Cisplatin Metabolites Research Articles

Urine Periostin Level and Renal Function in Malignancy Patients Treated with High-Dose Cisplatine

Cisplatin has been used extensively as a cancer treatment. Nephrotoxicity, which is assessed by blood urea levels, blood creatinine, and estimated glomerular filtration rate (eGFR), is caused by cisplatin metabolites that build up in the kidneys. Because of these indicators' numerous flaws, optimal biological markers are required. One of the key mediators of inflammatory processes, such as kidney fibrosis and inflammation, is periostin. In cancer patients undergoing high-dose cisplatin therapy, the purpose of this study is to ascertain how urine periostin changes and how it relates to kidney function. This cross-sectional study was carried out at the National Center General Hospital of Cipto Mangunkusumo's medical hematology-oncology outer clinic and medical hematology-oncology ward on the eighth floor starting in November 2019 and ending when the minimum sample was obtained through consecutive sampling. Data was analyzed by IBM SPSS Statistics for Windows version 23.0 based on the research objective. Of the 37 responders, 70.3% were men, 29.7% were between the ages of 41 and 50, 78.4% were married, 59.5% had completed high school, 37.8% were employed, 59.5% had NPC, and 64.9% had a Karnofsky score of 80. Between before and one week following chemotherapy II, the respondents' blood creatinine and urea levels rose. The eGFR value has also decreased. Periostin levels, on the other hand, tended to rise one week following treatment III after declining during chemotherapy I and II (p value>0.05). Urine periostin levels and other kidney function indicators did not significantly correlate (p>0.05), according to the correlation test, and several domains had negative directions. The correlation coefficient values were modest (r = 0.017-0.254). There is a changing of urine periostin level of malignant patients receiving high dose cisplatin therapy which increase after the third chemotherapy. No significant correlation was found between periostin levels and kidney function in malignant patients with high-dose cisplatin therapy.

Incorporation of N7-Platinated Guanines into Thermus Aquaticus (Taq) DNA Polymerase: Atomistic Insights from Molecular Dynamics Simulations.

In this work, we elucidated some key aspects of the mechanism of action of the cisplatin anticancer drug, cis-[Pt(NH3)2Cl2], involving direct interactions with free nucleotides. A comprehensive in silico molecular modeling analysis was conducted to compare the interactions of Thermus aquaticus (Taq) DNA polymerase with three distinct N7-platinated deoxyguanosine triphosphates: [Pt(dien)(N7-dGTP)] (1), cis-[Pt(NH3)2Cl(N7-dGTP)] (2), and cis-[Pt(NH3)2(H2O)(N7-dGTP)] (3) {dien = diethylenetriamine; dGTP = 5'-(2'-deoxy)-guanosine-triphosphate}, using canonical dGTP as a reference, in the presence of DNA. The goal was to elucidate the binding site interactions between Taq DNA polymerase and the tested nucleotide derivatives, providing valuable atomistic insights. Unbiased molecular dynamics simulations (200 ns for each complex) with explicit water molecules were performed on the four ternary complexes, yielding significant findings that contribute to a better understanding of experimental results. The molecular modeling highlighted the crucial role of a specific α-helix (O-helix) within the fingers subdomain, which facilitates the proper geometry for functional contacts between the incoming nucleotide and the DNA template needed for incorporation into the polymerase. The analysis revealed that complex 1 exhibits a much lower affinity for Taq DNA polymerase than complexes 2-3. The affinities of cisplatin metabolites 2-3 for Taq DNA polymerase were found to be quite similar to those of natural dGTP, resulting in a lower incorporation rate for complex 1 compared to complexes 2-3. These findings could have significant implications for the cisplatin mechanism of action, as the high intracellular availability of free nucleobases might promote the competitive incorporation of platinated nucleotides over direct cisplatin attachment to DNA. The study's insights into the incorporation of platinated nucleotides into the Taq DNA polymerase active site suggest that the role of platinated nucleotides in the cisplatin mechanism of action may have been previously underestimated.

Antiproliferative Activity of Pt(IV) Conjugates Containing the Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) Ketoprofen and Naproxen †.

Cisplatin and several non-steroidal anti-inflammatory drugs (NSAIDs) have been proven to act synergistically or at least additively on several tumor cell lines. Dual-action cisplatin-based Pt(IV) combos containing ketoprofen and naproxen offer good antiproliferative performance on a panel of human tumor cell lines, including a malignant pleural mesothelioma (MPM) one, a very chemoresistant tumor. The main reason of the increased activity relies on the enhanced lipophilicity of these Pt(IV) conjugates that in turn promotes increased cellular accumulation. A quick Pt(IV)→Pt(II) reduction generates the active cisplatin metabolite. The NSAID adjuvant action seems to be almost independent from cyclooxygenase-2 (COX-2) expression in the tumor cells under investigation (lung A-549, colon HT-29, HCT 116, SW480, ovarian A2780, and biphasic MPM MSTO-211H), but it seems to rely (at least in part) on the activation of the NSAID activated gene, NAG-1 (a member of the transforming growth factor beta, TGF-β, superfamily), which has been suggested to be involved in NSAID antiproliferative activity.

Cisplatin-induced renal injury is independently mediated by OCT2 and p53.

Tubular secretion of cisplatin is abolished in mice deficient for the organic cation transporters Oct1 and Oct2 (Oct1/2(-/-)mice), and these animals are protected from severe cisplatin-induced kidney damage. Since tubular necrosis is not completely absent in Oct1/2(-/-)mice, we hypothesized that alternate pathways are involved in the observed injury. Studies were done in wild-type, Oct1/2(-/-), or p53-deficient animals, all on an FVB background, receiving cisplatin intraperitoneally at 15 mg/kg. Cisplatin metabolites were analyzed using mass spectrometry, and gene expression was assessed using Affymetrix microarrays and RT-PCR arrays. KEGG pathway analyses on kidneys from mice exposed to cisplatin revealed that the most significantly altered genes were associated with the p53 signaling network, including Cdnk1a and Mdm2, in both wild-type (P = 2.40 × 10(-11)) and Oct1/2(-/-)mice (P = 1.92 × 10(-8)). This was confirmed by demonstrating that homozygosity for a p53-null allele partially reduced renal tubular damage, whereas loss of p53 in Oct1/2(-/-)mice (p53(-/-)/Oct1/2(-/-)) completely abolished nephrotoxicity. We found that pifithrin-α, an inhibitor of p53-dependent transcriptional activation, inhibits Oct2 and can mimic the lack of nephrotoxicity observed in p53(-/-)/Oct1/2(-/-)mice. These findings indicate that (i) the p53 pathway plays a crucial role in the kidney in response to cisplatin treatment and (ii) clinical exploration of OCT2 inhibitors may not lead to complete nephroprotection unless the p53 pathway is simultaneously antagonized.

Antiproliferative activity of Pt(IV)-bis(carboxylato) conjugates on malignant pleural mesothelioma cells

The bifunctional Pt(IV) conjugate cis,cis,trans-diamminedichloridobis(valproato)platinum(IV), based on the cisplatin square-plane with two axial valproato (2-propylpentanoate, VPA) ligands, has been re-synthesized with a modified procedure and its biological activity was compared with that of its isomer cis,cis,trans-diamminedichloridobis(n-octanoato)platinum(IV). Both complexes showed a striking cytotoxic effect (in the micro or sub-micromolar range) on various human carcinoma cell lines (namely ovarian, colon, breast and lung cancer), and, in particular, on cells derived from malignant pleural mesothelioma. This remarkable activity is due to the action of the cisplatin metabolite only, generated by the intracellular Pt(IV)→Pt(II) reduction, which concentration is greatly increased by the enhanced cellular accumulation of the original, highly lipophilic Pt(IV)-bis(carboxylato) complexes. The two axial VPA ligands are released in a too low concentration to act as histone deacetylase inhibitor (HDACI), as VPA works in the millimolar range, so that no synergism can be claimed. Moreover, n-octanoic acid is substantially deprived of any HDACI propensity.

Uptake of Cisplatin and its Metabolites Into Rat and Rabbit Renal Cortical Slices

The uptake of cisplatin and its metabolites into rat and rabbit renal cortical slices was investigated. Under anaerobic conditions the cisplatin uptake rate was significantly reduced by pretreatment with dinitrophenol and by organic cationic compounds (cimetidine, verapamil and quinidine). Verapamil competitively inhibited the uptake of cisplatin (apparent inhibitory constant (Ki) = 204.5 ± 43.0 μM). There was very little uptake of the cisplatin metabolite formed by complexation with glutathione. In addition, both aquated cisplatin and a metabolite formed by complexation with thiosulphate were taken up to a very low extent at 37°C compared with unchanged cisplatin. These results suggest that the intact chemical structure of cisplatin may be required for transport of cisplatin into the kidney and cisplatin may be partly transported via organic cation transport systems.

A simple and sensitive validated HPLC method for quantitative determination of cisplatin in human plasma

Cisplatin is an anti-tumor agent widely employed in cancer chemotherapy. A specific and selective method for the quantitative determination of cisplatin in human plasma and its applications to pharmacokinetic investigations is described. One simple ethanol-induced protein precipitation step followed by simple liquid–liquid extraction with chloroform is the only requirement as sample treatment. The resulting solution is injected into a Wakosil II (5 μm, 250 cm × 4.6 mm I.D.) analytical column. The mobile phase consisted of methanol:water:acetonitrile (40:30:30 v/v/v). The limit of quantitation was 1 μg/mL. The method showed good recovery (93.95%) and within batch recovery was 91.59–97.00%. At all levels intra- and inter-assay precision was lower than 7 and 10%, respectively. The intra- and inter-assay accuracy ranged from −2.7 to 2% and from −3.1 to 4.0%, respectively. The selectivity (discrimination between the parent drug and platinum containing species such as cisplatin metabolites), simplicity and spee...

SEC-ICP-MS and ESI-MS as tools to study the interaction between cisplatin and cytosolic biomolecules

Most of cisplatin’s citotoxic properties are due to the interaction of the drug with DNA. However, other biological molecules present in the cell cytosol, such as MT (metallothionein) and GSH (glutathione), are potential targets for cisplatin and have been related to its side-effects or with the cellular resistance mechanisms to the drug. Experiments simulating physiological conditions have been performed to study the specific cisplatin metabolites which interact with GSH and MT and to characterize the different drug–biomolecule adducts over time. A combination of size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) and electrospray ionization-mass spectrometry (ESI-MS) techniques has been used to provide sensible multi-elemental detection and structural information of the species of interest. Time dependent transformation of 10 μM cisplatin at neutral pH (7.4) produces different concentrations of the mono-aquo and oligomeric derivatives, as could be confirmed by ESI-MS. No di-aquo derivative was seen to be produced under these conditions at any of the incubation times used. Cisplatin and the oligomeric derivative were incubated with GSH and MT at different drug:biomolecule ratios. Adducts from cisplatin–GSH (1:500) and from cisplatin–MT (1:10) incubations were characterized by SEC-ICP-MS. While both GSH and MT reacted with cisplatin producing different compounds, only GSH reacted with the oligomeric derivative of cisplatin. SEC-ICP-MS experiments showed that, under neutral pH conditions, Cd atoms remained bound to the cisplatin:MT adducts, but Zn atoms were lost. Results were compared with those obtained by in vitro and in vivo experiments with rat kidney, liver and inner ear cytosols.

Cisplatin-Induced Toxicity Is Associated with Platinum Deposition in Mouse Kidney Mitochondria in Vivo and with Selective Inactivation of the α-Ketoglutarate Dehydrogenase Complex in LLC-PK1Cells

The anticancer drug cisplatin is nephrotoxic and neurotoxic. Previous data support the hypothesis that cisplatin is bioactivated to a nephrotoxicant. The final step in the proposed bioactivation is the formation of a platinum-cysteine S-conjugate followed by a pyridoxal 5'-phosphate (PLP)-dependent cysteine S-conjugate beta-lyase reaction. This reaction would generate pyruvate, ammonium, and a highly reactive platinum (Pt)-thiol compound in vivo that would bind to proteins. In this work, the cellular location and identity of the PLP-dependent cysteine S-conjugate beta-lyase were investigated. Pt was shown to bind to proteins in kidneys of cisplatin-treated mice. The concentration of Pt-bound proteins was higher in the mitochondrial fraction than in the cytosolic fraction. Treatment of the mice with aminooxyacetic acid (AOAA, a PLP enzyme inhibitor), which had previously been shown to block the nephrotoxicity of cisplatin, decreased the binding of Pt to mitochondrial proteins but had no effect on the amount of Pt bound to proteins in the cytosolic fraction. These data indicate that a mitochondrial enzyme catalyzes the PLP-dependent cysteine S-conjugate beta-lyase reaction. PLP-dependent mitochondrial aspartate aminotransferase (mitAspAT) is a mitochondrial enzyme that catalyzes beta-elimination reactions with cysteine S-conjugates of halogenated alkenes. We reasoned that the enzyme might also catalyze a beta-lyase reaction with the cisplatin-cysteine S-conjugate. In this study, mitAspAT was stably overexpressed in LLC-PK(1) cells. Cisplatin was significantly more toxic in confluent monolayers of LLC-PK(1) cells that overexpressed mitAspAT than in control cells containing vector alone. AOAA completely blocked the cisplatin toxicity in confluent mitAspAT-transfected cells. The Pt-thiol compound could rapidly bind proteins and inactivate enzymes in close proximity of the PLP-dependent cysteine S-conjugate beta-lyase. Treatment with 50 or 100 microM cisplatin for 3 h, followed by removal of cisplatin from the medium for 24 h, resulted in a pronounced loss of alpha-ketoglutarate dehydrogenase complex (KGDHC) activity in both mitAspAT-transfected cells and control cells. Exposure to 100 microM cisplatin resulted in a significantly greater loss of KGDHC activity in the cells overexpressing mitAspAT than in control cells. Aconitase activity was diminished in both cell types, but only at the higher level of exposure to cisplatin. AspAT activity was also significantly decreased by cisplatin treatment. By contrast, several other enzymes (both cytosolic and mitochondrial) involved in energy/amino acid metabolism were not significantly affected by cisplatin treatment in the LLC-PK(1) cells, whether or not mitAspAT was overexpressed. The susceptibility of KGDHC and aconitase to inactivation in kidney cells exposed to cisplatin metabolites may be due to the proximity of mitAspAT to KGDHC and aconitase in mitochondria. These findings support the hypothesis that a mitochondrial cysteine S-conjugate beta-lyase converts the cisplatin-cysteine S-conjugate to a toxicant, and the data are consistent with the hypothesis that mitAspAT plays a role in the bioactivation of cisplatin.

The electroreceptor organ of the catfish, Ictalurus melas, as a model for cisplatin-induced ototoxicity

The ototoxic side-effects of the anti-cancer drug cisplatin ( cis-diaminedichloroplatinum) have been widely investigated. However, the exact site of action remains unclear. In this study, the electroreceptor organ of the freshwater catfish Ictalurus melas is used as a model for examining the acute effects of cisplatin. The sensory cells in the electroreceptor organ are homologous to the inner hair cells in the cochlea of mammals. The effects of cisplatin administration can be investigated by in vivo recording of the spike trains from the electroreceptor organ primary afferents. Exposure of electroreceptor organs to 330 μM cisplatin for 1 h causes the spontaneous activity to drop, the overall sensitivity to diminish and the shape of the frequency characteristics to change. These effects persist in the week after administration. Control levels have returned at day 22. These results demonstrate an acute and, with considerable hysteresis, reversible cisplatin effect on the electroreceptor organs, which is to a large extent consistent with the cisplatin-induced effects in isolated hair cells in mammals. The time-course of the effect supports the hypotheses that ion channels are blocked immediately by cisplatin administration, and that cisplatin metabolites disturb enzymatic cellular processes.

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 μMof 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/106cells) after addition of 0.33, 1.6, and 2.5 mMof 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.

Cisplatin metabolites and their toxicity on isolated cochlear outer hair cells in vitro.

The exact mechanism of ototoxicity by cisplatin remains obscure. In the present study, the toxicity of cisplatin metabolites was evaluated using isolated outer hair cells of guinea pig cochlea. Cisplatin metabolites were produced by reaction of cisplatin and S9 fraction from guinea pig liver after administration with phenobarbital. The viability of the outer hair cells did not decrease after the incubation in non-metabolized cisplatin solutions or S9 fraction only, demonstrating the absence of toxic effect from non-metabolized cisplatin and the absence of intrinsic toxic substances in S9 fraction. However, metabolized cisplatin reacted with S9 fraction at 37 degrees C significantly decreased the viability. Cisplatin reacted with heat-treated S9 fraction did not produce a cytotoxic effect, suggesting an enzymatic basis for the formation of metabolites. When NADP was omitted, outer hair cells remained viable, suggesting the significant role of oxygenases in the metabolism of cisplatin. The existence of a toxic metabolites could account for the poor correlation between drug peak level and pathologic damage to the inner ear, and a delay in the onset of ototoxicity could represent the time necessary for induction of the metabolizing enzymes or for reaching a critical point in toxicity by metabolites.

Pharmacokinetics and toxicity of intraperitoneal cisplatin combined with regional hyperthermia.

Hyperthermia (HT) potentiates in vitro cytotoxicity of cisplatin, providing a rationale for HT enhancement of cisplatin effect in vivo. In this study, regional abdominal HT was combined with intraperitoneal (IP) cisplatin in canines to characterize temperature distributions, as well as pharmacokinetics and toxicity of IP cisplatin with and without HT. Cisplatin (65 mg/m2) in normal saline was administered IP with a two-hour dwell time in ten Beagle dogs. Five of the ten dogs were randomly selected to receive concurrent regional microwave-producing HT at approximately 41.5 degrees C (IP) for a 60-minute period. Systemic temperatures in heated animals ranged from 37 degrees C to 40 degrees C; IP temperatures ranged from 39 degrees C to 44 degrees C. Initial IP temperatures ranged from 39 degrees C to 44 degrees C. Initial IP cisplatin concentrations were ten to 22 times greater than serum levels; the IP drug half-lives were 133 +/- 9 minutes and 68 +/- 15 minutes in heated and unheated dogs, respectively (P less than .001). Total concentrations of serum and urine cisplatin did not differ between the heated and unheated controls. The area under the concentration v time curve for free, ultrafilterable cisplatin in serum in units of percent minutes was 40 +/- 8 in heated and 60 +/- 7 in unheated controls (P = .006). Except for transient nausea and vomiting, no evidence of serious toxicity was observed in serum chemistries or histopathologic sections at 21 days post-treatment. Experiments involving in vitro incubation of cisplatin in normal saline were performed as a function of saline temperature; these showed that the amount of reactive cisplatin metabolites formed increased linearly with temperature by approximately 30% from 38 degrees C to 44 degrees C. This study supports the hypothesis that, with IP temperature elevation, there is an increased rate of generation and retention of reactive metabolites of cisplatin in the peritoneal cavity relative to unheated controls. In spite of these differences in pharmacokinetics, no significant toxicity was encountered. This study provides a model for treatment of IP malignancy such as ovarian carcinoma with IP cisplatin and regional HT.

A study of the protective effect of chloride salts on cisplatin nephrotoxicity

In rats NaCl and NH 4Cl (25 mmoles/kg, p.o.) were found to be equally effective at preventing nephrotoxicity when administered to rats 90 min before cisplatin (5 mg/kg, i.p.) but (NH 4) 2SO 4 did not protect. The severity of nephrotoxicity, taken as the maximum elevation in blood urea concentration, showed a high degree of correlation with urinary chloride concentration, but not with urinary pH or volume. Sodium chloride did not protect against nephrotoxicity when administered 3 or 24 hr after cisplatin. Sodium chloride showed protection against nephrotoxicity caused by cisplatin metabolites only at low doses of platinum. For animals pretreated with NaCl (25 mmoles/kg) or water p.o. the urinary excretion of total platinum, cisplatin and six of the seven metabolites separated by hplc was not significantly different between the two treatments during the 0–5 hr period post dosing. However, one metabolite, possibly a nephrotoxic hydrolysis product, was excreted in significantly smaller amounts in the urine of animals pretreated with NaCl (P < 0.05). Furthermore, in all cisplatin treated animals the amount of this species excreted correlated with the severity of nephrotoxicity. Whilst this suggests that chloride ions may protect against the nephrotoxicity of cisplatin by inhibiting its rate of metabolism this metabolite accounts for only 2.5% of the platinum excreted. Furthermore, the data do not exclude the possibility that NaCl prevents cisplatin-induced nephrotoxicity by preventing renal ischaemia, which may normally follow cisplatin treatment, or that the renal uptake or transport of platinum may be inhibited by NaCl.

Cisplatin metabolites in plasma, a study of their pharmacokinetics and importance in the nephrotoxic and antitumour activity of cisplatin

For rats dosed with cisplatin the rate of appearance in plasma of ultrafilterable metabolites containing platinum has been investigated using HPLC. At least seven species containing platinum in addition to cisplatin are present 15 min following injection i.p. of 15 mg kg −1. Unchanged cisplatin has been almost completely eliminated from the plasma within 3 hr of dosing; however, metabolite species are still present. The same metabolite species form when cisplatin is incubated in vitro with plasma although in different proportions. After incubation for 24 hr at 37% a mixture of metabolites is produced which contains less than 4% cisplatin. This mixture, when injected i.p. into rats, is nephrotoxic at doses of platinum at which cisplatin is not. The mixture of metabolites has considerably less antitumour activity than cisplatin when tested against the mouse L1210 leukemia assay. Although no metabolite species has been unequivocally identified we present evidence which suggests that amongst the principle metabolite species are an hydrolysis product and methionine substitution products of cisplatin. A mixture of cisplatin methionine substitution complexes showed neither antitumour nor nephrotoxic properties. However, an hydrolysis product was shown to be nephrotoxic at a dose of platinum at which cisplatin is not. The work reported here is the first direct experimental demonstration that cisplatin metabolites are more nephrotoxic but less effective antitumour agents than the parent compound.

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