Michèle Masquelier

Elevated uptake of low density lipoprotein by drug resistant human leukemic cell lines

Overexpression of a 170kD membrane glycoprotein, P-glycoprotein (Pgp), which acts as an energy dependent efflux pump for cytotoxic drugs is believed to be one of the factors that is responsible for clinical drug resistance. Recent studies suggest that Pgp is also responsible for the intracellular transport of cholesterol from the plasma membrane to the endoplasmic reticulum. Leukemic cells from patients with acute myelogenous leukemia have an elevated uptake of low density lipoprotein (LDL) when compared with white blood cells from healthy individuals. Since elevated LDL receptor expression and multidrug resistance are both common events in leukemic cells, we investigated LDL receptor expression in sensitive and drug resistant human leukemic cell lines. We found a 2- to 10-fold higher uptake of LDL in five out of five drug resistant K562 cell lines. All three drug resistant HL60 cell lines studied also had higher uptake than the parental cells. The LDL receptor expression in vincristine resistant Pgp positive K562 cells was less sensitive to downregulation by sterols than in parental cells. There was no selective effect of the Pgp inhibitor PSC-833 or other Pgp modulators on LDL receptor activity in Pgp positive cells. Since also resistant Pgp, multidrug resistance protein 1, and breast cancer resistance protein negative cells exhibited an elevated LDL receptor activity, we conclude that overexpression of these proteins is not the mechanism behind the elevated LDL uptake in the drug resistant leukemic cell lines. The findings are of interest for the concept of using lipoproteins as carriers of cytotoxic drugs in cancer treatment.

Low Density Lipoprotein as a Carrier of Cytostatics in Cancer Chemotherapy: Study of Stability of Drug-carrier Complexes in Blood

Abstract Several solid tumour and leukemia cell types have a higher low density lipoprotein (LDL) uptake than the corresponding normal cells. We are investigating the possibilities to use LDL as a drug carrier to increase the selectivity of antineoplastic drugs in cancer chemotherapy. We have developed a method to incorporate lipophilic cytotoxic agents without interfering with the in vitro and in vivo properties of LDL. In this study, we examined the stability of some drug-LDL complexes in blood and plasma as this is an important prerequisite to achieve a selective therapy. The in vitro dialysis of N-trifluoroacetyl-adriamycin-14-valerat-LDL (AD-32-LDL) against plasma revealed a slow dissociation of the complex. The same method showed a fast and total leakage of paclitaxel from paclitaxel—LDL into the plasma chamber. The dissociation of paclitaxel was confirmed by an autoradiographic study of the distribution of paclitaxel—LDL in tumour-bearing mice. In patients with leukemia the rapid plasma dissociation of AD-32 from LDL illustrated a much higher in vivo instability of this complex. With this method, cholesteryl-linoleate only could be incorporated into LDL in a stable manner as shown by dialysis and autoradiography results. The incorporation of cytotoxic drug derivatives, containing lipophilic anchors, is now under study in order to obtain LDL complexes with better plasma stability.

Fluconazole-induced intoxication with phenytoin in a patient with ultra-high activity of CYP2C9

PurposeThe cytochrome P450 enzyme CYP2C9 metabolizes several important drugs, such as warfarin and oral antidiabetic drugs. The enzyme is polymorphic, and all known alleles, for example, CYP2C9*2 and*3, give decreased activity. Ultra-high activity of the enzyme has not yet been reported.MethodsWe present a patient with Behçet’s disease who required treatment with high doses of phenytoin. When fluconazole, a potent inhibitor of CYP2C9, was added to the treatment regimen, the patient developed ataxia, tremor, fatigue, slurred speech and somnolence, indicating phenytoin intoxication. On suspicion of ultra-high activity of CYP2C9, a phenotyping test for CYP2C9 with losartan was performed.ResultsThe patient was shown to have a higher activity of CYP2C9 than any of the 190 healthy Swedish Caucasians used as controls.ConclusionsOur finding of an ultrarapid metabolism of losartan and phenytoin may apply to other CYP2C9 substrates, where inhibition of CYP2C9 may cause severe adverse drug reactions.

Plasma stability and cytotoxicity of lipophilic daunorubicin derivatives incorporated into low density lipoproteins

The selective targeting of antineoplastic drugs to tumours by incorporation in low density lipoproteins (LDL) is an attractive possibility if the drug-LDL complex remains stable in the circulation and is taken up by the tumour. In previous studies we have shown that vincristine- and N-trifluoroacetyladriamycin-14-valerate-LDL complexes were unstable in vivo. We synthesized five N-substituted lipophilic derivatives of daunorubicin and studied their incorporation into LDL. Three out of five daunorubicin derivatives incorporated successfully into LDL. In vitro these complexes were more cytotoxic towards LDL receptor positive Chinese hamster ovary cells than LDL receptor negative cells. Non-specific cytotoxicity was explained by slow dissociation of the drug-LDL complex in plasma. Our results underline the importance of careful studies of plasma stability when investigating lipoproteins and other carriers in drug targeting.

Uptake of anthracyclines in vitro and in vivo in acute myeloid leukemia cells in relation to apoptosis and clinical response

AimsTo study anthracycline-induced apoptosis in leukemic cells isolated from patients with acute myelogenous leukemia (AML) in vitro and to compare intracellular anthracycline concentrations causing apoptosis in vitro with those obtained in vivo during anthracycline treatment.MethodsMononuclear blood cells from AML patients were isolated before (n = 20) and after anthracycline infusion (n = 24). The pre-treated cells were incubated in vitro with daunorubicin (DNR) and/or idarubicin (IDA). Anthracycline concentrations were determined by high-performance liquid chromatography, and apoptosis was detected by propidium iodine staining using a flow cytometer.ResultsThere was a clear concentration–response relationship between intracellular anthracycline levels and apoptosis albeit with a large interindividual variation. Intracellular levels >1200 μM always led to high apoptosis development (>60%) in vitro. The intracellular concentrations of DNR in vivo (n = 24) were more than tenfold lower than the concentrations needed to induce effective apoptosis in vitro, although a significant relation between in vivo concentrations and clinical remission was found. We also found a significant relation between apoptosis induction in leukemic cells by IDA in vitro and clinical remission.ConclusionsOur results indicate that intracellular anthracycline levels in vivo are suboptimal and that protocols should be used that increase intracellular anthracycline levels.

Daunorubicin Metabolism in Leukemic Cells Isolated from Patients with Acute Myeloid Leukemia

BACKGROUND Anthracyclines like daunorubicin (DNR) are important drugs in the treatment of acute myeloid leukaemia (AML). In vitro studies have shown that cellular metabolism of anthracyclines could play a role in drug resistance. Currently, it is not known what enzyme is responsible for anthracycline metabolism in leukemic cells. AIMS To study C-13 reduction of DNR to daunorubicinol (DOL) in leukemic cells isolated from patients with AML and to determine the most important enzyme involved. METHODS Mononuclear blood cells from 25 AML patients were isolated at diagnosis and used in a metabolic assay to determine the % DOL formed. mRNA and western blot analysis were performed on the 2 most likely candidates for anthracycline metabolism; carbonyl reductase 1 (CR1) and aldoketoreductase 1A1 (AKR1A1). DNR and DOL concentrations were determined by HPLC. RESULTS We found a large interindividual variation (up to 47-fold) in leukemic cell DNR metabolism. The specific CR1 inhibitor zeraleone analogue 5 significantly inhibited DNR metabolism with a mean inhibitory effect of 68 %. No correlation between mRNA levels of the enzymes and metabolism were found. Cellular DNR metabolism correlated significantly with CR1 protein expression, determined by western blot, (p < 0.05, R2 = 0,229) while no significant correlation was found with AKR1A1 protein expression. CONCLUSIONS DNR metabolism in AML cells shows a pronounced interindividual variability. Our results support that CR1 is the most important enzyme for conversion of DNR to DOL in AML cells. This information could in the future be used to genotype CR1 and possibly help to individualise dosing.

Quantification of valproic acid in dried blood spots

Abstract Background. Therapeutic drug monitoring (TDM) of the antiepileptic drug valproic acid (VPA) is recommended in patients with multiple drug therapy or with concomitant disabilities to ensure treatment efficacy and avoid adverse reactions in both adults and children. The use of sampling techniques compatible with home sampling, such as dried blood spot sampling could potentially facilitate this for patients. Aim. To assess the usefulness of a bioanalytical method for quantification of VPA in dried blood spots. Materials and methods. Quantification was based on liquid chromatography-mass spectrometry (LC-MS), both for the DBS method and the plasma-based reference method. Results. The method was validated in the range 10–1200 μmol/L. Total imprecision ranged from 4.9–8.9 (%CV) and accuracy was within ± 14%. Conclusion. The validated method has potential for evaluation in therapeutic drug monitoring in combination with home sampling of DBS. The impact of spot size can be controlled through acceptance criteria and hematocrit in the range 30–60% can be accepted in sampling. Comparison of VPA levels between plasma and whole blood cannot be done without considering the blood-plasma ratio.

Acute Myelogenous Leukemia Cells Secrete Factors that Stimulate Cellular LDL Uptake via Autocrine and Paracrine Mechanisms

Leukemic cells isolated from most patients with acute myelogenous leukemia (AML) have higher low density lipoprotein (LDL) uptake than normal mononuclear blood cells. Little is known, however, about the mechanism behind the elevated LDL uptake. We investigated if AML cells secrete factors that stimulate cellular LDL uptake. Mononuclear blood cells were isolated from peripheral blood from 42 patients with AML at diagnosis. Cellular LDL uptake was determined from the degradation rate of 125I-labelled LDL. Conditioned media from AML cells stimulated the LDL degradation in the leukemic cell lines KG1 and HL60, and in isolated AML cells. The stimulatory effect correlated with the LDL degradation in the AML cells directly after isolation from blood. Conditioned media also autostimulated LDL degradation in the AML cells themselves. Concentrations of IL-6 and IL-8 in AML cell conditioned media correlated with the LDL degradation in AML cells directly after isolation from blood. Addition of R-TNF-α, but not IL-6 or IL-8, stimulated LDL degradation in HL60, KG1, and AML cells. The LDL degradation in AML cells could be inhibited by a LDL receptor blocking antibody. AML cells secrete factors that stimulate LDL uptake in a paracrine and autocrine pattern which open up therapeutic possibilities to inhibit the uptake of LDL by administration of antibodies to these factors.