A rapid reversed phase high performance liquid chromatographic method for determination of etoposide (VP-16) in human plasma.

A rapid, simple and sensitive isocratic High Performance Liquid Chromatography (HPLC) method was developed to measure the concentration of etoposide in plasma samples with UV detection at 220 nm. The method uses a Bondapac C18 column at 60 degrees C. The mobile phase consists of Methanol: water (45:55 v/v) at a flow rate of 2.8 ml/min. Phenacetin was used as an internal standard. The plasma samples were extracted using ether with the organic layer evaporated under nitrogen. The residue was dissolved in 200 microl methanol with 20 microl injected into the HPLC column. The extraction method showed a recovery of 91.5+/-3% for etoposide. In this system, the retention time of phenacetin and etoposide were 3.3 and 4.4 min, respectively. The limit of detection of etoposide in plasma is 20 ng/ml and the limit of quantitation is 40 ng/ml. This analytical method has very good reproducibility (8.1% between-day variability at a concentration of 50 ng/ml). It is a fast, sensitive and economic method applicable for clinical and pharmacokinetic studies.

A rapid reversed phase high performance liquid chromatographic method for the determination of docetaxel (Taxotere) in human plasma using a column switching technique.

A rapid, simple and sensitive isocratic high performance liquid chromatography (HPLC) method was developed to measure the concentration of docetaxel in plasma samples with UV detection at 227 nm. The method uses a column switching technique with an Ultrasphere C18 column (75 x 4.6 mm ID, 3 mu, Altex, USA) as clean-up column and a CSC-nucleosil C8 column (150 x 4.6 mm ID, 5 mu, CSC, Montreal, Canada) as the analytical column. The mobile phase consisted of Phosphate buffer (30 mM, pH = 3)-acetonitrile (47:53, v/v) with the flow rates of 1.1 and 1.3 ml min-1 for clean-up and analytical columns, respectively. Paclitaxel was used as an internal standard. The plasma samples were extracted using a solid phase extraction method with Ammonium acetate (30 mM, pH = 5)-acetonitrile (50:50, v/v) as final eluent. The extraction method showed a recovery of 92% for docetaxel. In this system, the retention times of docetaxel and Paclitaxel were 7.2 and 8.5 min, respectively. The detection limit of docetaxel in plasma is 2.5 ng ml-1. This analytical method has a very good reproducibility (7.2% between-day variability at a concentration of 10 ng ml-1). It is applicable in clinical and pharmacokinetic studies.

In vitro carboplatin-mesna interaction in aqueous solution, human plasma and urine.

The chemotherapeutic benefit of synergistic drug combinations and higher drug dosages has generated interest in the application of these regimens to cancer patients. A major obstacle in the application of these strategies to the treatment of cancer is the dose-limiting toxicities of drug combinations. Sodium 2-mercaptoethane-sulfonate (mesna), a chemoprotective drug, may reduce the nephrotoxicity of carboplatin [CBDCA, paraplatin, JM-8, cis-diammine (1,1-cyclobutane dicarboxylato) platinum II] when administered in combination chemotherapy. The purpose of this study was to evaluate, compare and contrast in vitro the interaction of mesna with carboplatin in aqueous solution, human plasma and urine. Carboplatin and mesna were incubated separately and together at clinically relevant concentrations in plasma and urine. The concentration of carboplatin was assayed by HPLC, and the decay of carboplatin alone and in combination with mesna was compared. The incubation of carboplatin with mesna in human plasma up to 8 days did not result in a statistically significant interaction: the half-life of carboplatin in plasma when it was combined with mesna was 1.62 +/- 0.08 (SE) days compared to 1.85+/- 0.04 (SE) days for carboplatin by itself. The incubation of drugs in fresh human urine up to 15 days gave a half-life of 3.43+/- 0.8 (SE) days for carboplatin alone and 2.78 +/- 0.7 (SE) days for carboplatin when it was combined with mesna. Our results show that carboplatin and mesna do not significantly interact in plasma. Although a statistically significant difference between the half-life of carboplatin and the half-life of the carboplatin/mesna combination is detected in urine, it is not likely to be clinically significant, as there is no significant interaction detected in the first 48 h). It is thus unlikely that mesna would substantially affect the pharmacokinetics of carboplatin when both are given together to patients as part of combination chemotherapy regimens.

Evaluation of apoptosis in four human tumour cell lines with differing sensitivities to cisplatin.

Four human tumour cell lines were evaluated for their ability to undergo apoptosis when subjected to cisplatin or hyperthermia treatment. In an ovarian carcinoma line (A2780s) and its derivative cisplatin resistant line (A2780cp) the variation in response was expressed for both the colony survival endpoint and the apoptosis endpoint. Apoptosis was measured by the number of floating cells, DNA agarose gels, and electron microscopy. In fact, cisplatin resistance was expressed to a higher level for apoptosis, than colony survival in the A2780cp cell line compared to the A2780s line. The melanoma cell line (Sk Mel-3) also showed induced apoptosis by cisplatin treatment while the glioma line (U87MG) showed little to no apoptosis in response to cisplatin treatment. Hyperthermia (43 degrees C for 1 hour) induced apoptosis in the human melanoma cell line but not in the glioma cell line. These data indicate that, while both cisplatin and hyperthermia can induce apoptosis in human tumour cell lines, the degree of induction is highly cell line dependent.

Effects of PALA on the pharmacokinetics of 5-fluorouracil.

N-(phosphonacetyl)-L-aspartate (PALA) modulates the activity of 5-fluorouracil (5-FU) by inhibiting pyrimidine biosynthesis. A cross-over study was conducted to determine whether PALA affects the pharmacokinetic parameters of 5-FU in patients given 5-FU/folinic acid (FA). Six patients (3 males, 3 females) aged 63 4.3 (mean SD) years (body surface area of 1.84 18 m2) with metastatic colorectal carcinoma were given two courses of treatment. The treatment consisted of 250 mg/m2 of PALA on day 1 followed by 20 mg/m2 FA and 400 mg/m2 5-FU (5 min i.v. bolus injection) on days 2-5 in one cycle of treatment (PALA+). In another treatment cycle, these doses of 5-FU and FA were given for all 5 days without PALA (PALA-). The two courses were given four weeks apart. It was determined by random selection whether the course with PALA was given before or after the course without PALA. Blood samples were collected over a period of three hours, starting from the beginning of 5-FU infusion on days 2 and 5 of both courses. Plasma concentrations of 5-FU were determined by an HPLC technique. Pharmacokinetic parameters were calculated using a non-compartmental model. While there were no significant differences between pharmacokinetic parameters in the PALA+ vs PALA- courses, there was a trend towards a decreasing area under the curve (AUC) and increasing clearance (Cl) in PALA+ courses of treatment.

Cytotoxicity, accumulation, and efflux of cisplatin and its metabolites in human ovarian carcinoma cells.

Cisplatin (CP) is one of the most useful antineoplastic drugs. When CP is dissolved in human plasma, different metabolites are formed. Using the OV 2008 human ovarian cancer cell line, we examined the relative cytotoxicities of CP and its metabolites (aquated CP [AP], monomethionine CP [MP], bismethionine CP [BP], and a mixture of CP metabolites in ultrafiltrated human plasma [UP]) in vitro. By clonogenic assay, cell survival (percent of mean +/- SE) of OV 2008 cells exposed for 1 hr to 6.7 microM of CP was 9.8 +/- 0.7 and for its equal platinum contents of AP, MP, BP, and UP solutions were 3.3 +/- 0.7, 9.8 +/- 0.9, 15.9 +/- 1.1, 76.8 +/- 2.1, and 13.1 +/- 0.7, respectively. AP was the most cytotoxic species, and BP was the least cytotoxic species. Cellular platinum uptake (ng/10(6) cells) after addition of 0.33, 1.6, and 2.5 mM of each species for 1 hr was measured and a strong correlation was found between cytotoxicity of each CP metabolite and its corresponding cellular platinum (Pt) uptake (r = 0.997). There was a strong correlation between cytotoxicity of the CP metabolites and their DNA binding. With fractionation of these cells into DNA, nucleoplasm and cytoplasm, the highest platinum content was found on DNA. The most important factor that seems to be responsible for the cytotoxicities of the different CP metabolites is the amount of their associated intracellular accumulation, and particularly the degree of their binding to DNA.

The integrity of cisplatin in aqueous and plasma ultrafiltrate media studied by 195Pt and 15N nuclear magnetic resonance.

195Pt and 15N nuclear magnetic resonance (NMR) was used to study the chemical equilibria of cisplatin in water and plasma ultrafiltrate (PUF). Cisplatin was found to be stable for at least 2, but no longer than 5 months in a reconstituted clinical formulation, as determined by 195Pt NMR. In aqueous solution, the cis-PtCl2(NH3)2 195Pt and 15N NMR signal intensities decreased with time and the formation of [PtCl(H2O)(NH3)2]+ at PH values of 3.0, 6.5, 7.5 and 9.5 was observed within 24 h of sample preparation. In addition, [Pt(H2O)2(NH3)2]++ was observed at pH 3.0, and [PtCl(OH)(NH3)2] and [Pt(OH)2(NH3)2] were observed at pHs 7.5 and 9.5. During incubation of PUF with cisplatin for 35 h, 15N NMR signals for at least eight cisplatin derivatives appeared at different times, whereas only four were observed by 195Pt NMR. With our NMR protocols, the detection limit for quantifiable cisplatin derivatives is estimated at 500 microM using 195Pt NMR and < or = 200 microM using 15N NMR. In addition to providing useful information about the chemical stability of cisplatin and derivatives formed in aqueous solution, these magnetic resonance techniques, particularly 15N NMR, can provide useful information about the metabolism of cisplatin in biological regimes.

Pressure tuning infrared spectroscopic study of cisplatin-induced structural changes in a phosphatidylserine model membrane.

The dynamic effect of cis-diamminedichloroplatinum(II) (DPP) and its aquated metabolite (DDP-OH) on a dimyristoylphosphatidylserine (DMPS) model membrane was investigated by pressure tuning vibrational spectroscopy. The native species (DDP-Cl) and the aquated species (DPP-OH) were both observed to bind to the carboxylate group of the serine as evidenced by a frequency shift of 1622-1620 cm-1. However, only DDP-OH was observed to bind to the phosphate group (PO(-)2). The binding of either drug to DMPS resulted in an increased pressure required to halt the reorientational fluctuations of the acyl chains, indicating that the distance between the chains were increased. The two drugs did not partition into the matrix of the hydrophobic section in the model membrane. Collectively, these data suggest that DDP-Cl and DDP-OH are capable of binding to the polar head group of DMPS, resulting in an enlargement of the area of the head and a subsequent increase in the intermolecular distance between the acyl chains.