Immunohistochemical localization of OCT2 in the cochlea of various species.

OBJECTIVE: To locate the organic cation transporter 2 (OCT2) in the cochlea of three different species and to modulate the ototoxicity of cisplatin in the guinea pig by pretreatment with phenformin, having a known affinity for OCT2. STUDY DESIGN: Immunohistochemical and in vivo study. METHODS: Sections from the auditory end organs were subjected to immunohistochemical staining in order to identify OCT2 in cochlea from untreated rats, guinea pigs, and a pig. In the in vivo study, guinea pigs were given phenformin intravenously 30 minutes before cisplatin administration. Electrophysiological hearing thresholds were determined, and hair cells loss was assessed 96 hours later. The total amount of platinum in cochlear tissue was determined using mass spectrometry. RESULTS: Organic cation transporter 2 was found in the supporting cells and in type I spiral ganglion cells in the cochlea of all species studied. Pretreatment with phenformin did not reduce the ototoxic side effect of cisplatin. Furthermore, the concentration of platinum in the cochlea was not affected by phenformin. CONCLUSIONS: The localization of OCT2 in the supporting cells and type I spiral ganglion cells suggests that this transport protein is not primarily involved in cisplatin uptake from the systemic circulation. We hypothesize that OCT2 transport intensifies cisplatin ototoxicity via transport mechanisms in alternate compartments of the cochlea. LEVEL OF EVIDENCE: N/A.

Cisplatin and oxaliplatin are toxic to cochlear outer hair cells and both target thioredoxin reductase in organ of Corti cultures.

CONCLUSION: Inhibition of thioredoxin reductase (TrxR) may be a contributing factor in cisplatin-induced ototoxicity. Direct exposure of organ of Corti to cisplatin and oxaliplatin gives equal loss of hair cells. OBJECTIVES: Platinum-containing drugs are known to target the anti-oxidant selenoprotein TrxR in cancer cells. Two such anti-cancer, platinum-containing drugs, cisplatin and oxaliplatin, have different side effects. Only cisplatin induces hearing loss, i.e. has an ototoxic side effect that is not seen after treatment with oxaliplatin. The objective of this study was to evaluate if TrxR is a target in the cochlea. Loss of outer hair cells was also compared when cisplatin and oxaliplatin were administered directly to the organ of Corti. METHODS: Organ of Corti cell culture was used for direct exposure to cisplatin and oxaliplatin. Hair cells were evaluated and the level of TrxR was assessed. Immunohistochemical staining for TrxR was performed. An animal model was used to evaluate the effect on TrxR after treatment with cisplatin and oxaliplatin in vivo. RESULTS: Direct exposure of cochlear organotypic cultures to either cisplatin or oxaliplatin induced comparable levels of outer hair cell loss and inhibition of TrxR, demonstrating that both drugs are similarly ototoxic provided that the cochlea becomes directly exposed.

Cochlear pharmacokinetics of cisplatin: an in vivo study in the guinea pig.

OBJECTIVES/HYPOTHESIS: Cisplatin produces toxic lesions to outer hair cells (OHCs) in the cochlear base but not in the apex. The objective of this study was to compare the pharmacokinetic profile of cisplatin in scala tympani (ST) perilymph in the cochlear base and apex, respectively. STUDY DESIGN: In vivo animal study. METHODS: Forty-seven guinea pigs were given an intravenous bolus injection of an ototoxic dose of cisplatin. Ten to 240 minutes after cisplatin was given, blood, cerebrospinal fluid (CSF), and ST perilymph were aspirated within the same target time. ST perilymph was aspirated from the basal turn and from the apex of the cochlea by two different sampling techniques. Liquid chromatography with postcolumn derivatization was used for quantitative determination of the parent drug. RESULTS: Ten minutes after administration, the concentration of cisplatin in ST perilymph was 4-fold higher in the basal turn of the cochlea than in the apex. At 30 minutes, the drug concentrations did not differ. At 60 minutes, the level of cisplatin in ST perilymph and blood UF was equivalent. The perilymph-blood ratio increased thereafter with time. CONCLUSION: The pharmacokinetic findings of an early high concentration of cisplatin in the base of the cochlea and delayed elimination of cisplatin from ST perilymph compared to blood might correlate to the cisplatin-induced loss of OHCs in the base of the cochlea.

Quantitative liquid chromatographic determination of intact cisplatin in blood with microwave-assisted post-column derivatization and UV detection.

The anticancer agent cisplatin (cis-diamminedichloroplatinum(II), cis-[PtCl2(NH3)2]) easily undergoes ligand-exchange reactions, resulting in mainly inactive Pt complexes. This paper presents a method for selective analysis of intact cisplatin in blood using LC and UV detection. Blood samples (hematocrit: 0.22-0.52) were spiked with cisplatin (final concentrations: 2.48 × 10⁻7 M-9.90 × 10⁻6 M) and subjected to centripetal ultrafiltration. The blood ultrafiltrate was separated (loop volume: 5 µl) with a porous graphitic carbon column and a mobile phase of HEPES-buffer (pH 9.3). Prior to UV detection (344 nm), the eluate was mixed with sodium N,N-diethyldithiocarbamate (DDTC) in a microwave field (115 °C) in order to improve the UV absorptivity. Cisplatin eluted as a Pt-DDTC complex after 11.8 min. The peak area was influenced primarily by the hematocrit, the DDTC concentration, and the temperature and residence time in the microwave cavity. The method was robust and sensitive provided preparing a fresh DDTC solution each day and, at the end of a day's run, destroying DDTC remaining in the system. It offers the main advantages of high selectivity, sensitivity, and robustness, minimal sample processing, and the possibility to use small sample volumes.

Prevention of cisplatin-induced hearing loss by administration of a thiosulfate-containing gel to the middle ear in a guinea pig model.

PURPOSE: Thiosulfate may reduce cisplatin-induced ototoxicity, most likely by relieving oxidative stress and by forming inactive platinum complexes. This study aimed to determine the concentration and protective effect of thiosulfate in the cochlea after application of a thiosulfate-containing high viscosity formulation of sodium hyaluronan (HYA gel) to the middle ear prior to i.v. injection of cisplatin in a guinea pig model. METHODS: The release of thiosulfate (0.1 M) from HYA gel (0.5% w/w) was explored in vitro. Thiosulfate in the scala tympani perilymph of the cochlea 1 and 3 h after application of thiosulfate in HYA gel to the middle ear was quantified with HPLC and fluorescence detection. Thiosulfate in blood and CSF was also explored. The potential otoprotective effect was evaluated by hair cell count after treatment with thiosulfate in HYA gel applied to the middle ear 3 h prior to cisplatin injection (8 mg/kg b.w.). RESULTS: HYA did not impede the release of thiosulfate. Middle ear administration of thiosulfate in HYA gel gave high concentrations in the scala tympani perilymph while maintaining low levels in blood, and it protected against cisplatin-induced hair cell loss. CONCLUSION: HYA gel is an effective vehicle for administration of thiosulfate to the middle ear. Local application of a thiosulfate-containing HYA gel reduces the ototoxicity of cisplatin most likely without compromising its antineoplastic effect. This provides a minimally invasive protective treatment that can easily be repeated if necessary.

Is Platinum Present in Blood and Urine from Treatment Givers during Hyperthermic Intraperitoneal Chemotherapy?

Background. In selected patients with peritoneal carcinomatosis (PC) originating from colorectal cancer (CRC) the high dosage of oxaliplatin (460 mg/m(2)) is recommended for hyperthermic intraperitoneal chemotherapy (HIPEC), which may be a health risk to those administering the drug. The aim of this study was to determine the risk of platinum (Pt) exposure for the two main people handling and administering the cytotoxic agent during HIPEC. Methods. Samples of blood and urine were collected from one male surgeon and one female perfusionist during oxaliplatin-based HIPEC treatment with open abdomen coliseum technique on six consecutive patients with PC from CRC. Results. All blood samples analysed were below the detection limit of <0.05 nmol/L Pt, and the urine samples were all below the detection limit of <0.03 nmol/L Pt. Conclusions. There appears to be little or no risk of Pt exposure during HIPEC when the recommended protective garment is used and the safety considerations are followed.

New insights into the biotransformation and pharmacokinetics of oxaliplatin.

Oxaliplatin is used primarily in the treatment of metastatic colorectal cancer. In this minireview, we discuss potentially important biotransformation pathways in light of its short elimination half-life in vivo. We also highlight new information achieved using a selective analytical technique to measure intact oxaliplatin in pharmacokinetic studies (comprising intravenous, intraperitoneal, and intrahepatic administration) and compare to results obtained by measurements of total platinum. The use of selective analytical techniques is strongly recommended giving kinetic parameters of the parent compound and not only to a complex mixture of platinum containing endogenous compounds.

Cisplatin and oxaliplatin toxicity: importance of cochlear kinetics as a determinant for ototoxicity.

BACKGROUND: Cisplatin is a cornerstone anticancer drug with pronounced ototoxicity, whereas oxaliplatin, a platinum derivative with a different clinical profile, is rarely ototoxic. This difference has not been explained. METHODS: In HCT-116 cells, cisplatin (20 microM)-induced apoptosis was reduced by a calcium chelator from 9.9-fold induction (95% confidence interval [CI] = 8.1- to 11.7-fold), to 3.1-fold induction (95% CI = 2.0- to 4.2-fold) and by superoxide scavenging from 9.3-fold (95% CI = 8.8- to 9.8-fold), to 5.1-fold (95% CI = 4.4- to 5.8-fold). A guinea pig model (n = 23) was used to examine pharmacokinetics. Drug concentrations were determined by liquid chromatography with post-column derivatization. The total platinum concentration in cochlear tissue was determined by inductively coupled plasma mass spectrometry. Drug pharmacokinetics was assessed by determining the area under the concentration-time curve (AUC). Statistical tests were two-sided. RESULTS: In HCT-116 cells, cisplatin (20 microM)-induced apoptosis was reduced by a calcium chelator from 9.9-fold induction (95% confidence interval [CI] = 8.1- to 11.7-fold to 3.1-fold induction) (95% CI = 2.0- to 4.2-fold) and by superoxide scavenging (from 9.3-fold, 95% CI = 8.8- to 9.8-fold, to 5.1-fold, 95% CI = 4.4- to 5.8-fold). Oxaliplatin (20 microM)-induced apoptosis was unaffected by calcium chelation (from 7.1- to 6.2-fold induction) and by superoxide scavenging (from 5.9- to 5.6-fold induction). In guinea pig cochlea, total platinum concentration (0.12 vs 0.63 microg/kg, respectively, P = .008) and perilymphatic drug concentrations (238 vs 515 microM x minute, respectively, P < .001) were lower after intravenous oxaliplatin treatment (16.6 mg/kg) than after equimolar cisplatin treatment (12.5 mg/kg). However, after a non-ototoxic cisplatin dose (5 mg/kg) or the same oxaliplatin dose (16.6 mg/kg), the AUC for perilymphatic concentrations was similar, indicating that the two drugs have different cochlear pharmacokinetics. CONCLUSION: Cisplatin- but not oxaliplatin-induced apoptosis involved superoxide-related pathways. Lower cochlear uptake of oxaliplatin than cisplatin appears to be a major explanation for its lower ototoxicity.

Oxaliplatin neurotoxicity--no general ion channel surface-charge effect.

BACKGROUND: Oxaliplatin is a platinum-based chemotherapeutic drug. Neurotoxicity is the dose-limiting side effect. Previous investigations have reported that acute neurotoxicity could be mediated via voltage-gated ion channels. A possible mechanism for some of the effects is a modification of surface charges around the ion channel, either because of chelation of extracellular Ca2+, or because of binding of a charged biotransformation product of oxaliplatin to the channel. To elucidate the molecular mechanism, we investigated the effects of oxaliplatin and its chloride complex [Pt(dach)oxCl](-) on the voltage-gated Shaker K channel expressed in Xenopus oocytes. The recordings were made with the two-electrode and the cut-open oocyte voltage clamp techniques. CONCLUSION: To our surprise, we did not see any effects on the current amplitudes, on the current time courses, or on the voltage dependence of the Shaker wild-type channel. Oxaliplatin is expected to bind to cysteines. Therefore, we explored if there could be a specific effect on single (E418C) and double-cysteine (R362C/F416C) mutated Shaker channels previously shown to be sensitive to cysteine-specific reagents. Neither of these channels were affected by oxaliplatin. The clear lack of effect on the Shaker K channel suggests that oxaliplatin or its monochloro complex has no general surface-charge effect on the channels, as has been suggested before, but rather a specific effect to the channels previously shown to be affected.

High concentrations of thiosulfate in scala tympani perilymph after systemic administration in the guinea pig.

CONCLUSION: High concentrations of the antioxidant thiosulfate reach scala tympani perilymph after i.v. administration in the guinea pig. Thiosulfate concentrations in perilymph remain elevated longer than in blood. This warrants further studies on the possibility of obtaining otoprotection by thiosulfate administration several hours before that of cisplatin without compromising the anticancer effect caused by cisplatin inactivation in the blood compartment. OBJECTIVE: Thiosulfate may reduce cisplatin-induced ototoxicity, presumably by oxidative stress relief and formation of inactivate platinum complexes. This study aimed to explore to what extent thiosulfate reaches scala tympani perilymph after systemic administration in the guinea pig. MATERIALS AND METHODS: Scala tympani perilymph (1 microl) was aspirated from the basal turn of each cochlea up to 3 h after thiosulfate administration (103 mg/kg b.w., i.v.). Blood samples were also taken. Thiosulfate was quantified by HPLC and fluorescence detection. RESULTS: Substantial thiosulfate concentrations were found in perilymph. The area under the concentration-time curve for thiosulfate in perilymph and blood was 3100 microMxmin and 6300 microMxmin, respectively. The highest thiosulfate concentrations in perilymph were found at the first sampling at about 10 min. Due to a more rapid elimination from blood, perilymph concentrations exceeded those of blood towards the end of the experiment.

Kinetics of Cisplatin and its monohydrated complex with sulfur-containing compounds designed for local otoprotective administration.

The anticancer drug cisplatin can cause permanent inner ear damage. We have determined the second-order degradation rate constant, k(Nu), of cisplatin and its more toxic monohydrated complex (MHC) in the presence of each of the sulfur-containing nucleophiles N-acetyl-l-cysteine, l-cysteine methyl ester, 1,3-dimethyl-2-thiourea, d-methionine, and thiosulfate, compounds that are under evaluation for local administration to prevent cisplatin-induced ototoxicity. MHC was isolated from a hydrolysis solution of cisplatin using liquid chromatography (LC). The degradations were evaluated by measuring the disappearance of MHC and cisplatin at 37 degrees C and pH 7.4 in the presence of each of the nucleophiles using LC and photometric detection. The k(Nu) of MHC and of cisplatin was 0.044 M(-1)sec(-1) and 0.012 M(-1)sec(-1) with N-acetyl-l-cysteine, 0.24 M(-1)sec(-1) and 0.067 M(-1)sec(-1) with l-cysteine methyl ester, 0.16 M(-1)sec(-1) and 0.074 M(-1)sec(-1) with 1,3-dimethyl-2-thiourea, 0.070 M(-1)sec(-1) and 0.069 M(-1)sec(-1) with d-methionine, and 3.9 M(-1)sec(-1) and 0.091 M(-1)sec(-1) with thiosulfate, respectively. Our results suggest that thiosulfate, as being the strongest nucleophile, is a promising candidate for local application in order to reduce the inner ear content of MHC and cisplatin. However, otoprotection is a multifactorial event, and it remains to be established how important nucleophilicity is for the effectiveness of the protecting agent.

An evaluation of pharmacist contribution to an oncology ward in a Swedish hospital.

AIM: The aim of this project was to establish the importance of a pharmacist in the health-care team in improving drug use in an oncology ward in the Department of Oncology, Karolinska University Hospital, Stockholm, Sweden. METHODS AND PATIENTS: The pharmacist participated in the medical round in the mornings and worked as a member of the health-care team. Drug-related problems (DRPs) were identified by drug chart reviews based on data from medical files, laboratory tests and interviews with patients and/or relatives. A questionnaire to physicians and nurses was used to evaluate their experiences of the pharmacist's contribution to the oncology ward. RESULTS: In total, 114 DRPs were identified in 58 patients. For each DRP, the pharmacist gave proposals for solutions. Sixty-eight suggestions out of 114 (59.6%) were implemented by the physician. Two suggestions (1.8%) were partly followed. For 32 suggestions (28.0%) it was unclear if they had caused any change in medication. Twelve suggestions (10.5%) were not followed. Most of the physicians and nurses acknowledged the pharmacist's contribution to improved drug use in the ward. CONCLUSION: A pharmacist can improve drug use in an oncology ward as a member of the health-care team. The pharmacist contributes with a systematic focus on the patient from a drug perspective.

Oxaliplatin degradation in the presence of important biological sulphur-containing compounds and plasma ultrafiltrate.

Oxaliplatin undergoes extensive non-enzymatic chemical transformation in the body. Complexes with sulphur-containing compounds have previously been found in plasma from patients treated with oxaliplatin. We have studied the kinetics for the reactions between oxaliplatin and cysteine, methionine, and glutathione, by determination of the degradation of oxaliplatin using liquid chromatography with UV-detection. We also studied the degradation of oxaliplatin in plasma ultrafiltrate (PUF). For the degradation of oxaliplatin in the presence of glutathione, methionine, and cysteine, the second-order rate constants were 4.7M(-1)min(-1) (95% confidence interval [C.I.], 4.4-5.0M(-1)min(-1)), 5.5M(-1)min(-1) (95% C.I., 5.2-5.7M(-1)min(-1)), and 15M(-1)min(-1) (95% C.I., 14-17M(-1)min(-1)), respectively. The reaction rate was much faster than previously reported kinetics for cisplatin. The degradation rate of oxaliplatin in PUF was biphasic. The rate constant for the first phase varied from 9.5x10(-3) to 0.13min(-1) and for the second phase from (1.7 to 1.8)x10(-3)min(-1) in PUF from five healthy volunteers. The first hours of the degradation of oxaliplatin in PUF are accounted for by the degradation of oxaliplatin in a cocktail of sodium chloride and sulphur-containing compounds at physiological plasma concentrations. In conclusion, the rate of the reaction of oxaliplatin with three sulphur-containing compounds was faster for oxaliplatin than what is previously known for cisplatin. This may be important with respect to differences in the cellular effects of cisplatin and oxaliplatin treatment.

Inhibition of thioredoxin reductase but not of glutathione reductase by the major classes of alkylating and platinum-containing anticancer compounds.

Mammalian thioredoxin reductase (TrxR) is important for cell proliferation, antioxidant defense, and redox signaling. Together with glutathione reductase (GR) it is the main enzyme providing reducing equivalents to many cellular processes. GR and TrxR are flavoproteins of the same enzyme family, but only the latter is a selenoprotein. With the active site containing selenocysteine, TrxR may catalyze reduction of a wide range of substrates, but can at the same time easily be targeted by electrophilic compounds due to the extraordinarily high reactivity of a selenolate moiety. Here we addressed the inhibition of the enzyme by major anticancer alkylating agents and platinum-containing compounds and we compared it to that of GR. We confirmed prior studies suggesting that the nitrosourea carmustine can inhibit both GR and TrxR. We next found, however, that nitrogen mustards (chlorambucil and melphalan) and alkyl sulfonates (busulfan) efficiently inhibited TrxR while these compounds, surprisingly, did not inhibit GR. Inhibitions were concentration and time dependent and apparently irreversible. Anticancer anthracyclines (daunorubicin and doxorubicin) were, in contrast to the alkylating agents, not inhibitors but poor substrates of TrxR. We also found that TrxR, but not GR, was efficiently inhibited by both cisplatin, its monohydrated complex, and oxaliplatin. Carboplatin, in contrast, could not inhibit any of the two enzymes. These findings lead us to conclude that representative compounds of the major classes of clinically used anticancer alkylating agents and most platinum compounds may easily target TrxR, but not GR. The TrxR inhibition should thereby be considered as a factor that may contribute to the cytotoxicity seen upon clinical use of these drugs.

High-dose Cisplatin with amifostine: ototoxicity and pharmacokinetics.

OBJECTIVES/HYPOTHESIS: Ototoxicity is a common side effect of high-dose cisplatin treatment. Thiol-containing chemoprotectors ameliorate cisplatin ototoxicity under experimental conditions. The trial was initiated to test the efficacy of amifostine protection in high-dose cisplatin treatment (125-150 mg/m) for metastatic malignant melanoma, to correlate the ototoxic outcome with cisplatin pharmacokinetics, and to evaluate the importance of using a selective analytical method for the quantification of cisplatin. STUDY DESIGN: Prospective study of 15 patients with stage IV malignant melanoma. METHODS: Clinical follow-up of therapeutic response, pure-tone audiometry, and analysis of cisplatin and its monohydrated complex in blood ultrafiltrate by liquid chromatography with postcolumn derivatization were performed. Ultrafiltered blood platinum was analyzed by inductively coupled plasma mass spectrometry. RESULTS: Ototoxicity and gastrointestinal toxicity were the most prominent side effects. Three patients ultimately required hearing aids. All patients had audiometric changes at one or more frequencies after the second treatment course, and all but one patient reported auditory symptoms. No correlation was found between hearing loss and blood cisplatin pharmacokinetics. Platinum levels determined by inductively coupled plasma mass spectrometry were higher than total platinum levels calculated from cisplatin and monohydrated complex concentrations obtained by liquid chromatography analysis. CONCLUSION: Ototoxicity was unacceptable despite amifostine treatment. Cisplatin pharmacokinetics during the first treatment course were not predictive of hearing loss. Amifostine caused a lowering of dose-normalized area under the concentration-time curve for cisplatin and monohydrated complex. Use of the unselective inductively coupled plasma mass spectrometry analysis leads to an overestimation of active drug. Selective analysis of cisplatin is especially important when evaluating cisplatin pharmacokinetics during chemoprotector treatment.

Antitumor efficacy and acute toxicity of the novel dipeptide melphalanyl-p-L-fluorophenylalanine ethyl ester (J1) in vivo.

The novel alkylating dipeptide melphalanyl-p-L-fluorophenylalanine ethyl ester (J1) was evaluated for acute toxicity and antitumor activity in mice, with melphalan as a reference. To determine a safe and tolerable dose for efficacy studies the acute toxicity following intravenous injection in the tail vein was monitored using a 14-day schedule with up to four doses. The highest tested dose, 25 micromoles/kg, was considered close to this level, with minor effects on body weight gain but significant effects on hematological parameters. Melphalan and J1 appeared equitoxic with no statistically significant differences. Subsequently a mouse hollow fiber model was employed with subcutaneous implantation of fibers containing human tumor cells. Three different human tumor cell lines as well as two samples of primary human tumor cells (ovarian carcinoma and chronic lymphatic leukemia) were used as tumor models. At the dose level tested there was a marked and statistically significant decrease in both T-cell leukemia CCRF-CEM and small cell lung cancer NCI-H69 tumor cell growth and viability in response to J1 as compared with both placebo and melphalan treated groups. In primary ovarian carcinoma cells only J1 treatment resulted in significant tumor regression (net cell kill). In summary the results indicate that, despite an expected short half time in the blood circulation, the promising in vitro data from the previous studies of J1 seems translatable into the in vivo situation. At equal doses of alkylating units J1, compared to melphalan, was more active in the mouse hollow-fiber model, but showed similar general toxicity.

Oxaliplatin degradation in the presence of chloride: identification and cytotoxicity of the monochloro monooxalato complex.

PURPOSE: To study the degradation of oxaliplatin in chloride media and evaluate the cytotoxicity of oxaliplatin in normal and chloride-deficient medium. METHODS: The products of the reaction of oxaliplatin with chloride were separated on a Hypercarb S column with a mobile phase containing 40% methanol in 0.05 M ammonia and subjected to electrospray ionization mass spectrometry. The cytotoxicity of oxaliplatin in normal and chloride-deficient medium was evaluated by 30-min incubations on human colon adenocarcinoma cells (HT-29). RESULTS: We identified a new intermediate degradation product, the monochloro monooxalato complex ([Pt(dach)oxCl]-) and the final product. the dichloro complex (Pt(dach)Cl2), by liquid chromatography-mass spectrometry. [Pt(dach)oxCl]- was found as the negative ion, M-, at m/z 431, and the positive ion, [M+2H]+, m/z 433. Pt(dach)Cl2 was found as the negative ion, [M-H]-, m/z 377, and the positive ion, [M+NH4]+, m/z 396. The fast initial degradation of oxaliplatin can be coupled to the fast formation of [Pt(dach)oxCl]-. In the cytotoxic assay, the cell survival was not affected by the chloride levels. CONCLUSIONS: [Pt(dach)oxCl]-, a new transformation product of oxaliplatin, has been identified. Its in vitro cytotoxic effect does not appear to exceed that of oxaliplatin.