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Fluorescence microscopy has emerged as an attractive technique to probe the intracellular processing of Pt-based anticancer compounds. Herein, we reported the first through-bond energy transfer (TBET) fluorescent probe NPR1 designed for sensitive detection and quantitation of Pt II complexes. The novel TBET probe was successfully applied for ratiometric fluorescence imaging of anticancer Pt II complexes such as cisplatin and JM118 in cells. Capitalizing on the ability of the probe to discriminate between Pt II complexes and their Pt IV derivatives, the probe was further applied to study the activation of Pt IV prodrug complexes that are known to release active Pt II species after intracellular reduction., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Quantitative screening for potential drug-protein binding is an essential step in developing novel metal-based anticancer drugs. ICP-MS approaches are at the core of this task; however, many applications lack in the capability of large-scale high-throughput screenings and proper validation. In this work, we critically discuss the analytical figures of merit and the potential method-based quantitative differences applying four different ICP-MS strategies to ex vivo drug-serum incubations. Two candidate drugs, more specifically, two Pt(IV) complexes with known differences of binding affinity towards serum proteins were selected. The study integrated centrifugal ultrafiltration followed by flow injection analysis, turbulent flow chromatography (TFC), and size exclusion chromatography (SEC), all combined with inductively coupled plasma-mass spectrometry (ICP-MS). As a novelty, for the first time, UHPLC SEC-ICP-MS was implemented to enable rapid protein separation to be performed within a few minutes at > 90% column recovery for protein adducts and small molecules. Graphical abstract Quantitative screening for potential drug-protein binding is an essential step in developingnovel metal-based anticancer drugs.

Purpose: The aim of this pilot study was to evaluate surface contamination by platinum drugs in the environment of patients in ICUs and wards treated by hyperthermic intraperitoneal chemotherapy (HIPEC)., Methods: The monitoring included 12 HIPEC treatments from four hospitals during the following 3 days after perfusion. A total of 33 urine and 33 drainage fluids from HIPEC patients and 160 wipe samples from several surfaces (urine/drainage bags, floors, gloves) were taken during the study period., Results: In urine, the highest platinum concentrations were measured on the first day after perfusion. Median platinum concentrations were 1260 ng/ml for patients after cisplatin perfusion and 11,000 ng/ml for oxaliplatin treatment. Concentrations decreased until day three to 413 ng/ml cisplatin and 529 ng/ml oxaliplatin, respectively. In drainage liquids, platinum concentrations were generally lower. Platinum concentrations from surfaces of bags and floors ranged from 0.01 to 439 pg/cm(2) (median: urine bag 2.77 pg/cm(2), drainage bag 0.22 pg/cm(2), floor left 0.14 pg/cm(2), floor right 0.24 pg/cm(2)), with the highest contamination found on the outer surface of the urine bags. Samples from nurses' protective gloves ranged between 0.03 and 12 pg/cm(2) (median: 0.2 pg/cm(2))., Conclusions: High platinum-drug concentrations in urine and drainage liquids are the main source of contamination. Therefore, safe handling of these liquids is the best way to avoid cross-contamination on surfaces in wards and ICUs. Our results show that it is possible to take care of HIPEC patients without high contaminations during the first 3 days.

We have developed six dihydroxidoplatinum(IV) compounds with cytotoxic potential. Each derived from active platinum(II) species, these complexes consist of a heterocyclic ligand (HL) and ancillary ligand (AL) in the form [Pt(HL)(AL)(OH)2](2+), where HL is a methyl-functionalised variant of 1,10-phenanthroline and AL is the S,S or R,R isomer of 1,2-diaminocyclohexane. NMR characterisation and X-ray diffraction studies clearly confirmed the coordination geometry of the octahedral platinum(IV) complexes. The self-stacking of these complexes was determined using pulsed gradient stimulated echo nuclear magnetic resonance. The self-association behaviour of square planar platinum(II) complexes is largely dependent on concentration, whereas platinum(IV) complexes do not aggregate under the same conditions, possibly due to the presence of axial ligands. The cytotoxicity of the most active complex, exhibited in several cell lines, has been retained in the platinum(IV) form., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Dicycloplatin is a new generation supramolecular platinum-containing anti-cancer drug. Due to its structure, it is difficult to differentiate dicycloplatin from physical mixtures of carboplatin and cyclobutane dicarboxylate, and confounding results may arise during drug characterization. To solve this problem, this study aims to provide a reliable and reproducible standard for the determination of dicycloplatin. A simple method for dicycloplatin quality control has been developed using X-ray powder diffraction (XRPD) and high performance liquid chromatography (HPLC). XRPD allowed the control of impurities and dissociation of the dicycloplatin active ingredient to less than 1%, and HPLC allowed the monitoring and control of the relative molar ratio of carboplatin and cyclobutane dicarboxylate within the purity range. The study proved for the first time that the dicycloplatin supramolecule is substantially different from a physical mixture of carboplatin and cyclobutane dicarboxylate.

The aim of this study was to evaluate air and surface contaminations, and internal contamination of healthcare workers during open-abdomen HIPEC using oxaliplatin. Platinum (Pt) was measured in urine of exposed workers and in multiple air and surface samples. Three successive HIPEC procedures were investigated in each of the two hospitals participating in the study. Analysis of air samples did not detect any oxaliplatin contamination. Heavy contamination of the operating table, the floor at the surgeon's feet, and the surgeon's overshoes were observed. Hand contamination was observed in surgeons using double gloves for intra-abdominal chemotherapy administration, but not in those using three sets of gloves. Pt was not detected in urine samples obtained after HIPEC (<5 ng/L). The main risk of HIPEC is related to direct or indirect skin exposure and can be prevented by correct use of adapted protective equipment.

The cytotoxic, platinum-based anticancer drugs, cisplatin, carboplatin and oxaliplatin, enter the aquatic environment largely in municipal wastes via excretion from outpatients undergoing chemotherapy. The environmental behaviour, effects and fate of these drugs are, however, unknown. In this study, the adsorption of the drugs to untreated and chemically modified (oxide-free and organic-free) sediment was examined in both river water and low salinity (S=3.2) estuarine water in order to determine the nature and extent of their interactions with suspended particles. In all cases, adsorption isotherms were linear, and the slopes of the relationships, or distribution coefficients (KDs), ranged from about 10(2) to 10(3) ml g(-1). Overall, adsorption decreased in the order: cisplatin>carboplatin>oxaliplatin; in river water and: cisplatin>carboplatin, oxaliplatin; in estuarine water. There was no clear dependence of adsorption on sediment treatment but, for all sediment types, both cisplatin and carboplatin adsorption was greater in river water than in estuarine water. Qualitatively, these observations are consistent with the rates of formation of reactive, aquated degradation products and the dependencies of these rates on aqueous chloride concentration. We predict that during transport through an estuarine turbidity maximum (of suspended sediment concentration=1 g L(-1)), up to about 45% of cisplatin and 35% of carboplatin are filtered out from the aqueous phase but that no more than 7% of oxaliplatin is retained., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A micellar electrokinetic chromatography (MEKC) method was developed for the determination of cisplatin, carboplatin, and oxaliplatin in pharmaceutical formulations. The background electrolyte consisted of a phosphate buffer (pH 7.0; 25 mM) with sodium dodecyl sulfate (80 mM). The applied voltage was 30 kV and the sample injection was performed in the hydrodynamic mode. All analyses were carried out in a fused silica capillary with an internal diameter of 50 μm and a total length of 64.5 cm. The detection of target compounds was performed at 200 nm. Under these conditions, a complete separation of cisplatin, carboplatin and oxaliplatin was achieved in less than 10 min. The MEKC-UV method was validated and trueness values between 99.7% and 100.8% were obtained with repeatability and intermediate precision values of 0.7-1.4% and 1.1-1.7%, respectively for the three drugs. This method was found appropriate for controlling pharmaceutical formulations containing platinum complexes and successfully applied in quality control at the Geneva University Hospitals., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A capillary electrophoresis-based method to characterize a PEGylated liposomal drug formulation of the anti-cancer agent oxaliplatin was developed. Pharmaceutical characterization in terms of determination of the free and total oxaliplatin concentrations in the liposomal formulation was successfully performed allowing calculation of the percentage of encapsulated drug and encapsulation efficiency. The trapping efficiency was likewise calculated. The capillary electrophoresis method allowed liposome characterization in the intended formulation media (sucrose solution with low electrolyte concentration), and the attained results were consistent with inductively coupled plasma mass spectrometry measurements. Accelerated drug leakage studies were initiated by the sonication of the PEGylated formulation, using an ultrasound probe, subsequently the drug leakage was determined by capillary electrophoresis. The results obtained with the PEGylated liposomes demonstrate that capillary electrophoresis may be a useful tool for the characterization of liposomal drug formulations., (Copyright © 2011 Elsevier B.V. All rights reserved.)

CIEF with whole column imaging detection (WCID) was used to investigate the interaction of platinum-based anticancer drugs, cis-platinum(II) diamine dichloride (cisplatin) and [SP-4-2-{1R-trans)]-(1,2-cyclohexanediamine-N,N')[ethanedioata(2-)-O,O']platinum (oxaliplatin), with human hemoglobin A(0) (Hb). This technique facilitates the investigation and characterization of the formation of adducts between drugs and proteins. Cisplatin and oxaliplatin were mixed with the target protein at different concentrations (0:1, 1:1, 1:10, 1:50, and 1:100), and the reaction mixtures were incubated for 0, 0.5, 1, 12, 24, 48, and 72 h at 37 degrees C in a water-bath. The focused Hb-drug adduct profiles were imaged by WCID. At higher drug to protein molar ratios (for both oxaliplatin and cisplatin), the results exhibit significant changes in the peak shapes and heights, which may indicate the destabilization of the protein. However, the conformational change was less evident at lower molar ratios. In addition, a major pI shift was observed for the oxaliplatin reaction mixtures (for 1:10, 1:50, and 1:100 ratios). In comparison with previously reported findings obtained by other analytical methods, conclusions were drawn about the validity of CIEF as a simple and convenient method for the investigation of protein-drug interactions. These results may provide useful information for further understanding the activity and toxicity of these chemotherapeutic drugs and improving their clinical performance.

Metal-based anticancer agents are frequently used in the treatment of a wide variety of cancer types. The monitoring of these anticancer agents in biological samples is important to understand their pharmacokinetics, pharmacodynamics, and metabolism. In addition, determination of metals originating from anticancer agents is relevant to assess occupational exposure of health care personnel working with these drugs. The high sensitivity of inductively coupled plasma mass spectrometry (ICP-MS) has resulted in an increased popularity of this technique for the analysis of metal-based anticancer drugs. In addition to the quantitative analysis of the metal of interest in a sample, ICP-MS can be used as an ultrasensitive metal selective detector in combination with speciation techniques such as liquid chromatography. In the current review we provide a systematic survey of publications describing the analysis of platinum- and ruthenium-containing anticancer agents using ICP-MS, focused on the determination of total metal concentrations and on the speciation of metal compounds in biological fluids, DNA- and protein-adducts, and environmental samples. We conclude that ICP-MS is a powerful tool for the quantitative analysis of metal-based anticancer agents from multiple sample sources., ((c) 2008 Wiley Periodicals, Inc.)

Antitumoral Pt-containing drugs present side effects like nephrotoxicity and ototoxicity. Several systematic experiments have been carried out with Wistar rats treated with cisplatin, carboplatin, and oxaliplatin to study Pt-drugs accumulation and elimination, and Pt-biomolecule distribution in the cells and cytosols of ear, kidney, and liver. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis shows a cisplatin accumulation capability between oxaliplatin (the highest) and carboplatin (the lowest). The maximum concentration of Pt in all the organs studied was achieved around the first week after cisplatin treatment. During the first 30 days, the elimination was very fast, decreasing in the subsequent 60 days in all the organs. Analysis of cytosols by liquid chromatography (LC)-ICP-MS showed an analogous behavior. In most samples, the distribution of the three drugs in the cellular and cytosolic fractions was similar for all the tissues. For kidney and ear, approximately 60% and 30%, respectively, of the metal accumulated was present in the cytosol, the cytosolic fractions smaller than 50 KDa being especially important. Cisplatin-biomolecule interaction strength under denaturing conditions was evaluated by LC-ICP-MS and showed a quite strong bond.

The present work focuses on the fate of two cancerostatic platinum compounds (CPC), cisplatin and carboplatin, as well as of two inorganic platinum compounds, [PtCl(4)](2-) and [PtCl(6)](2-) in biological wastewater treatment. Laboratory experiments modelling adsorption of these compounds onto activated sludge showed promising specific adsorption coefficients K(D) and K(OC) and Freundlich adsorption isotherms. However, the adsorption properties of the investigated substances were differing significantly. Adsorption decreased following the order cisplatin>[PtCl(6)](2-)>[PtCl(4)](2-)>carboplatin. LogK(D)-values were ranging from 2.5 to 4.3 , logK(OC) from 3.0 to 4.7. A pilot membrane bioreactor system (MBR) was installed in a hospital in Vienna and fed with wastewater from the oncologic in-patient treatment ward to investigate CPC-adsorption in a sewage treatment plant. During three monitoring periods Pt-concentrations were measured in the influent (3-250 microg l(-1) Pt) and the effluent (2-150 microgl(-1) Pt) of the treatment plant using ICP-MS. The monitoring periods (duration 30d) revealed elimination efficiencies between 51% and 63% based on averaged weekly input-output budgets. The derived logK(D)-values and logK(OC)-values ranged from 2.4 to 4.8 and from 2.8 to 5.3, respectively. Species analysis using HPLC-ICP-MS proofed that mainly carboplatin was present as intact drug in the influent and--due to low logK(D)--in the effluent of the MBR.

Platinum originating from the excreted cancerostatic platinum compounds (CPC) cisplatin, carboplatin and oxaliplatin was monitored over a period of 28 days in the wastewater of the oncologic ward of the Vienna University Hospital. Concentration levels ranging from 4.7 to 145 microg L(-1) were measured by inductively coupled plasma mass spectrometry (ICP-MS). An average ratio of weekly drug emission/drug consumption of 0.27+/-0.12 was assessed. Model studies were carried out for fundamental understanding of CPC interaction with the solid phases present at different stages of the water cycle. Wastewater and activated sludge were spiked with CPC at concentration levels as found in the sewer of the oncologic ward. The platinum concentration remaining in the tested solution was measured after 24 h of incubation. Depending on pH, the three substances exhibited considerably different adsorption rates in wastewater. At pH 7, cisplatin was adsorbed by 88%, whereas only 26% of carboplatin and 54% of oxaliplatin were removed from the aqueous phase. Adsorption by activated sludge was higher, less affected by pH variation and comparable for all investigated CPC (96% for cisplatin, 70% for carboplatin and 74% for oxaliplatin at pH 6.8). In a next step, the dependence of CPC adsorption was tested for wastewater and activated sludge of different sampling sites. Strong variations were found only for wastewater, whereas activated sludge showed more consistent elimination rates (average values: cisplatin 92%, carboplatin 72%, and oxaliplatin 78%). These findings indicate that the major part of the excreted CPC is adsorbed by the solid phase in the water cycle and is thus expected to be removed from the wastewater by sewage treatment plants.

We report the interaction of intact human holo-transferrin (holo-Tf) with oxaliplatin (an anticancer drug), and the characterization of a complex composed of (1:1) intact holo-Tf and the parent oxaliplatin molecule using nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS). The molecular weight of this complex was determined to be 80,077 Da, which was an increase of 397 mass units compared to the protein alone (79,680 Da), suggesting that a parent drug molecule was bound to the intact protein. We further examined the interaction between the intact protein and oxaliplatin using size-exclusion high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICPMS). The protein complex and free oxaliplatin were separated by HPLC and quantitatively determined by simultaneous monitoring of both 195Pt and 56Fe using ICPMS. The HPLC/ICPMS detected both Pt and Fe signals at retention time of 2.6 min, identifying the protein-drug complex. The Fe signal at 2.6 min did not change with the increase in incubation time of the reaction mixture containing holo-Tf and oxaliplatin, while the Pt signal at the same retention time increased over time, further demonstrating that the formation of this complex does not affect the protein-bound Fe. The binding constant of the (1:1) intact human holo-Tf-oxaliplatin complex was determined to be 7.7x10(5) M-1. Both nanoESI-MS and HPLC/ICPMS results support that the holo-Tf and parent oxaliplatin molecules form complexes through non-covalent binding, suggesting that holo-Tf may be a useful carrier for oxaliplatin delivery., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

AP 5280 is a novel polymer-conjugated platinum anticancer agent showing promising in vitro and in vivo activity against solid tumors. The aim of this study was to develop a parenteral pharmaceutical dosage form for phase I clinical trials. AP 5280 drug substance was characterized by using a wide range of analytical techniques and showed excellent solubility in water. However, as aqueous solutions of AP 5280 proved to be labile upon sterilization by moist heat, it was decided to develop a lyophilized dosage form. Initially, glass vials were used as primary packaging, but this led to a high breakage rate, which could be completely prevented by the use of CZ resin vials. Stability studies to date show that the lyophilized product in glass vials is stable for at least 12 months when stored at 2-8 degrees C in the dark and the lyophilized product in CZ resin vials is stable for at least 6 months under these conditions. Photostability testing revealed photolability of AP 5280 drug substance and lyophilized product in both types of primary container, necessitating storage in the dark. The first clinical experiences indicate that the proposed formulation is fully applicable for use in the clinical setting.

A rapid and sensitive RP-HPLC method with UV detection for routine control of oxaliplatin in a pharmaceutical formulation (Eloxatin) was developed. Quantitation was accomplished with the internal standard method. The procedure was validated by linearity (correlation coefficient=0.999948), accuracy, robustness and intermediate precision. Experimental design was used during validation to calculate method robustness and intermediate precision. For robustness test three factors were considered: percentage v/v of acetonitrile, flow rate and temperature; an increase in the flow rate results in a decrease of the drug found concentration, while the percentage of organic modifier and temperature have no important effect on the response. For intermediate precision measure the considered variables were: analyst, equipment and days. The RSD value (2.27%, n=24) indicated a good precision of the analytical method.

Cis-dichloroamine (4-amino-2, 2, 6, 6-tetramethylpiperidine-1-oxyl) platinum (II) (abbreviated to HJ5) is a novel platinum anticancer compound which has prospects for research and application. In order to establish a conventional analytical method, an HPLC has been studied. Under the conditions of acetonitrile-water (10:90, volume ratio) as mobile phase, detection wavelength of 210 nm, a Phenomenex column C18(150 mm x 3.9 mm i.d., 10 microns). The linear equation, recovery and accuracy of the method were determined with external standard method. The HPLC has satisfactory resolution between the peaks of HJ5 and impurities. The peak areas of HJ5 were linear to its amounts detected and the detection limit was 0.07 microgram. The hydration of HJ5 can be inhibited effectively by NaCl solution. In a 9 g/L NaCl solution, the stability can be kept for more than 2 hours which is enough for analysis.

A survey of investigations performed over the last decade concerning analytical, clinical and pharmacological data on cancer chemotherapy with Pt-based drugs is reported. From this standpoint discussion focuses on clinical studies aimed at evaluating therapeutic response and toxicity during regional and systemic treatment of cisplatin against solid tumors in adults as well as in children. Concurrent treatments with locoregional hyperthermia in the case of limb tumors or with radiotherapy in the case of lung carcinoma are also dealt with.

A sensitive postcolumn derivatization/UV detection system has been developed for HPLC analysis of antitumor divalent and quadrivalent platinum complexes. It is based on the derivatization of platinum complexes by reaction with sodium bisulfite to corresponding product(s) which has enhanced absorptivity at 280-300 nm. Platinum complexes examined in this study were cisplatin, carboplatin and oxaliplatin (divalent platinum complexes) and oxoplatin and tetraplatin (quadrivalent ones). The proposed detection system was sensitive to all these complexes. Under the detection conditions optimized for individual complexes, the HPLC gave linear relationships between the complex concentration and the peak height. Detection limits at 290 nm with 100 microliters injection were 20 nM for cisplatin, 40 nM for oxoplatin, 60 nM for carboplatin and tetraplatin and 100 nM for oxaliplatin (S/N = 3 at 0.005 AUFS). The proposed system was successfully applied for the determination of cisplatin and oxoplatin in plasma and urine. Pharmacokinetic behavior of oxoplatin and its reduced product cisplatin following a single intravenous injection of oxoplatin in rabbits has been discussed.

The use of ICP-MS and its various sample introduction techniques has great potential in extending the time scale of study for the pharmacokinetics of platinum containing anti-tumour drugs. Electrothermal vaporization ICP-MS can be used to measure very small samples with very low concentrations. Speciation studies of platinum in tissues can be carried out with HPLC-ICP-MS over longer time periods. Finally, LA-ICP-MS is potentially useful for studying the spatial distribution of platinum in tissues and tumours.

Quantification and identification of platinum drugs and their metabolites in biological samples has always been difficult because these compounds are thermally unstable, non-volatile and insoluble. We have demonstrated that electrospray ionization mass spectrometry can be a valuable technique for direct mass spectral analysis of platinum anticancer agents and for obtaining structural information as a result of fragmentation. Full-scan spectra were obtained with approximately 10 pmol samples. These results show the potential of applying this technique in pharmacokinetic studies of platinum anticancer drugs.

Ormaplatin is a racemic, platinum(IV), anti-cancer drug which is currently involved in phase 1 clinical trials. Characterizing the purity of ormaplatin represents an analytical challenge for several reasons. These include the lack of solution stability of ormaplatin, its process impurities and solution decomposition products, the strong concentration and pH dependence of various decomposition pathways and solution equilibria and the absence of chromatographic reproducibility. Two independent types of aqueous decomposition pathways have been observed. The first pathway involves the generation of soluble PtIV containing species which remain in equilibrium with the parent material in solution. The second pathway involves the generation of PtII containing materials which tend to be less soluble. The most interesting material in this latter class is probably the chloride bridged mixed valence material which has been characterized structurally by X-ray diffraction. Liquid chromatography and sample handling procedures have been developed which allow for the accurate determination of bulk drug purity, as well as, an understanding of numerous reversible equilibria.

A selective and convenient high-performance liquid chromatographic assay was developed for ethylenediamineplatinum(II) malonate (JM-40) in plasma ultrafiltrate and urine. A mu Porasil silica column (30 cm) was used with acetonitrile-water (90:10, v/v) as the mobile phase and the elution of compounds was monitored by ultraviolet absorbance at 214 nm. A linear dynamic range of at least three decades (1-1000 micrograms/ml) was achieved. The detection limit in plasma ultrafiltrate was 0.35 micrograms/ml. The stability of JM-40 was determined in 0.9% sodium chloride, 5% glucose, plasma ultrafiltrate and urine. More stable drug solutions were obtained with 5% glucose than with 0.9% sodium chloride. JM-40 was also determined in plasma ultrafiltrate and urine samples of one patient receiving short-term infusions of the drug. In plasma ultrafiltrate unmetabolized JM-40 was detected during the first 5 h after administration and had a half-life of 21.3 +/- 1.6 min. The parent drug was excreted in the urine in rapidly decreasing amounts. Eighteen per cent of the dose was recovered as unmetabolized drug during the first 6 h.

A survey of investigations performed by our group over the last few years whose goal was to obtain analytical, clinical and pharmacokinetic data concerning cancer chemotherapy with Pt-based drugs is reported. From this standpoint the use of inductively coupled-plasma atomic emission spectrometry for determining Pt levels in biological samples is discussed, particularly as regards: 1) the amelioration of sample introduction procedures into the torch in the case of micro-sampling and 2) the investigation of plasma drug distribution by means of liquid chromatography techniques. Clinical studies evaluated therapeutic response and toxicity during regional and systemic treatments with Cisplatin and Carboplatin against solid tumors in adults as well as in children. Several pharmacokinetic parameters such as plasma half-lives for free and protein-bound drug, tissues exposure as determined by AUC and urinary excretion are examined.

Several anti-cancer agents are administered by continuous infusion in an attempt to improve the therapeutic index obtained with solid tumours. In Exeter this approach, combined with the use of ambulatory infusion pumps, forms the basis of a home oncology programme in which a complete course of medication in pre-filled syringes is supplied on an out-patient basis. It is essential to ensure that the drug remains stable during storage prior to use and also during infusion where the temperature of the drug solution in a holster-worn ambulatory pump can reach 37 degrees C. In this study the stability of a carboplatin infusion (20 mg in 2 ml) in pre-filled syringes under storage and in-use conditions was determined using a stability-indicating HPLC assay. There was no loss of carboplatin from pre-filled syringes stored at 4 degrees C for 5 days. At 37 degrees C, the loss of carboplatin was 3.1% over 24 h.

Iproplatin is structurally unique among the platinum (Pt) agents in the clinic because it is a quadrivalent complex. On the basis of the redox parameters for the Pt(IV) and Pt(II) oxidation states in a chloride system, it has been suggested that Pt(IV) complexes will be reduced to Pt(II) complexes in a biological environment. To test this hypothesis, uptake and metabolism studies of [14C]-iproplatin were carried out in L1210 cells. The L1210 cells raised in DBA2/J mice were incubated in vitro with 50 and 100 microM [14C]-iproplatin at 37 degrees C in Hanks' balanced salt solution, and total uptake and radioactivity associated with acid-insoluble fractions were measured for up to 3 h. Under these conditions, the uptake of iproplatin was linear with time and increased with increasing concentrations of iproplatin in the medium. At all times measured, greater than 35% of radioactivity was associated with the acid-insoluble fraction, suggesting binding to macromolecules. The [14C]-labelled compounds in neutralized acid extracts of cells were separated by reverse-phase high-performance liquid chromatography (HPLC). Three labelled compounds were detected; based on chromatographic elution times, they appeared to be iproplatin, cis-dichloro-bis-isopropylamine platinum(II) (CIP), the reduction product of iproplatin, and a third compound more polar than iproplatin and CIP. The finding of free CIP and the macromolecular binding of radioactivity in the cells suggests that iproplatin is reduced intracellularly.

Specific, sensitive and reproducible high-performance liquid chromatographic procedures were developed for the quantitative analysis of carboplatin in human plasma and urine. Plasma and urine were ultrafiltered with an Amicon Centrifree micropartition system, and samples were injected onto a LiChrosorb diol column. The mobile phase was CH3CN/H2O (92:8, v/v) for plasma and CH3CN/0.015% H3PO4 (89:11, v/v) for urine. The effluents were monitored at 229 nm. Carboplatin eluted by 10 min. The detector response was linear from 0.5 (plasma) or 5 (urine) to 500 micrograms/ml. The lower limit of quantification was 1.0 micrograms/ml plasma and 5.0 micrograms/ml urine. Constituents in plasma and urine, and possible degradation products (cyclobutane mono- and dicarboxylic acids) did not interfere. Within-day precision was less than 4% for plasma and 9% and 4% for urine concentrations of 40 and 401 micrograms/ml, respectively. Within-day accuracy was 96% or greater for both matrices. Carboplatin was not bound to the Centrifree membrane and recovery was 94% for plasma and 96% for urine. The storage stability of carboplatin in water, plasma, plasma ultrafiltrate, and urine and the extent of binding to human plasma proteins were evaluated. The percentage of carboplatin reversibly bound to plasma proteins was minimal (less than or equal to 10%) over a range of 1-50 micrograms/ml. In human plasma at 37 degrees C the drug was stable for about 2 h, but then degraded with a half-life of 32 h. Carboplatin had limited stability in water, plasma, and urine stored at -25 degrees C. Biological samples, therefore, should be stored frozen and analyzed within a week of collection to obtain valid results.

The synthesis, physical properties, and antitumor activity of the cis-, d,l-trans-, d-trans-, and l-trans- stereoisomers of 1,2-diaminocyclohexane tetrachloroplatinum(IV) are described. The objective of the study was to produce a platinum complex with activity against cisplatin-resistant tumor cells and with suitable pharmaceutical properties for formulation development. The isomers had the following solubilities in saline: cis-, 2 mg/ml; d,l-trans-, 6.5 mg/ml; and d-trans- and l-trans-, 15-16 mg/ml. The four complexes showed slightly better activity than cisplatin against the ip implanted murine L1210 leukemia. In contrast to cisplatin, all complexes produced significant increases in life span against L1210/cisplatin, a subline of L1210 with acquired resistance to cisplatin. However, the cis- isomer was less active against L1210/cisplatin. The d,l-trans- isomer (tetraplatin) was selected for further studies based on greater ease for large-scale synthesis. It showed superior activity to cisplatin against P388/cisplatin and like cisplatin showed significant and reproducible activity against the ip implanted B16 melanoma, ip implanted M5076 sarcoma, ip implanted P388 leukemia, and MX-1 human breast xenograft implanted under the renal capsule. Purity and stability (greater than 24 hours in saline) were evaluated by high-performance liquid chromatography and found to be suitable for development of a parenteral dosage form. Preliminary studies in a rat model (to be reported elsewhere) showed it to be less nephrotoxic than cisplatin on a molar basis and worthy of further study.

We have previously shown that a significant portion of the total platinum in the plasma of patients receiving iproplatin is protein-bound. We have also identified cis-dichloro-bis-isopropylamine platinum(II) (CIP) as a major metabolite of iproplatin. To understand the nature of the bound platinum, we carried out in vitro comparative protein-binding studies for iproplatin and CIP. These studies indicate that when CIP is incubated in plasma, protein binding occurs, with a 2.7-h half-life for the disappearance of CIP; the parent complex does not bind and is stable in plasma for at least 48 h. The time dependence of protein binding with CIP suggests the formation of other chemical species from CIP that may be responsible for the observed protein binding. The results indicate that in patients receiving the drug, the reduction of iproplatin to CIP must take place intracellularly and that CIP or its protein-binding derivatives must efflux from the cells into the plasma. Efflux studies carried out to explore this possibility with cells in the whole blood showed that iproplatin was taken up into cells, but the efflux of protein-binding iproplatin metabolites did not occur. To understand further the nature of the metabolites of iproplatin, we carried out 195Pt-NMR (nuclear magnetic resonance) studies with urine from two patients who received a high dose of iproplatin (500 mg/m2). The predominant signals from the 195Pt-NMR corresponded to the divalent platinum complexes and not to quadrivalent complexes, indicating that the iproplatin metabolites in urine are divalent in nature.

Various His-Pt(II) coordination compounds were prepared by reaction of K2PtCl4 or cis-[Pt(NH3)2Cl2](cis-DDP) with His and analyzed by 1H and 13C NMR spectroscopy, electrophoresis, and ion-exchange chromatography. His may be coordinated to Pt by the imidazol iminogroup and/or the alpha-aminogroup; the carboxygroup remains always free. Both bidentate as well as monodentate ligands were identified. Cis-DDP reacts with His to give a mixture of compounds where all these possibilities are present: cis-diamine-(histidine-N,N-)Pt(II) and three different types of cis-diammine-bis(histidine). HCl trans cleavage of compounds with bidentate His ligands leads to a mixture of two compounds having His ligated to Pt by an amino or imin group. The methods applied are suitable for analyzing reactions of His with cis-DDP under model conditions similar to physiological conditions.

A reversed-phase high-performance liquid chromatographic analysis was developed for aqua[1,1-bis(aminomethyl)cyclohexane]sulphatoplatinum(II) (spiroplatin) and its hydrolysis and oligomerization products. The platinum complexes were detected by differential pulse amperometry at a hanging mercury drop electrode, at -540 mV vs. Ag/AgCl. The limit of detection was 0.05 microM. Aqueous solutions of spiroplatin appeared to contain the diaqua, monoaquamonosulphato, monoaquamonochloro, dichloro and hydroxo-bridged dimer complexes of [1,1-bis(aminomethyl)cyclohexane]platinum(II) in mutual equilibrium. The equilibrium shifts after dilution in infusion fluids were studied. Detection of these platinum(II) complexes in untreated human plasma ultrafiltrate and urine demonstrated the selectivity of the described analysis.

The optical properties of the DNA complexes with the compounds of bivalent platinum were studied. The compounds differed by the nature of the anionic and neutral ligands and their spatial arrangement about the platinum atom. It was shown that the same as cis-[Pt (NH3)2Cl2] the platinum compounds with the biological activity, i.e. [Pt (en) Cl2], cis-[PtNH3 (Bz) Cl2] and cis-[Pt (NH3)2NO2Cl] induced at low values of r (a ratio of the number of the platinum moles added to the number of the DNA nucleotide moles in the solution) an increase in the amplitude of the positive band in the spectrum of the circular dichroism (CD) of the linear DNA and a marked decrease in the amplitude of the negative band in the spectrum of the CD of the liquid crystalline microphase of DNA formed in the presence of polyethyleneglycol. By the character of the action on the CD spectrum of the linear and condensed DNA [Pt (tetrameen)Cl2] which had no selective antimitotic effect might be referred to the above platinum compounds. Trans-[Pt (NH3)2NO2Cl], [PtNH3PyCl2], cis-[Pt (NH3)2(NO2)2] and [Pt (NH3)3Cl]Cl having no biological activity either induced only a decrease in the amplitude of the positive band in the CD spectrum of the linear DNA or had no effect on the CD spectrum. The effect of these compounds on the CD spectrum of the liquid crystalline microphase of DNA was slightly pronounced or not observed.