Antonieta Sauerteig

Expression of drug resistance-associated mdr-1, GST π, and topoisomerase II genes during cell cycle traverse

The expression of drug resistance-associated mdr-1, GST pi, and topoisomerase II genes was analyzed in cell cycle phase enriched populations of doxorubicin-resistant murine leukemic P388/R-84 cells. Flow cytometric analysis of bromodeoxyuridine (BrdU) incorporation and staining with anti-BrdU antibodies was used to confirm the purity of cell cycle phase enriched populations obtained by centrifugal elutriation. Doxorubicin (DOX) and daunorubicin (DNR) accumulation was significantly lower in S-phase cells, and coincubation with verapamil (VPL) or chlorpromazine (CPZ) enhanced DOX and DNR accumulation more in S-phase than in G1- and G2/M-phase cells. While the cellular content of mdr-1 and topoisomerase II mRNAs changed, GST pi mRNA content remained constant during the cell cycle. S-phase cells had about 3-fold higher mdr-1 mRNA content than G1- and G2/M-phase cells. In G1 cells, P-glycoprotein expression, as determined by C219 monoclonal antibody, was 12% less than that of S and G2/M cells. Topoisomerase II mRNA content increased with the progression of cell cycle and peaked in G2/M cells. These observations suggest that cell cycle stage related changes in expression of drug resistance markers may have a major bearing on chemosensitivity of drug-resistant cells.

Flow cytometric analysis of the multiple drug resistance phenotype

Laser flow cytometry is increasingly used for quantitation of cellular fluorescent drug retention, effect of efflux blockers and for expression of drug resistance related cellular surface markers. Several intrinsic and extrinsic factors can affect the results obtained from drug retention functional assays and lead to artifacts. In the present study, we have used a panel of well-characterized parental and drug resistant cell lines, fluorochromes and efflux blockers to identify the possible sources of artefacts in flow cytometric analysis of the multiple drug resistance phenotype.

Prochlorperazine as a doxorubicin-efflux blocker: phase I clinical and pharmacokinetics studies.

SummaryDoxorubicin (DOX) efflux in drug-resistant cells is blocked by phenothiazines such as trifluoperazine (TFP) and prochlorperazine (PCZ) in vitro. The present phase I study was conducted in 13 patients with advanced, incurable, nonhematologic tumors to determine whether PCZ plasma levels high enough to block DOX efflux could be achieved in vivo. The treatment schedule consisted of prehydration and i. v. administration of 15, 30, 50, and 75 mg/m2 PCZ followed by a standard dose of 60 mg/m2 DOX. The hematologic toxicities attributable to DOX were as expected and independent of the PCZ dose used. Toxicities attributable to PCZ were sedation, dryness of the mouth, cramps, chills, and restlessness. The maximal tolerated dose (MTD) of PCZ in this schedule was 75 mg/m2. Pharmacokinetic analysis indicated a large interpatient variation in peak plasma PCZ levels that ranged from 95 to 1100 ng/ml. The three plasma half-lives of PCZ were:t1/2α (±SE), 20.9±5.3 min;t1/2β, 1.8±0.3 h; andt1/2γ, 21.9±5.3 h. The volume of distribution (Vd), total clearance (ClT), and area under the curve (AUC) for PCZ were 2254±886 l/m2, 60.2±13.5 l m−2h−1, and 1624±686 ng ml−1 h, respectively. DOX retention in tumor cells retrieved from patients during the course of therapy indicated the appearance of cells with enhanced DOX retention. The combination of DOX and high-dose i. v. PCZ appeared to be safe, well tolerated, and active in non-small-cell lung carcinoma.

MDR-1 gene expression, anthracycline retention and cytotoxicity in human lung-tumor cells from refractory patients

SummaryLung-tumor cells from pleural effusion of four refractory patients and in cell lines established from them were analyzed for anthracycline retention, cytotoxicity, and MDR-1 gene and P-glycoprotein expression. Murine leukemic P388 and doxorubicin-resistant P388/R84 lines were used as controls. The 50% growth-inhibitory concentration (IC50) for doxorubicin among lung-tumor lines varied from 0.16 to 0.31 μM in soft agar. Heterogeneity in doxorubicin or daunorubicin retention and response to the efflux-blocking action of 25 μm prochlorperazine was noted in pleural effusion of FCCL-1,-4, and-8. Among the cell lines established, an efflux-blocking effect in a subpopulation was noticed only in FCCL-1 and-4. Although the MDR-1 gene was present in all cell lines, including P388, its expression was pronounced only in P388/R84 and FCCL-1. In situ hybridization of antisense RNA probe to tumor cells showed high heterogeneity for MDR-1 message in the human lung-tumor cells as compared with the murine cells. Northern and slot blot hybridization confirmed in situ hybridization in lines with high levels of MDR-1 expression. The synthesis of MDR-1 mRNA and P-glycoprotein in tumor lines was correlated. The results suggest that because of extensive tumor-cell heterogeneity in human tumors, monitoring of MDR expression by in situ hybridization, quantitation of P0glycoprotein content by laser flow cytometry (and/or immunohistochemical methods), and drug efflux (by laser flow cytometry) may be the best ways to monitor multidrug resistance in human tumors.

Phase I and pharmacokinetics studies of prochlorperazine 2-h i.v. infusion as a doxorubicin-efflux blocker

In an earlier phase I study, we reported that the maximal tolerated dose (MTD) of prochlorperazine (PCZ) given as a 15-min i.v. infusion was 75 mg/m2. The highest peak plasma PCZ concentration achieved was 1100 ng/ml. The present study was conducted to determine if PCZ levels high enough to block doxorubicin (DOX) efflux in vitro could be achieved and sustained in vivo by increasing the duration of i.v. infusion from 15 min to 2 h. The treatment schedule consisted of i.v. prehydration with at least 500 ml normal saline (NS) and administration of a fixed standard dose of 60 mg/m2 DOX as an i.v. bolus over 15 min followed by i.v. doses of 75, 105, 135, or 180 mg/m2 PCZ in 250 ml NS over 2 h. The hematologic toxicities attributable to DOX were as expected and independent of the PCZ dose. Toxicities attributable to PCZ were sedation, dryness of mouth, anxiety, akathisia, hypotension, cramps, and confusion. The MTD of PCZ was 180 mg/m2. Large interpatient variation in peak PCZ plasma levels (91–3215 ng/ml) was seen, with the plasma half-life (t1/2α) being approximately 57 min in patients given 135–180 mg/m2 PCZ. The volume of distribution (Vd), total clearance (ClT), and area under the curve (AUC) were 350.1±183.8 l/m2, 260.7±142.7 l m2 h−1 and 1539±922 ng ml h−1, respectively, in patients given 180 mg/m2 PCZ and the respective values for patients receiving 135 mg/m2 were 48.9±23.76 l/m2, 33.2±2.62 l m2 h−1, and 4117±302 ng ml h−1. High PCZ plasma levels (>600 ng/ml) were sustained in all patients treated with 135 mg/m2 PCZ for up to 24 h. DOX plasma elimination was biphasic at 135 and 180 mg/m2 PCZ, and a>10-ng/ml DOX plasma level was maintained for 24 h. Partial responses were seen in three of six patients with malignant mesothelioma, in two of ten patients with non-small-cell lung carcinoma, and in the single patient with hepatoma. Our data show that PCZ can be safely given as a 2-h infusion at 135 mg/m2 with clinically manageable toxicities. The antitumor activity of the combination of DOX and PCZ needs to be confirmed in phase II trials.

Flow Cytometric Monitoring of Drug Resistance in Human Solid Tumors

Resistance to cancer chemotherapy continues to be a major hurdle in successful management of refractory human malignancies. Drug resistance may be intrinsic or acquired after chemotherapy. Several well-known extracellular factors such as drug metabolism and pharmacokinetics may be responsible for failure of chemotherapy. However, a major reason for drug resistance resides at the cellular level and often involves cellular mechanisms which under normal conditions may have other protective and important biological roles. Tumor cell resistance is believed to be multifactorial involving altered drug transport (influx, retention and efflux), and biochemical mechanisms such as xenobiotic detoxification/ alternate metabolic pathways, and altered targets (1–3). Multiple drug resistance (MDR) has been recently described as a phenomenon in which tumor cells are resistant to a variety of unrelated natural products such as alkaloids and antibiotics used as cancer chemotherapeutic agents (1,3).