Methyl-beta-cyclodextrin and doxorubicin pharmacokinetics and tissue concentrations following bolus injection of these drugs alone or together in the rabbit.

The purpose of this work was to determine the pharmacokinetics and the tissue concentrations of methyl-beta-cyclodextrin (MEBCD) and doxorubicin (DOX) in rabbits following administration of MEBCD and DOX, alone or in combination. MEBCD (200 mg/kg) and DOX (1 mg/kg) were intravenously injected to white New Zealand rabbits and blood samples were obtained over a 48-h period after administration. After this period, administration was repeated and animals were killed 1, 2 or 4 h after injection. Heart, liver and kidney were then removed. MEBCD and DOX analysis in plasma and tissues was performed using two HPLC methods with fluorimetric detection. MEBCD pharmacokinetic profile was consistent with a two-compartment model (t1/2 alpha: 30 min; t1/2 beta: 7 h). Co-administration with DOX did not modify the main pharmacokinetic parameters of MEBCD. However, C5 min, t1/2 alpha, t1/2 beta and AUCinfinity were decreased by the co-administration of DOX with MEBCD compared to DOX alone. Assays of excised tissues showed that DOX enhanced the cardiac, renal and hepatic concentrations of MEBCD. On the other hand, MEBCD did not alter the cardiac distribution of DOX, while renal and hepatic distribution profiles were modified. In this study, the pharmacokinetic parameters of MEBCD injected intravenously were determined for the first time. DOX did not enhance MEBCD pharmacokinetic profile but MEBCD reduced the distribution half-life of DOX. Tissue determination showed that MEBCD did not enhanced the cardiac accumulation of DOX, which is auspicious for further in vivo experiments using the co-administration of DOX and MEBCD.

Antiproliferative effect of methyl-beta-cyclodextrin in vitro and in human tumour xenografted athymic nude mice.

The anti-tumour activity of methyl-beta-cyclodextrin (MEBCD), a cyclic oligosaccharide known for its interaction with the plasma membrane, was investigated in vitro and in vivo and compared with that of doxorubicin (DOX) in the human tumour models MCF7 breast carcinoma and A2780 ovarian carcinoma. In vitro proliferation was assessed using the MTT assay. In vivo studies were carried out using xenografted Swiss nude mice injected weekly i.p. with MEBCD at 300 or 800 mg kg(-1) or DOX at 2 mg kg(-1), during 2 months. Under these conditions, MEBCD was active against MCF7 and A2780 cell lines and tumour xenografts. For each tumour model, the tumoral volume of the xenografted mice treated with MEBCD was at least twofold reduced compared with the control group. In the MCF7 model, MEBCD (800 mg kg(-1)) was more active than DOX (2 mg kg(-1)). After 56 days of treatment with MEBCD, no toxicologically meaningful differences were observed in macroscopic and microscopic parameters compared with controls. The accumulation of MEBCD in normal and tumour tissues was also assessed using a chromatographic method. Results indicated that after a single injection of MEBCD, tumour, liver and kidneys accumulated the highest concentrations of MEBCD. These results provided a basis for the potential therapeutic application of MEBCD in cancer therapy.

In vitro modulation of doxorubicin and docetaxel antitumoral activity by methyl-beta-cyclodextrin.

Methyl-beta-cyclodextrin (MEBCD) was investigated for its effect on the antitumoral activity of various antineoplastic agents (doxorubicin (DOX), docetaxel (DXL), 5-fluorouracil (5-FU) and cisplatin (CDDP)) in three different human parental sensitive cancer cell lines (K562 S, MCF7 S and A2780 S) and their multidrug resistant variant sublines (K562 R, MCF7 R and A2780 R). At non-cytotoxic concentrations, MEBCD was able to increase significantly DOX and DXL cytotoxic activity in all the cell lines tested. The sensitisation ratios (IC50 drug control/IC50 drug-MEBCD treated) ranged from 3l1 to 14.3. Moreover, intracellular DOX accumulation, determined by high-performance liquid chromatography, was also increased when cells were treated with MEBCD combined with DOX (approximately 2-3 fold). The effects of MEBCD in resistant sublines were greater than in their parental sensitive cell lines. Other experiments demonstrated that the action of the MEBCD was independent of DOX. These data provided a basis for the potential therapeutic application of MEBCD in cancer therapy.

Tumor cell membrane as a potential target for methyl-beta-cyclodextrin.

The purpose of this work was to determine the role of methyl-beta-cyclodextrin (MEBCD) in combination with doxorubicin (DOX) on DOX intracellular accumulation and efflux, in comparison to verapamil in a sensitive parental and multidrug-resistant human cancer cell line (HL-60 S and HL-60 R). Moreover, cell membrane and nuclear modifications induced by MEBCD were investigated. At concentration of 10 mumol for 10(6) cells, MEBCD combined with doxorubicin (DOX), was able to significantly enhance the intracellular concentration of DOX in HL-60 S and HL-60 R cell lines during the period of exposure. In the resistant subline, MEBCD activity was higher than that of verapamil. Moreover, treatment of cells with MEBCD resulted in a modification in cell membrane integrity and cell morphology, but had no own activity in the distribution of the cells within cell cycle.

Pharmacokinetics of 5-fluorouracil (5-FUra) in patients with metastatic colorectal cancer receiving 5-FUra bolus plus continuous infusion with high dose folinic acid (LV5FU2).

The pharmacokinetics of 5-fluorouracil (5-FUra) were investigated in 16 patients with metastatic colorectal cancer receiving high-dose folinic acid (LV 200 mg/m2) followed by 5-FUra bolus (400 mg/m2) and continuous infusion (600 mg/m2) on days 1 and 2. Quantitation of unchanged drug was assessed by a highly specific high-performance liquid chromatographic method. The concentrations of 5-FUra at the end of the loading dose averaged 30.7 +/- 13.2 micrograms/ml (i.e., 236 microM). The steady-state plasma concentration averaged 0.31 +/- 0.11 microgram/ml (i.e., 2.4 microM). 5-FUra plasma levels declined rapidly after the end of infusion with an apparent elimination half-life of 7.08 +/- 3.21 minutes. Clearance ranged from 776 to 3023 ml/min/m2. Large patient-to-patient variations in plasma 5-FUra concentrations were observed. No toxicity greater than WHO grade 2 was seen. One patient experienced grade 1 stomatitis and two others experienced grade 1 and 2 myelosuppression. One patient developed diarrhoea and another suffered asthenia. Nausea and vomiting were observed in 5 patients.

High-performance liquid chromatographic assay for methyl-beta-cyclodextrin in plasma and cell lysate.

This paper describes a high-performance liquid chromatographic method with fluorescence detection for the analysis of methyl-beta-cyclodextrin (MEBCD) in plasma and cell lysate, after in situ complexation with 1-naphthol. The size-exclusion HPLC column packed with TSK 3000 SW gel, was equilibrated with an eluent mixture composed of methanol and purified water (2:98, v/v) containing 10(-4) M 1-naphthol as a fluorophore. The detection is based on fluorescence enhancement caused by the formation of inclusion complexes and was performed at 290 and 360 nm for excitation and emission, respectively. The method involved a simple treatment of the samples with chloroform. Daunorubicin was used as internal standard. Limits of quantitation were 0.8 microM in plasma and 0.5 microM in cell lysate. Detection limits of 0.5 microM (50 pmol) and 0.3 microM (30 pmol) were obtained for MEBCD in the two media, respectively. Linear detection response was obtained for concentrations ranging from 1 to 100 microM in plasma and cell lysate. Recovery from plasma proved to be more than 40%. Precision, expressed as C.V. was in the range of 4 to 11%. Accuracy ranged from 89 to 105%.

Determination of 5-fluorouracil and its main metabolites in plasma by high-performance liquid chromatography: application to a pharmacokinetic study.

This paper describes a relatively simple and sensitive high-performance liquid chromatographic assay (HPLC) with ultraviolet absorbance detection for 5-fluorouracil (5-FUra) and its two main metabolites, 5-fluorouridine (5-FUrd) and 5-fluoro-2'-deoxyuridine (5-FdUrd), in plasma. In this study, two plasma clean-up procedures involving addition of internal standard, solid-phase and liquid-liquid extractions have been developed. A reversed-phase Kromasil C18 column was used. The detection was performed at 268 nm for 5-FUra and at 275 nm for the two metabolites. Linear detection responses were obtained for concentrations ranging from 25 to 1000 ng/ml. The average recovery from plasma was 35, 42 and 48% for 5-FUra, 5-FUrd and 5-FdUrd, respectively. Precision, expressed as C.V., ranged from 2.7 to 13% and the mean recovery from 94 to 105%. The limits of quantitation and detection of the three analytes were 20 and 10 ng/ml, respectively. The method was used to monitor the pharmacokinetic profile of 5-FUra and its two metabolites in patients with metastatic colorectal cancer.

Methyl-beta-cyclodextrin in HL-60 parental and multidrug-resistant cancer cell lines: effect on the cytotoxic activity and intracellular accumulation of doxorubicin.

The purpose of this work was to determine the role of methyl-beta-cyclodextrin (MEBCD) in combination with doxorubicin (DOX) on the cellular proliferation of a sensitive parental and a multidrug-resistant human cancer cell line (HL-60 S and HL-60 R) and to study the effect of MEBCD on DOX intracellular accumulation. The cytotoxicity of DOX at five concentrations (50-50,000 nM) was evaluated with or without the coadministration of four fixed noncytotoxic concentrations of MEBCD (100, 200, 500, and 1,000 microM). Intracellular DOX concentrations were determined by a high-performance liquid chromatography (HPLC) method with fluorescence detection. MEBCD applied at 500 and 1000 microM in combination with doxorubicin (DOX) significantly potentiated the activity of DOX used alone on both sensitive and multidrug-resistant cell lines; 50% growth-inhibitory (IC50) ratios (IC50 MEBCD-DOX/IC50 DOX) were about 3:4 and 1.6:4 for HL-60 S and HL-60 R, respectively. Moreover, intracellular DOX accumulation, determined by HPLC during 6 h of drug exposure, was about 2-4 times higher for cells treated with MEBCD in combination with DOX than in those treated with DOX alone. Similar results were obtained using other paired MCF 7 sensitive and resistant cell lines. Correlation between these results and an MEBCD-cell membrane interaction was discussed. These initial data provide a basis for the potential therapeutic application of MEMBCD in cancer therapy.

High-performance liquid chromatographic assay for melphalan in human plasma. Application to pharmacokinetic studies.

This paper describes a simple, rapid and reproducible high-performance liquid chromatographic method (HPLC) with ultraviolet absorbance detection for the analysis of melphalan in plasma. The HPLC column was an Ultrasphere ODS (5 microns) and the eluent was composed of methanol, purified water and acetic acid (49.5:49.5:1, v/v). The detection was performed at 261 nm. The method involved a simple treatment of the samples with methanol. The propylparaben was used as internal standard. Linear detection response was obtained for concentrations ranging from 50 to 2500 ng/ml. Recovery from plasma proved to be more than 90%. Precision, expressed as C.V., was in the 0.5 to 9% range. Accuracy ranged from 95 to 102%. This method was used to determine the pharmacokinetic parameters of melphalan following high-dose (140 mg/m2) intravenous administration in patients with advanced malignancies undergoing peripheral blood hematopoietic progenitor-cell transplantation.

High-performance liquid chromatographic determination of tazobactam and piperacillin in human plasma and urine.

A high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) absorbance was developed for the analysis of piperacillin-tazobactam (tazocillin), in plasma and urine. The detection was performed at 218 nm for tazobactam and 222 nm for piperacillin. The procedure for assay of these two compounds in plasma and of piperacillin in urine involves the addition of an internal standard (ceftazidime for tazobactam and benzylpenicillin for piperacillin) followed by a treatment of the samples with acetonitrile and chloroform. To quantify tazobactam in urine, diluted samples were analysed using a column-switching technique without internal standard. The HPLC column, LiChrosorb RP-select B, was equilibrated with an eluent mixture composed of acetonitrile-ammonium acetate (pH 5). The proposed technique is reproducible, selective, and reliable. The method has been validated, and stability tests under various conditions have been performed. Linear detector responses were observed for the calibration curve standards in the ranges 5-60 micrograms/ml for tazobactam, and 1-100 micrograms/ml for piperacillin and spans what is currently though to be the clinically relevant range for tazocillin concentrations in body fluids. The limit of quantification was 3 micrograms/ml for tazobactam and 0.5 microgram/ml for piperacillin in plasma and urine. Extraction recoveries from plasma proved to be more than 85%. Precision, expressed as C.V., was in the range 0.4-18%.