically binds to the polylactosamine moiety of Pgp170 at the first loop of Pgp. Other targets in the membrane may exist in close proximity to Pgp170, such as conA-reactive glycoproteins with terminal mannosyl residues. WGA-reactive N-acetyl glucosamine residues can also be modified resulting in conformational changes in trans-membrane regions of the ABC transporter. Our results demonstrate that MDR can be reversed by interaction of various compounds with Pgp or by modification of the membrane structure around the Pgp.     

Effect of cyclosporin A and analogues on cytosolic calcium and vasoconstriction: possible lack of relationship to immunosuppressive activity.

1. The full therapeutic potential of the main immunosuppressive drug, cyclosporin A (CsA), is limited because of its side effects, namely nephrotoxicity and hypertension. Several lines of evidence suggest that the origin of both side effects could be CsA-induced vasoconstriction. However, the underlying molecular mechanisms are not well understood. 2. Diameter measurements of rat isolated mesenteric arteries showed an increase in noradrenaline- and [Arg]8vasopressin-induced vasoconstriction when arteries were pretreated with CsA. 3. Measurements in cultured vascular smooth muscle cells (VSMC) of either cytosolic calcium concentration or of 45Ca2+ efflux showed that CsA potentiated the calcium influx to several vasoconstrictor hormones: [Arg]8vasopressin, angiotensin II, endothelin-1 and 5-hydroxytryptamine. On the other hand, 45Ca2+ efflux in response to thapsigargin, which depletes calcium from intracellular pools, was not potentiated by CsA. 45Ca2+ uptake was not altered by CsA or by any of the analogues tested. 4. Time-course studies in cultured VSMC showed that maximal CsA-induced Ca2+ potentiation occurred after ca. 20 h and this effect was reversed over approximately the next 20 h. 5. To investigate the possible role played by the known intracellular targets of CsA, namely cyclophilin and calcineurin, CsA derivatives with variable potencies with respect to their immunosuppressive activity, were tested on the calcium influx to [Arg]8vasopressin. Derivatives devoid of immunosuppressive activity (cyclosporin H, PSC-833) potentiated calcium signalling, while the potent immunosuppressant, FK520, a close derivative of FK506, and MeVal4CsA, an antagonist of the immunosuppressive effect of CsA did not. The latter compound was unable to reverse the calcium potentiating effect of CsA. 6. Our results show that CsA increases the calcium influx to vasoconstrictor hormones in smooth muscle cells, which presumably increases vasoconstriction. Loading of the intracellular calcium pools appears not to be involved. Experiments with derivatives of CsA and FK520 suggest that interactions with cyclophilins and calcineurin are not the mechanism involved. This indicates, for the first time, that the immunosuppressive activity can be dissociated from the calcium potentiating effect of CsA in vascular smooth muscle.     

Reversal of P-glycoprotein-mediated multidrug resistance by protopanaxatriol ginsenosides from Korean red ginseng

The overexpression of P-glycoprotein (Pgp) or the multidrug resistance-associated protein (MRP) confers multidrug resistance (MDR) to cancer cells. MDR cells can be sensitized to anticancer drugs when treated concomitantly with an MDR modulator. In this study, we investigated whether or not ginseng saponins could reverse MDR mediated by Pgp or MRP. The chemosensitization and drug accumulation effects of ginseng saponins such as the total saponin, protopanaxadiol ginsenosides (PDG), protopanaxatriol ginsenosides (PTG), ginsenosides-Rb 1, -Rb 2, -Rc, -Rg 1 and -Re were tested on the daunorubicin- and doxorubicin-resistant acute myelogenous leukemia sublines (AML-2/D100 and AML-2/DX100), which overexpress Pgp and MRP, respectively. PTG showed cytotoxicity in both sublines and was able to reverse resistance in the AML-2/D100 subline in a concentration-dependent manner. Conversely, other ginseng saponins at concentrations less than 300 microg/mL showed neither cytotoxicity nor chemosensitizing activity in both resistant sublines. Flow cytometry analysis showed that the effect of PTG (100 microg/mL) on drug accumulation of daunorubicin in the AML-2/D100 subline was 2-fold higher than that observed in the presence of verapamil (5 microg/mL) and 1.5 times less than cyclosporin A (3 microg/mL). The maximum non-cytotoxic concentrations of PTG did not appear to increase the Pgp levels, which is in contrast to verapamil and cyclosporin A. PTG at 200 microg/mL or more completely inhibited the azidopine photolabeling of Pgp. The results suggest that PTG has a chemosensitizing effect on Pgp-mediated MDR cells by increasing the intracellular accumulation of drugs through direct interaction with Pgp at the azidopine site. In addition, PTG may have a beneficial effect on cancer chemotherapy with respect to the possibility of long-term use without the concern of Pgp activation.     

Modulation by LY335979 of P-glycoprotein function in multidrug-resistant cell lines and human natural killer cells

Resistance to chemotherapy by some human tumors may be due to overexpression of membrane-associated transport proteins. The best characterized of these is the multidrug resistance (MDR) transporter, P-glycoprotein (Pgp). The aim of this study was to measure the inhibitory effects of a potent new MDR modulator, (2R)-anti-5-(3-[4-(10,11-difluoromethanodibenzo-suber-5-yl) piperazin-1-yl]-2-hydroxypropoxy)quinoline trihydrochloride (LY335979), in the drug-resistant cell line HL60/VCR and in normal, human CD56(+) lymphocytes. We used flow cytometric methods to detect the accumulation of rhodamine 123 and daunorubicin, fluorescent MDR substrates, in these cells. Our results indicate that LY335979 was 500-1500 times more potent than cyclosporin A or verapamil in restoring Pgp substrate accumulation in the MDR cell line HL60/VCR. Moreover, LY335979 could effectively block Pgp function on isolated CD56(+) lymphocytes (IC(50) = 1.2 nM) or CD56(+) lymphocytes in whole blood (IC(50) = 174 nM). We conclude that LY335979 is among the most potent Pgp inhibitors described and that it maintains significant potency in whole-human blood. These latter findings are important for establishing the dosing regimens of LY335979 for future clinical studies.     

Reversal of multidrug resistance by the P-glycoprotein modulator, LY335979, from the bench to the clinic

Multidrug resistance may be conferred by P-glycoprotein (Pgp, ABCB1) or the multidrug resistance associated protein (MRP). These membrane proteins are members of the ATP binding cassette transporter superfamily and are responsible for the removal from the cell of several anticancer agents including doxorubicin. Modulators can inhibit these transporters. LY335979 is among the most potent modulators of Pgp with a Ki of 59 nM. LY335979 is selective for Pgp, and does not modulate MRP-mediated resistance by MRP1 (ABCC1) and MRP2 (ABCC2). LY335979 significantly enhanced the survival of mice implanted with Pgp-expressing murine leukemia (P388/ADR) when administered in combination with either daunorubicin, doxorubicin or etoposide. Coadministration of LY335979 with paclitaxel compared to paclitaxel alone significantly reduced the tumor mass of the Pgp-expressing UCLA-P3.003VLB lung carcinoma in a xenograph model and delayed the development of tumors in mice implanted with the parental drug-sensitive UCLA-P3 tumor. LY335979 was without significant effect on the pharmacokinetics of these anticancer agents. This may be due impart to its poor inhibition of four major cytochrome P450 isozymes important in metabolizing doxorubicin and other oncolytics. The selectivity and potency of this modulator allows the clinical evaluation of the role of Pgp in multidrug resistance. LY335979 is currently in clinical trials.     

3'-O, 4'-O-aromatic acyl substituted 7,8-pyranocoumarins: a new class of P-glycoprotein modulators

Objectives P‐glycoprotein (Pgp) overexpression in tumour cells leads to multidrug resistance (MDR) and causes failure in cancer chemotherapy. We have previously identified (±)‐praeruptorin A (PA) as a potential lead compound for Pgp modulators. In this study we investigated the MDR‐reversing activities of PA derivatives. Methods Series 7,8‐pyranocoumarins with various C‐3′ and C‐4′ side chains had been semi‐synthesized and their MDR‐reversing activity was investigated in Pgp‐overexpressing MDR tumour cell line HepG2/Dox and in a KB V1 xenograft animal model. Key findings All 7,8‐pyranocoumarins exhibited equal or higher activity in modulating Pgp. DCK ( 12 ), DMDCK ( 15 ), 16 , 21 , 23 and 24 at 4 µm achieved 91%～99% decrease in IC50 value (concentration inhibiting cell growth by 50%) of anticancer agents vinblastine, doxorubicin, puromycin and paclitaxel, and were more active than others. DMDCK also remarkably enhanced the growth inhibitory effect of paclitaxel on KB V1 xenografts (P < 0.05), showing a potency required for clinical usage. Mechanistic studies suggested that these 7,8‐pyranocoumarins might reverse Pgp‐MDR through directly binding to substrate binding site(s) or allosteric site(s) on Pgp therefore impairing Pgp‐mediated drug transport. Conclusions Results from the study suggested that 3′‐O, 4′‐O‐aromatic acyl substituted 7,8‐pyranocoumarins could serve as a new class of Pgp modulator. Acyls play an important role in maintaining and enhancing the Pgp‐modulating ability of pyranocoumarins. 3,4‐Dimethoxyl substituted aromatic acyls, bearing a methoxy that might interact with Pgp as hydrogen bond accepter, were shown to be the most potent for reversing MDR.     

Biochemical and pharmacological properties of an allosteric modulator site of the human P-glycoprotein (ABCB1)

The drug-transport function of the human P-glycoprotein (Pgp or ABCB1) is inhibited by a number of structurally unrelated compounds, known as modulators or reversing agents. Among them, the thioxanthene derivative flupentixol inhibits Pgp-mediated drug transport by an allosteric mechanism. Unlike most other Pgp modulators, the cis isomer of flupentixol [cis-(Z)-flupentixol] facilitates interaction of Pgp with its transport-substrate [125I]iodoarylazidoprazosin (or [125I]IAAP), yet inhibits transport. In this study, we show that the flupentixol site acts as a common site of interaction for the tricyclic ring-containing modulators thioxanthenes and phenothiazines. The allosteric stimulation of [125I]IAAP binding to Pgp occurs independent of the phosphorylation status of the transporter. Stimulation is retained in purified Pgp reconstituted into proteoliposomes, suggesting no involvement of any other cellular protein in the phenomenon. However, perturbation of the lipid environment of the reconstituted Pgp by nonionic detergent octylglucoside abolishes stimulation by cis-(Z)-flupentixol of [125I]IAAP binding. Extensive trypsin digestion of the [125I]IAAP-labeled Pgp generates a 5.5 kDa fragment with 80% of the stimulated level of labeling associated with it. Sensitivity to inhibition by transport-substrate vinblastine and competitive modulator cyclosporin A suggests that the elevated level of [125I]IAAP binding to the fragment represents a functionally relevant interaction with the substrate site of Pgp. In summary, we demonstrate that allosteric modulation by cis-(Z)-flupentixol is mediated through its interaction with Pgp at a site specific for tricyclic ring-containing Pgp modulators of thioxanthene and phenothiazine backbone, independent of other cellular components and the phosphorylation status of the protein.     

P-glycoprotein efflux pump expression and activity in Calu-3 cells

The purpose of this work was to determine if the sub-bronchial epithelial cell model, Calu-3, expresses the functionally active P-glycoprotein (Pgp) efflux pump. Calu-3 cells express lower levels of Pgp than both Caco-2 and A549 cells as determined by Western Blot analysis. In Calu-3 cells, accumulation of the Pgp substrates rhodamine 123 (Rh123) and calcein acetoxymethyl ester (calcein-AM) was increased in the presence of the specific Pgp inhibitors cyclosporin A (CsA), vinblastine, and taxol. Significant inhibition of Pgp activity was not observed until after 2 h in both cell lines. The organic anion/multidrug resistance associated protein-1 (MRP1) inhibitors, probenecid and indomethacin, did not affect Rh123 accumulation, whereas an increase in calcein accumulation was observed by both agents. The metabolic inhibitor sodium azide decreased the efflux of Rh123 out of Calu-3 cells to the same degree as CsA, supporting inhibition of an active, efflux pathway. The basolateral-to-apical transport of Rh123 was significantly higher than that in the reverse direction, indicating a secretory pathway of efflux that was inhibited 25-fold by CsA. Basolateral-to-apical transport of Rh123 was inhibited slightly with both MRP1 inhibitors; however, no significant effect of Rh123 net secretion was observed. Mixed inhibitor studies demonstrated that Rh123 efflux was mainly Pgp mediated. These results support an energy-dependent Pgp efflux pump pathway that is sensitive to inhibition with CsA in Calu-3 cells.     

In vivo effects of cyclosporin A and ketoconazole on the pharmacokinetics of representative substrates for P-glycoprotein and cytochrome P450 (CYP) 3A in rats

In this study, the in vivo effects of cyclosporin A (CsA) and ketoconazole (KCZ), which are used as inhibitors of P-glycoprotein (Pgp) and cytochrome P450 (CYP) 3A, respectively, on the pharmacokinetics of rhodamine 123 (Rho123), nelfinavir (NFV) and erythromycin (EM) were evaluated in rats. The biliary excretion clearance (Clbile) of a known Pgp substrate, Rho123, after intravenous pretreatment with CsA or KCZ (0-20 mg/kg after i.v. administration) showed maximum reduction by 85.6 or 54.1%, respectively, suggesting that the inhibitory potency of KCZ is about half that of Pgp in the liver. Without pretreatment with CsA or KCZ, the clearance ratio of Clbile relative to the total body clearances of Rho123, NFV and EM was 10.5, 0.07 and 31.1%, respectively. After CsA pretreatment, these ratios decreased markedly in a manner dependent on the dose of CsA, while after CZ pretreatment the clearance ratios of NFV and EM increased significantly in a manner dependent on the dose of KCZ. However, in the liver, the contribution of Pgp to the changes in the pharmacokinetic parameters of Rho123, NFV and EM after intravenous administration was much less than that of CYP3A. The portal levels of Rho123 and EM but not NFV after intra-loop administration in the presence of 10 microM CsA in the jejunal loop increased significantly, while in the presence of 25 microM KCZ in the jejunal loop, the portal levels of those substrates showed no notable change as compared to the control levels. In conclusion, KCZ had dual potency to inhibit CYP3A and Pgp, and its inhibitory potency for Pgp was half that of CsA in the rat liver. In addition, metabolism via CYP3A contributed more significantly to the clearance of these substrates that did excretion via Pgp in the liver. In the small intestine, the contribution of Pgp is a more important factor in determining the oral bioavailability of EM than metabolism via CYP enzymes. The elimination of NFV is mainly dependent on liver metabolism via CYP3A, and the Pgp efflux mechanism in the liver and intestine did not contribute as importantly to the oral bioavailability of NFV under in vivo conditions, although NFV has been demonstrated to be a substrate of Pgp under in vitro conditions.     

The role of the 8-18 helix of CGRP8-37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions

1. The role of individual residues in the 8-18 helix of CGRP(8-37) in promoting high-affinity binding to CGRP(1) receptors expressed on rat L6 and human SK-N-MC cells has been examined. The relative potencies of various derivatives were estimated from their ability to inhibit the human alphaCGRP-mediated increase in cyclic AMP production and the binding of [(125)I]-human alphaCGRP. 2. Arg(11) and Arg(18) were replaced by serines to give [Ser(11,18)]CGRP(8-37). These bound with pKi values <6 to SK-N-MC cells and had apparent pA(2) values of 5.81+/-0.04 and 5.31+/-0.11 on SK-N-MC and L6 cells. CGRP(8-37) had a pKi of 8.22 on SK-N-MC cells and pK(b) values on the above cell lines of 8.95+/-0.04 and 8.76+/-0.04. 3. The arginines were replaced with glutamic acid residues. [Glu(11)]CGRP(8-37) had a pK(b) of 7.14+/-0.14 on SK-N-MC cells (pKi=7.05+/-0.05) and 6.99+/-0.08 on L6 cells. [Glu(18)]CGRP(8-37) had a pK(b) of 7.10+/-0.0.08 on SK-N-MC cells (pKi=6.91+/-0.23) and 7.12+/-0.09 on L6 cells. 4. Leu(12), Leu(15) and Leu(16) were replaced by benzoyl-phenylalanine (bpa) residues. On SK-N-MC cells, the apparent pA(2) values of [bpa(12)]-, [bpa(15)]- and [bpa(16)]CGRP(8-37) were respectively 7.43+/-0.23, 8.34+/-0.11 and 5.66+/-0.16 (pKi values of 7.14+/-0.17, 7.66+/-0.21 and <6): on L6 cells they were 7.96+/-0.36, 8.28+/-0.21 and 6.09+/-0.04 (all n=3). 5. It is concluded that the Arg(11) and Arg(18) are involved in specific electrostatic interactions with other residues, either on the CGRP(1) receptors or elsewhere on CGRP(8-37). Leu(16) is in a conformationally restricted site when CGRP(8-37) binds to CGRP(1) receptors, unlike Leu(12) and Leu(15).