Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmembrane ion gradients

A new method, based on the ion-translocating properties of the ionophores nigericin and A23187, is described for loading large unilamellar vesicles (LUVs) with the drugs vincristine and ciprofloxacin. LUVs composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (55:45 mol/mol) or sphingomyelin (SPM)/Chol (55:45 mol/mol) exhibiting a transmembrane salt gradient (for example, internal solution 300 mM MnSO4 or K2SO4; external solution 300 mM sucrose) are incubated in the presence of drug and, for experiments involving divalent cations, the chelator EDTA. The addition of ionophore couples the outward movement of the entrapped cation to the inward movement of protons, thus acidifying the vesicle interior. External drugs that are weak bases can be taken up in response to this induced transmembrane pH gradient. It is shown that both nigericin and A23187 facilitate the rapid uptake of vincristine and ciprofloxacin, with entrapment levels approaching 100% and excellent retention in vitro. Following drug loading, the ionophores can be removed by gel exclusion chromatography, dialysis, or treatment with biobeads. In vitro leakage assays (addition of 50% mouse serum) and in vivo pharmacokinetic studies (in mice) reveal that the A23187/Mn2+ system exhibits superior drug retention over the nigericin/K+ system, and compares favorably with vesicles loaded by the standard DeltapH or amine methods. The unique features of this methodology and possible benefits are discussed.     

Effects of some antineoplastic drugs (vincristine, doxorubicin and epirubicin) on human platelet aggregation

The effects of some antineoplastic drugs (vincristine, doxorubicin and epirubicin) on collagen- and ADP-induced human platelet aggregation are investigated. Platelet rich plasma (PRP) and platelet poor plasma (PPP) from healthy male and female donors were used. The PRP was adjusted with analogous PPP to 300,000 platelets/microliters. Platelet aggregation was studied according to Born's turbidimetric technique using an Aggrecorder II PA 3220 with collagen at a concentration of 10 micrograms/ml and ADP at a concentration of 30 microM. Vincristine, doxorubicin and epirubicin significantly (p < 0.01) inhibited collagen- and ADP-induced platelet aggregation. The vincristine induced inhibition was higher than that induced by doxorubicin or epirubicin. The effects of doxorubicin and epirubicin were more intense on ADP-induced platelet aggregation than on the collagen induced one. Moreover, the doxorubicin inhibition of ADP-induced platelet aggregation was greater than the epirubicin one. In conclusion, our study shows that vincristine, doxorubicin and epirubicin inhibit human platelet aggregation. The present results may improve the therapeutic use of these drugs since it has been clearly shown that drugs with antiplatelet activity could block metastases.     

Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients

The uptake of the anticancer agent doxorubicin into large unilamellar vesicles (LUVs) exhibiting a transmembrane pH gradient (inside acidic) has been investigated using both kinetic and equilibrium approaches. It is shown that doxorubicin uptake into the vesicles proceeds via permeation of the neutral form and that uptake of the drug into LUVs with an acidic interior is associated with high activation energies (Ea) which are markedly sensitive to lipid composition. Doxorubicin uptake into egg-yolk phosphatidylcholine (EPC) LUVs exhibited an activation energy of 28 kcal/mol, whereas for uptake into EPC/cholesterol (55:45, mol/mol) LUVs Ea = 38 kcal/mol. The equilibrium uptake results obtained are analyzed in terms of a model which includes the buffering capacity of the interior medium and the effects of drug partitioning into the interior monolayer. From the equilibrium uptake behaviour, a doxorubicin partition coefficient of 70 can be estimated for EPC/cholesterol bilayers. For a 100 nm diameter LUV, this indicates that more than 95% of encapsulated doxorubicin is partitioned into the inner monolayer, presumably located at the lipid/water interface. This is consistent with 13C-NMR behaviour as a large proportion of the drug appears membrane associated after accumulation as reflected by a broadening beyond detection of the 13C-NMR spectrum. The equilibrium accumulation behaviour of a variety of other lipophilic amines is also examined in terms of the partitioning model.     

Comparison of adriamycin and derivatives uptake into large unilamellar lipid vesicles in response to a membrane potential

The uptake of adriamycin (ADM) and several derivatives into large unilamellar vesicles (LUV) displaying a transmembrane potential and having a lipid composition close to that of the inner mitochondrial membrane has been measured. Drug association to neutral liposomes, made of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (70:30, w/w) was shown to be potential-dependent: in the absence of potential, accumulation of drug was almost undetectable, whereas between 11 and 50 nmol of drug/mumol phospholipid, depending on the anthracycline used, was associated to LUV exhibiting a membrane potential after 1 h incubation. Association of drugs to LUV with a lipid composition closer to that of the inner mitochondrial (cardiolipin, CL, 20%; PC 50%; PE, 30%, w/w) and displaying a membrane potential is higher than with neutral vesicles (between 40 and 76 nmol of anthracycline/mumol phospholipid after 1 h incubation). Since it is known that ADM and derivatives have a high affinity for CL, a fraction of the associated drug may bind to CL on the outer side of the vesicles. This was confirmed by the fact that, in the absence of potential, between 40 and 56 nmol of anthracycline/mumol phospholipid was still associated to LUV containing CL. In order to discriminate between drug adsorbed at the surface of the LUV and drug accumulated inside the LUV, an anthracycline fluorescence quencher (I-) was used. It was shown on neutral LUV displaying a membrane potential, that between 55 and 81% of the associated drug is actually entrapped inside the vesicles, inaccessible to the quencher. These percentages decreased to between 41 and 68%, respectively, in the presence of LUV containing CL and exhibiting a membrane potential, whereas for LUV of the same composition but displaying no membrane potential almost all the associated drug is adsorbed on the outer face of the LUV, accessible to the quencher, and likely bound to CL. This study brings evidence that antitumour anthracyclines despite important structural homologies do not accumulate to the same extent into vesicles mimicking the lipid composition and the membrane potential of mitoplasts. This ability to reach the matrix compartment of mitochondria could partly explain the differences of cardiotoxicities associated to anthracyclines with closely related molecular structure.     

Correlations among Epworth Sleepiness Scale scores, multiple sleep latency tests and psychological symptoms

Mouse leukemic cell subline L1210/VCR exerts expressive multidrug resistance (MDR) that is mediated by P-glycoprotein. Cells originally adapted to vincristine are also extremely resistant to doxorubicin. Resistance to both vincristine and doxorubicin is connected with depression of drug uptake. While resistance of L1210 cells to vincristine could be reversed by verapamil as chemosensitizer, resistance of cells to doxorubicin was insensitive to verapamil. Action of verapamil (well-known inhibitor of PGP activity) on multidrug resistance was often used as evidence that MDR is mediated by PGP. From this point it may be possible that the resistance of L1210/VCR cells to vincristine is mediated by PGP and the resistance to doxorubicin is mediated by other PGP-independent system. Another and more probable explanation of different effect of verapamil on resistance of L1210/VCR cells to vincristine and doxorubicin may be deduced from the following fact: Using UV spectroscopy we found that doxorubicin dissolved in water buffered medium interacts effectively with verapamil. This interaction may be responsible for the decrease of concentration of both drugs in free effective form and consequently for higher survival of cells. In contrast to doxorubicin vincristine does not give any interaction with verapamil that is measurable by UV spectroscopy and resistance of L1210/VCR cells to vincristine may be fully reversed by verapamil.     

The overall partitioning of anthracyclines into phosphatidyl-containing model membranes depends neither on the drug charge nor the presence of anionic phospholipids

Anthracyclines are potent anticancer agents. Their use is limited by the problem of multidrug resistance (MDR) associated with a decreased intracellular accumulation of drug correlated with the presence, in the membrane of resistant cells, of the P-glycoprotein responsible for an active efflux of the drug. The activity of a drug depends upon its intracellular concentration which itself depends on the kinetics (a) of passive influx (b) of passive efflux and (c) of the P-glycoprotein-mediated efflux of drug across the cell membrane. The ability of an anthracycline to overcome MDR depends largely on the first point. The passive drug uptake is governed by their incorporation into the lipid matrix and both electrostatic and hydrophobic forces seem necessary for the stabilization of anthracyclines into lipid bilayers. The aim of the present study was to determine the relative importance of these two interactions. Using microspectrofluorometry and the observation that the fluorescence of anthracycline is enhanced when the dihydroanthraquinone part is embedded within the lipid bilayer, we have determined the partition coefficient (alternatively, the binding constant) of 12 anthracycline derivatives in large unilamellar vesicles. The anthracyclines were (a) doxorubicin, daunorubicin and idarubicin which, at pH 7.2, bear a single positive charge at the level of the amino group on the sugar, (b) their corresponding neutral 3'-hydroxy derivatives where the amino group in the sugar has been replaced by a hydroxyl, (c) the three 13-hydroxy derivatives, doxorubicinol, daunorubicinol and idarubicinol, (d) pirarubicin and (e) two permanently positively charged derivatives. The large unilamellar vesicles contained phosphatidylcholine with various amounts of phosphatidic acid which is negatively charged and of cholesterol. We came to the conclusion that the efficiency of drug incorporation in the bilayers depends neither on the presence of a positive charge on the drug nor on the presence of anionic phospholipid but on the hydrophobicity of the molecule: the neutral and the positively charged form have the same ability to partition into the bilayer. However, the percentage of each form present should depend on the electrostatic parameters.     

Preoperative evaluation of the chemosensitivity of breast cancer by means of double phase 99mTc-MIBI scintimammography

The chemosensitivity of breast cancer is important for its management, but it is difficult to evaluate preoperatively. Tc-99m hexakis-2-methoxyisobutylisonitrile (MIBI) scintimammography has been reported to indicate the expression of P-glycoprotein, which is one factor concerned with multidrug resistance. We developed a chemosensitivity assay by using surgical specimens to investigate whether 99mTc-MIBI scintimammography findings before the operation are related to chemosensitivity according to our assay. Fifteen patients with primary breast cancer were enrolled into the study. Early and delayed images were obtained at 10 and 120 minutes after intravenous injection of 99mTc-MIBI, respectively. Regions of interest were placed on the tumors and the contralateral healthy breasts in each patient to estimate 99mTc-MIBI uptake in the tumor, and retention indices were then calculated to assess the washout of 99mTc-MIBI. Chemosensitivity assay was performed by incubating surgical specimens with anticancer agents such as doxorubicin, epirubicin, pinorubicin, mitomycin C, cisplatin and 5-fluorouracil. 99mTc-MIBI washout on scintimammography was successfully related to inhibition ratios on chemosensitivity tests when compared with 99mTc-MIBI uptake by the tumor. In particular, high correlation coefficients were obtained between the retention index of 99mTc-MIBI and the inhibition ratios of doxorubicin (r = 0.75), epirubicin (r = 0.60) and pinorubicin (r = 0.62), but poor correlation was found for mitomycin C (r = 0.44) and cisplatin (r = 0.31). Our results indicate that the retention index of 99mTc-MIBI is closely correlated to chemosensitivity to anthracyclines, suggesting that double-phase scintimammography allows preoperative prediction of chemosensitivity of breast cancer.     

Decreased drug accumulation without increased drug efflux in a novel MRP-overexpressing multidrug-resistant cell line

KB/7D cells represent a multidrug-resistant subclone of human nasopharyngeal carcinoma KB cells generated by continuous exposure to the topoisomerase II inhibitor VP-16 (etoposide). KB/7D cells also show cross-resistance to doxorubicin and vincristine. Phenotypic traits of the cell line include a 2-fold decrease in topoisomerase II levels and a decrease in the uptake of VP-16 without an increase in the rate of drug efflux or expression of P-glycoprotein, suggesting a novel mechanism associated with the uptake of anticancer drugs. This study demonstrated that the multidrug-resistance associated protein (MRP) is overexpressed in KB/7D cells, and that the loss of resistance in revertant cells correlates with the loss of MRP. The resistance to VP-16 and doxorubicin could be overcome, partially, and resistance to vincristine could be overcome completely, by the L-enantiomer of verapamil, but not by the D-enantiomer or by BIBW 22 (4-[N-(2-hydroxy-2-methyl-propyl)-ethanolamino]-2,7-bis[cis-2,6-++ +dimethylmorpholino)-6-phenylpteridin), an inhibitor of MDR-1. L-Verapamil was shown to be significantly more potent than D-verapamil in modulating the accumulation defect in KB/7D cells towards doxorubicin, as measured by flow cytometry and confocal microscopy, and towards VP-16, as measured by increases in protein-linked DNA strand breaks. This suggests that KB/7D cells are multidrug resistant due to decreases in topoisomerase II levels and the overexpression of MRP, that MRP leads to a decrease in drug accumulation, and that L-verapamil can modulate the MRP-associated accumulation defect and drug-resistance phenotype. This contrasts with previous studies that suggest that MRP causes multidrug resistance by exporting cytotoxic drugs out of the cell and that did not show modulation of MRP by verapamil.     

A randomized comparison of the efficacy and toxicity of epirubicin and doxorubicin in the treatment of patients with non-Hodgkin's lymphoma

BACKGROUND: Combination chemotherapy consisting of methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisolone, and bleomycin (MACOP-B) has been frequently used for the treatment of non-Hodgkin's lymphoma. This randomized study was undertaken to assess the efficacy and toxicity of this regimen when either doxorubicin or epirubicin was used as the anthracycline drug. METHODS: Between April 1989 and December 1993, 211 previously untreated patients with intermediate grade and high grade non-Hodgkin's lymphoma were randomized to receive either doxorubicin (n=106) or epirubicin (n=105) with the MACOP-B regimen. These patients were followed through December 1996. Numerous clinical features predictive of response and survival were analyzed. Cardiac and noncardiac toxicity in the two treatment arms were compared. RESULTS: The median age of the patients was 48 years. Complete remission was experienced by 122 patients (58.3%); 62 patients (58.5%) achieved complete remission in the doxorubicin arm and 60 (58.1%) in the epirubicin arm. Response rates, time to treatment failure, relapse data, and overall survival were comparable between the two arms. Morbidity due to mucositis, vomiting, peripheral neuropathy, and cardiotoxicity were also comparable. The overall mortality was 10%. Mortality due to neutropenic sepsis was considerably higher among patients who received epirubicin (10 patients) than among those who received doxorubicin (5 patients). Cardiac evaluation revealed no difference in toxicity between the two arms. CONCLUSIONS: Epirubicin was as effective as doxorubicin in terms of patients' responses to therapy. There was no difference in cardiotoxicity between the two treatment arms. However, in this study, the mortality due to neutropenic sepsis was significantly higher among patients treated with epirubicin.     

Inhibition of anthracycline semiquinone formation by ICRF-187 (dexrazoxane) in cells

The formation of semiquinone free radicals of doxorubicin, epirubicin, daunorubicin, and idarubicin was measured by electron paramagnetic resonance (EPR) spectroscopy in hypoxic suspensions of chinese hamster ovary (CHO) cells. The amount of semiquinone produced was in the order idarubicin > doxorubicin > daunorubicin > epirubicin. The idarubicin semiquinone signal was both the fastest to be formed and to decay. Idarubicin, which was the most lipophilic of the anthracyclines studied, also displayed the fastest fluorescence-measured cellular uptake of drug. Thus, it was concluded that semiquinone formation was dependent upon the rate of cellular uptake. Lysed cell suspensions were also shown to be capable of producing the doxorubicin semiquinone in the presence of added NADPH. The cardioprotective agent dexrazoxane (ICRF-187) was observed to decrease the amount of doxorubicin semiquinone observed in cell suspensions. Dexrazoxane also decreased the amount of doxorubicin semiquinone observed in the NADPH-lysed cell suspension mixture. Neither bipyridine nor deferoxamine decreased NADPH-dependent doxorubicin semiquinone formation. These results suggest that dexrazoxane does not decrease doxorubicin semiquinone formation through an iron complex formed from hydrolyzed dexrazoxane. Dexrazoxane may be inhibiting an NADPH-dependent enzyme.     

In vitro and in vivo targeting of immunoliposomal doxorubicin to human B-cell lymphoma

The ability to selectively target liposomal anticancer drugs via specific ligands against antigens expressed on malignant cells could improve the therapeutic effectiveness of the liposomal preparations as well as reduce adverse side effects associated with chemotherapy. Long-circulating formulations of liposomes containing lipid derivatives of poly(ethyleneglycol) [sterically stabilized liposomes (SLs)] have been described previously, and new techniques have recently been developed for coupling monoclonal antibodies (Abs) at the poly(ethyleneglycol) terminus of these liposomes. Ab-targeted SLs [immunoliposomes (SILs)] containing entrapped anticancer drugs are predicted to be useful in the treatment of hematological malignancies such as B-cell lymphomas or multiple myeloma, in which the target cells are present in the vasculature. The specific binding, in vitro cytotoxicity, and in vivo antineoplastic activity of doxorubicin (DXR) encapsulated in SILs coupled to monoclonal Ab anti-CD19 (SIL[anti-CD19]) were investigated against malignant B cells expressing CD19 surface antigens. Binding experiments with SIL[anti-CD19] resulted in a 3-fold higher association of the SILs with a human CD19+ B lymphoma cell line (Namalwa) in comparison with nontargeted SLs. Using flow cytometry, fluorescently labeled SIL[anti-CD19] bound to B cells with no recognition of T cells in a mixture of B cells and T cells in culture. Nontargeted SLs demonstrated significantly lower recognition of either B cells or T cells. Targeted DXR-SIL[anti-CD19] displayed a higher cytotoxicity to B cells relative to DXR entrapped in nontargeted SLs. Therapeutic experiments in severe combined immunodeficient mice implanted with Namalwa cells by the i.v. or i.p. routes resulted in significantly increased effectiveness of DXR-SIL[anti-CD19] compared to similar amounts of free DXR, DXR-SL (no Ab), or isotype-matched nonspecific Abs attached to DXR-SL. Single doses (3 mg/kg) of DXR-SIL[anti-CD19] administered i.v. resulted in a significantly improved therapeutic benefit, including some long-term survivors. From our results, we infer that targeted anti-CD19 liposomes containing the anticancer drug DXR may be selectively cytotoxic for B cells and may be useful in the selective elimination of circulating malignant B cells in vivo.     

Characterization of vincristine transport by the M(r) 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione

The M(r) 190,000 multidrug resistance protein (MRP) confers resistance to a broad spectrum of natural product drugs. However, it has not been possible to demonstrate that MRP can actively transport unmodified forms of these compounds, although the protein has been shown to transport structurally diverse glutathione (GSH)- and glucuronide-conjugated molecules. Previously, we showed that ATP-dependent uptake of vincristine by MRP-enriched, inside-out membrane vesicles could be stimulated by physiological concentrations of GSH (Loe et al., J. Biol. Chem., 271: 9675-9682, 1996). We have now established that the ATP/GSH dependent vincristine uptake is both proportional to the level of MRP in the membrane vesicles and can be inhibited by monoclonal antibodies shown previously to inhibit transport of established MRP substrates, such as leukotriene C4. We also show that short-chain alkyl derivatives of GSH can stimulate drug uptake, which suggests that vincristine transport does not necessarily involve a change in redox state or glutathionylation of the protein. Furthermore, vincristine uptake is accompanied by ATP- and drug-dependent accumulation of GSH, which can also be stimulated to a lesser extent by vinblastine but not daunorubicin or doxorubicin. Although GSH or vincristine alone are very poor inhibitors of MRP-mediated transport of leukotriene C4, together they act as relatively potent competitive inhibitors. Overall, our data demonstrate that MRP can actively cotransport GSH and unmodified vincristine and that these compounds probably interact, either with the leukotriene C4 binding site(s) on the protein or with a mutually exclusive site.     

Handling of doxorubicin by the LLC-PK1 kidney epithelial cell line

The characteristics of doxorubicin handling have been studied in the cultured kidney epithelial cell line LLC-PK1, which has structure and function similar to those of renal tubular cells and expresses P-glycoprotein. The uptake of doxorubicin by LLC-PK1 cells was time dependent, reaching a steady state at about 4 hr, and reduced at low temperature; the initial uptake was saturable. The efflux of doxorubicin from LLC-PK1 cells was also temperature dependent but, even at 37 degrees C, a significant percentage of the drug remained associated with the cells after 180 min, which suggests a strong cellular binding, and the fluorescence microscopy revealed that the drug was concentrated in intracellular organelles. Substances that are substrates for P-glycoprotein, such as verapamil, vinblastine, vincristine and quinidine, significantly increased doxorubicin concentrations in LLC-PK1 cells. Similar results were obtained with the metabolic inhibitors sodium metavanadate and 2,4-dinitrophenol. On the other hand, the uptake was not affected by the classic organic cation transport drugs cimetidine, decynium 22 or decynium 24, nor by the organic anion drug probenecid. These results indicate that, in LLC-PK1 cells, doxorubicin enters by passive diffusion, is trapped in intracellular organelles and then is extruded from cells by a mechanism that probably involves P-glycoprotein. On the contrary, substances that interfere with the renal organic cation or anion secretory system have no effect on doxorubicin net accumulation in these cells.     

Flip-flop of doxorubicin across erythrocyte and lipid membranes

Doxorubicin, an anticancer drug, is extruded from multidrug resistant (MDR) cells and from the brain by P-glycoprotein located in the plasma membrane and the blood-brain barrier, respectively. MDR-type drugs are hydrophobic and, as such, enter cells by diffusion through the membrane without the requirement for a specific transporter. The apparent contradiction between the presumably free influx of MDR-type drugs into MDR cells and the efficient removal of the drugs by P-glycoprotein, an enzyme with a limited ATPase activity, prompted us to examine the mechanism of passive transport within the membrane. The kinetics of doxorubicin transport demonstrated the presence of two similar sized drug pools located in the two leaflets of the membrane. The transbilayer movement of doxorubicin occurred by a flip-flop mechanism of the drug between the two membrane leaflets. At 37 degrees, the flip-flop exhibited a half-life of 0.7 min, in both erythrocyte membranes and cholesterol-containing lipid membranes. The flip-flop was inhibited by cholesterol and accelerated by high temperatures and the fluidizer benzyl alcohol. The rate of doxorubicin flux across membranes is determined by both the massive binding to the membranes and the slow flip-flop across the membrane. The long residence-time of the drug in the inner leaflet of the plasma membrane allows P-glycoprotein a better opportunity to remove it from the cell.     

Kinetic analysis in living cells of the inhibition of the P-glycoprotein-mediated efflux of anthracyclines by vinca alkaloids

Cells that overexpress the mdr 1 gene have decreased steady-state accumulation and increased efflux of many anticancer drugs including anthracyclines and vinca alkaloids. The mechanism(s) of P-glycoprotein-mediated efflux of drugs is (are) still poorly understood. In an attempt to identify mechanism(s) by which multidrug resistance can be circumvented, the cellular accumulation has been examined of pirarubicin, doxorubicin and idarubicin alone and in conjunction with four vinca alkaloid derivatives--vinblastine, navelbine, vindesine and vincristine. The present study was performed using a spectrofluorometric method with which it is possible to follow continuously the uptake and release of fluorescent molecules by living cells, as the incubation of the cells with the drug proceeds. Erythroleukemia K562 cell lines were used. It has been shown that the P-glycoprotein-mediated efflux of these three anthracyclines can be inhibited by vinca alkaloids derivatives. At pH 7.2, 50% of the P-glycoprotein-mediated efflux of daunorubicin and idarubicin was inhibited by about 40 +/- 10 microM vinblastine and that of pirarubicin by 10 +/- 2 microM vinblastine. The vinblastine concentration required to inhibit 50% of the active efflux of these anthracyclines did not depend on the anthracycline concentrations used, indicating that the inhibition was non competitive. The ability of navelbine, vincristine and vindesine to inhibit the active efflux of pirarubicin was also checked; 15 +/- 3 microM navelbine are required to inhibit 50% of the active efflux but at concentrations lower than 100 microM, neither vincristine nor vindesine were able to inhibit this efflux, indicating that the vinca alkaloids compounds which are the most efficient are the most lipophilic. For the four vinca alkaloids, the concentration required to inhibit 50% of the efflux was lower as the pH was higher. A detailed kinetics analysis of the P-glycoprotein-mediated efflux of pirarubicin in the presence of vinblastine indicates a non competitive inhibition with K(I) = 12 +/- 2 microM.     

Control of testicular vasomotion by testosterone and tubular factors in rats

We have compared the pharmacological and molecular characteristics of 2 cell lines derived from the C6 rat glioblastoma, and selected for resistance either to doxorubicin (C6 0.5 line) or to vincristine (C6 IV line). Each line displays a preferential 400-fold resistance towards the drug used for selection, the C6 IV line being especially weakly resistant to doxorubicin (13-fold). Verapamil completely restored doxorubicin sensitivity in the C6 IV line as well as vincristine resistance in the C6 0.5 line, but could not completely reverse doxorubicin resistance in the C6 0.5 line or vincristine resistance in the C6 IV line. This suggests that specific mechanisms of resistance against each drug were added to a common P-glycoprotein-mediated multidrug-resistance mechanism. Doxorubicin efflux was total within 2 hr in the C6 IV line, whereas it remained 8 to 10% of drug in the C6 0.5 line 4 hr after drug removal, despite a more rapid efflux of the drug in the first 30 min. This 2-compartment behavior could be related to a special sub-cellular distribution of doxorubicin in C6 0.5 cells. Northern and Western blot analysis of the mdrI gene and of the P-glycoprotein expressed by the 2 resistant cell lines made it possible to quantify their degree of over-expression; when compared with the C6 wild strain, the C6 0.5 line over-expressed both the mdrI gene and the P-glycoprotein to a slightly higher level than the C6 IV line. Northern and Western blot analysis also suggested that C6 0.5 cell preferentially over-expressed the mdrIa gene, whereas the C6 IV cells preferentially over-expressed the mdrIb gene. This differential over-expression was confirmed after polymerase-chain-reaction amplification of the cDNA sequences transcribed from total RNA extracted from the 2 lines. It can be concluded therefore that the mdrIa gene product is more efficient than the mdrIb gene product in extruding anti-cancer drugs from the cells; and that the mdrIb gene product might preferentially extrude vincristine rather than doxorubicin.     

The effect of vincristine-polyanion complexes in STEALTH liposomes on pharmacokinetics, toxicity and anti tumor activity

Poly(ethylene glycol) (PEG)-derivatized liposome vehicles improve antitumor effectiveness of entrapped anthracyclines and vinca alkaloids. However, the plasma clearance of entrapped vincristine is substantially faster than the lipid phase or other entrapped aqueous markers, suggesting leakage out of the liposome during transit in the blood compartment. We tested the effect of altering the drug's in vivo leakage rate on pharmacokinetics, toxicity, and antitumor activity of entrapped drug in rodent models. Suramin, heparin, and dextran sulfate were tested for their ability to produce a precipitable complex in vitro. PEG-derivatized liposomes were prepared with the complexing agent inside, and vincristine was driven inside using an ammonium gradient. The resulting preparations were found to have plasma distribution half-lives significantly longer than the formulation without a complex-forming agent. There was no increase in acute lethality, and in the case of the suramin-vincristine complex, the acute lethality was significantly reduced at the highest does level. Anti-tumor activity against the mouse mammary carcinoma MC2 was tested in a multiple-dose study. Free vincristine did not affect the tumor growth rate significantly, but at the same dose level all PEG-coated liposome formulations inhibited tumor growth markedly. The suramin containing formulation was as effective as the formulation lacking polyanion, but the heparin and dextran sulfate containing formulations were less effective. Thus, compounds which form insoluble complexes with vincristine alter in vivo plasma distribution phase pharmacokinetics without increasing acute lethality, but without a corresponding increase in anti-tumor activity.     

Energetics and mechanism of drug transport mediated by the lactococcal multidrug transporter LmrP

The gene encoding the secondary multidrug transporter LmrP of Lactococcus lactis was heterologously expressed in Escherichia coli. The energetics and mechanism of drug extrusion mediated by LmrP were studied in membrane vesicles of E. coli. LmrP-mediated extrusion of tetraphenyl phosphonium (TPP+) from right-side-out membrane vesicles and uptake of the fluorescent membrane probe 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) into inside-out membrane vesicles are driven by the membrane potential (Deltapsi) and the transmembrane proton gradient (DeltapH), pointing to an electrogenic drug/proton antiport mechanism. Ethidium bromide, a substrate for LmrP, inhibited the LmrP-mediated TPP+ extrusion from right-side-out membrane vesicles, showing that LmrP is capable of transporting structurally unrelated drugs. Kinetic analysis of LmrP-mediated TMA-DPH transport revealed a direct relation between the transport rate and the amount of TMA-DPH associated with the cytoplasmic leaflet of the lipid bilayer. This observation indicates that drugs are extruded from the inner leaflet of the cytoplasmic membrane into the external medium. This is the first report that shows that drug extrusion by a secondary multidrug resistance (MDR) transporter occurs by a "hydrophobic vacuum cleaner" mechanism in a similar way as was proposed for the primary lactococcal MDR transporter, LmrA.     

Rapid recovery of a functional MDR phenotype caused by MRP after a transient exposure to MDR drugs in a revertant human lung cancer cell line

Prior studies have shown that, in some human tumour cells, increased expression of the multidrug resistance gene MDR1 can be induced in response to certain stress conditions such as a transient exposure to cytotoxic agents. Little is known about the possibility of increasing the expression of the recently cloned multidrug resistance-associated protein (MRP) in response to a transient exposure to cytotoxic drugs. In order to examine this possibility, we have used sensitive assays (RT-PCR, flow cytometry) and the sensitive large cell lung cancer cell line, COR-L23/P, and the revertant line (COR-L23/Rev), generated by growing the doxorubicin-selected, MRP-overexpressing resistant variant COR-L23/R without drug exposure for 24-28 weeks. COR-L23/Rev overexpresses MRP, but to a lesser extent than COR-L23/R. COR-L23/Rev rapidly recovered similar levels of MRP mRNA, protein expression, resistance and drug accumulation deficit as COR-L23/R after a 48-72 h exposure to cytotoxic concentrations of doxorubicin or vincristine but not cisplatin. The increase in MRP mRNA could only be detected 3 to 4 days after the transient exposure to drugs. However, when the parental line, COR-L23/P, was exposed to equitoxic doses of doxorubicin, vincristine or cisplatin, no increase in the levels of MRP mRNA could be observed at higher doses (5- to 10-fold the IC50) of doxorubicin or vincristine (but not of cisplatin), we detected a transient increase in the levels of MDR1 mRNA immediately after short-term exposure. In conclusion, we have shown that a human revertant lung cancer cell line (COR-L23/Rev) has the ability to recover quickly, similar levels of MRP expression and resistance as COR-L23/R after a transient exposure to the MDR-drugs doxorubicin and vincristine.     

Development of ciprofloxacin-loaded nanoparticles: physicochemical study of the drug carrier

This paper describes the optimization of the preparation of ciprofloxacin-loaded polyethylbutylcyanoacrylate (PEBCA) nanoparticles. The association of ciprofloxacin with nanoparticles was performed by emulsion polymerization, but successful entrapment was only obtained in the presence of acetone in the polymerization medium. This preparation process led to a stable ciprofloxacin nanoparticle suspension, with a mean size value twice as high as that obtained in the absence of drug, and an association efficiency of 82%. Moreover, the molecular weight value of ciprofloxacin nanoparticles was shown to be reduced as compared with unloaded nanoparticles. Drug release from the colloidal carrier in medium containing esterase was found to be very slow (a maximum of 51.5% after 48 h), suggesting that this release resulted from bioerosion of the polymer matrix. Interestingly, it was observed that 30.5% of the initial amount of ciprofloxacin was not detectable by HPLC analysis after nanoparticle preparation and corresponded either to ciprofloxacin covalently bound to PEBCA or to ciprofloxacin chemically degraded during the polymerization process. 19F-NMR analysis demonstrated that ciprofloxacin entrapped into nanoparticles was only in its neutral form. The measurements of molecular weight suggest the participation of the antibiotic as an anionic polymerization initiator, leading to the formation of a chemical bond between some of the drug and the polymer. These data allowed us to propose a model describing the association of ciprofloxacin with PEBCA nanoparticles obtained by emulsion polymerization.