Miranda Verschraagen

Simultaneous determination of intact cisplatin and its metabolite monohydrated cisplatin in human plasma.

Cisplatin is a cytotoxic platinum compound, used in the treatment of several solid tumors. Cisplatin and to a greater extent its hydrolysis product monohydrated cisplatin are responsible for side-effects like nephrotoxicity. A sensitive, accurate and precise method was developed to simultaneously determine cisplatin and monohydrated cisplatin in plasma. The compounds were separated by high-performance liquid chromatography and quantified by off-line furnace atomic absorption spectrophotometry. The linear ranges for cisplatin and monohydrated cisplatin in deproteinized plasma were 60-600 and 87.5-700 nM, respectively. From plasma, the mean recovery of cisplatin was 83.2% and that of monohydrated cisplatin 79.1%. The lower limits of quantification of cisplatin and monohydrated cisplatin in deproteinized plasma were 60 and 87.5 nM, respectively. Over the whole calibration range, the within- and between-day accuracy of intact cisplatin ranged from 100.7 to 111.4 and 94.8-102.0%, respectively. The within- and between-day accuracy of monohydrated cisplatin ranged from 107.1 to 113.3 and 101.4-104.9%, respectively. The within-day and between-day precision of cisplatin ranged from 3.4 to 11.5 and 7.3-10.3%, respectively. For monohydrated cisplatin, the within-day and between-day precision ranged from 3.7 to 6.2 and 5.6-7.9%, respectively. Currently, the developed assay has been implemented in pharmacokinetic studies of patients treated with cisplatin alone or in combination with other drugs.

Pharmacokinetics and preliminary clinical data of the novel chemoprotectant BNP7787 and cisplatin and their metabolites.

BNP7787 (disodium 2,2′‐dithio‐bis‐ethane sulfonate) is currently undergoing development as a chemoprotective agent to prevent common and serious cisplatin‐induced side effects. In the kidneys, intestine, and liver, BNP7787 is believed to undergo intracellular conversion into 2‐mercaptoethane sulfonate (mesna), which can locally inactivate toxic platinum species.

Phase I and pharmacokinetic study of the novel chemoprotector BNP7787 in combination with cisplatin and attempt to eliminate the hydration schedule.

BNP7787 (disodium 2,2′-dithio-bis-ethane sulphonate; Tavocept™) is a novel agent developed to protect against cisplatin (cis-diammine-dichloroplatinum(II))-associated chronic toxicities. In this study, we determined the recommended dose of BNP7787 when preceding a fixed dose of cisplatin, the pharmacokinetics (PKs) and the possible reduction of saline hydration. Patients with advanced solid tumours received BNP7787 in escalating doses of 4.1–41 g m−2 as a 15-min intravenous (i.v.) infusion followed by cisplatin 75 mg m−2 as a 60-min i.v. infusion together with pre- and postcisplatin saline hydration in a volume of 2200 ml; cycles were repeated every 3 weeks. PK was carried out using BNP7787, cisplatin and the combination. Twenty-five patients were enrolled in stage I of the study to determine the recommended dose of BNP7787. No dose-limiting toxicity was reached. The highest dose level of 41 g m−2 resulted in a low incidence of grade 2 toxicities, being nausea and vomiting, dry mouth or bad taste and i.v. injection site discomfort. Doses of BNP7787 ⩾18.4 g m−2 did not show a drug interaction between BNP7787 and cisplatin. In stage II of the study, patients received a fixed dose of BNP7787 of 18.4 g m−2 preceding cisplatin and were entered in prespecified reduced saline hydration steps. A total of 21 patients in cohorts of six to nine patients received reduced saline hydration of 1600 ml (step A), 1000 ml (step B) and 500 ml (step C). In step C, two out of six evaluable patients experienced grade 1 nephrotoxicity. Cisplatin acute toxicities in all 46 patients were as expected. Only five patients complained of paresthesias grade 1 and six developed slight audiometric changes. Partial tumour response was observed in four patients and stable disease in 15 patients. In conclusion, BNP7787 was tolerated well up to doses of 41 g m−2. The recommended dose of 18.4 g m−2 enabled safe reduction of the saline hydration schedule for cisplatin to 1000 ml. Further studies will assess whether BNP7787 offers protection against platinum-related late side effects.

Quantification of BNP7787 (dimesna) and its metabolite mesna in human plasma and urine by high-performance liquid chromatography with electrochemical detection.

A sensitive and accurate assay was developed and validated to determine BNP7787 (dimesna), a new protector against cisplatin-induced toxicities, and its metabolite mesna in plasma and urine of patients. Both analytes were measured as mesna in deproteinized plasma or in urine diluted with mobile phase using high-performance liquid chromatography with an electrochemical detector provided with a wall-jet gold electrode. The assays for BNP7787 and mesna in deproteinized plasma were linear over the range of 1.6-500 microM and 0.63-320 microM, respectively. In plasma, the mean recovery of BNP7787 over the whole concentration range was 100.6% and of mesna 94.6%. The lower limits of quantitation (LLQs) of BNP7787 and mesna in deproteinized plasma were 1.6 microM and 0.63 microM, respectively. For both compounds the within- and between-day accuracy and precision of the assay was better than 12%. The assays for BNP7787 and mesna in urine were linear over the range of 0.8-1200 microM and 0.63-250 microM, respectively. In urine, the mean recovery of BNP7787 over the whole concentration range was 94.1% and of mesna 93.1%. The LLQ of BNP7787 in urine was 0.8 microM and of mesna 1.6 microM. The within- and between-day accuracy and precision of the assay for BNP7787 and mesna was lower than 15%. The stability of mesna in urine increased with an increasing concentration of mesna, lower temperature and addition of EDTA (1 g/l) and hydrochloric acid (0.2 M). BNP7787 in urine was stable for at least 24 h at temperatures in the range of -20 degrees C up to 37 degrees C and independent of the concentration. The developed assays are currently applied for samples of patients with solid tumors participating in a phase I trial of BNP7787 in combination with cisplatin.

Quantification of mesna and total mesna in kidney tissue by high-performance liquid chromatography with electrochemical detection.

A sensitive and selective assay for the determination of mesna and total mesna in tissue was developed and validated. After a simple homogenization, extraction and deproteinization step, mesna could be measured immediately by HPLC with an electrochemical detector provided with a sensitive wall-jet gold electrode. Total mesna (i.e., free mesna and mesna present in mesna disulfides and mixed mesna disulfides) could be measured after pre-column reduction with sodium borohydride to free mesna. The lower limit of quantification of mesna and total mesna was for both compounds 10 nmol/g. The assays for mesna and total mesna in tissue were linear over the ranges of 10-3000 and 10-10000 nmol/g, respectively. The within-day and between-day precisions of both methods were better than 9%. The within-day and between-day accuracy of the mesna assay ranged from 103.7 to 113.6%, whereas the accuracies of the total mesna assay ranged from 97.8 to 106.7%. Mesna in an EDTA containing tissue homogenate or in deproteinized tissue homogenate stored at -80 degrees C was stable for at least 12 weeks. Total mesna was stable under all conditions measured. The developed assays will be applied for the determination of the distribution of mesna and total mesna in tissues of the rat after administration of mesna or BNP7787.

Pharmacokinetic behaviour of the chemoprotectants BNP7787 and mesna after an i.v. bolus injection in rats.

In preclinical studies, BNP7787 (disodium 2,2′-dithio-bis-ethane sulphonate), the disulphide form of mesna, has demonstrated selective protection against cisplatin-induced nephrotoxicity due to conversion into mesna inactivating toxic platinum species. Mesna (sodium 2-mercapto ethane sulphonate), however, can affect the antitumour activity of cisplatin, while BNP7787 does not interfere with the antitumour activity. To understand the difference in interference with cisplatin-induced antitumour activity between BNP7787 and mesna as well to characterise the selective nephroprotection by BNP7787, the pharmacokinetics of BNP7787 and mesna, each given i.v. 1000 mg kg−1, were determined in plasma, kidney, liver, red blood cells (RBC), skeletal muscle and tumour of Fischer rats bearing subcutaneously implanted WARD colon tumours. The following results were obtained: (1) high concentrations of BNP7787 and mesna were observed in the plasma and kidney after administration of BNP7787 or mesna, except for mesna in plasma after BNP7787 administration; (2) in all other sampled compartments, the AUC values of both compounds were at least 5.5-fold lower than the corresponding values in kidney; (3) the AUC of mesna in plasma after mesna administration was comparable to the AUC of mesna in kidney after a dose of BNP7787 that can completely prevent cisplatin-induced nephrotoxicity in rats; (4) the AUC of mesna in plasma was five-fold higher relative to the AUC of mesna following BNP7787 administration (P<0.01). In conclusion, the five-fold higher AUC of mesna in plasma after mesna administration and the fact that mesna is more reactive with (hydrated) cisplatin than its disulphide form BNP7787 represent a plausible explanation as to why mesna administration can reduce the antitumour activity of cisplatin. After BNP7787 administration, the distribution of BNP7787 and mesna was restricted to the kidney, which confirmed the selective protection of the kidney by BNP7787.