Amount Of Cisplatin Research Articles

Zein - laponite coacervate aided co-delivery of curcumin and cisplatin towards MDA-MB-231 breast cancer cells: Validating the concept

Cancer cell resistance towards drugs leads to limited drug efficacy and therefore, combination therapy of drugs has emerged as a solution. In addition to this, the importance of a carrier system for the co-delivery of drugs to improve the potential of anti-cancer drug efficacy cannot be overlooked. Nowadays, polymer-rich dense phases of liquid droplets, known as coacervates, have emerged as an appealing option to be utilised as drug carriers. Our previous study which reported the complexation between zein and laponite to form coacervates and their future prospect as dual drug carriers has been extended in this work. The present work has validated the usefulness of the synthesized coacervate as a dual-drug carrier for a hydrophilic drug (cisplatin) and a hydrophobic drug (curcumin) towards a breast cancer cell line (MDA-MB-231). The MTT assay and flow cytometry data for MDA-MB-231 cells suggested that the coacervate carrying dual drugs effectively transported anticancer drugs to the cancer cells, thereby arresting their cell cycle in the S phase (by neutral coacervates, X1) and in the G2/M phase (by charged coacervates, X2). Further, it is also revealed that the amount of cisplatin needed to achieve half maximal inhibitory concentration (IC50) was reduced by a maximum of 70% when flavonoid (curcumin) was used in combination with cisplatin due to synergistic effect.

PD53-04 BIODEGRADABLE DEVICE FOR CONTROLLED RELEASE OF CISPLATIN AS LOCAL CHEMOTHERAPY FOR BLADDER CANCER

You have accessJournal of UrologyCME1 May 2022PD53-04 BIODEGRADABLE DEVICE FOR CONTROLLED RELEASE OF CISPLATIN AS LOCAL CHEMOTHERAPY FOR BLADDER CANCER Gilad Amiel, Alexandra Bukchin-Tihomirov, Ahmed Kabha, Azik Hoffman, and Eyal Zussman Gilad AmielGilad Amiel More articles by this author , Alexandra Bukchin-TihomirovAlexandra Bukchin-Tihomirov More articles by this author , Ahmed KabhaAhmed Kabha More articles by this author , Azik HoffmanAzik Hoffman More articles by this author , and Eyal ZussmanEyal Zussman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002630.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The usual treatment for bladder tumors after local resection is instillation of chemotherapy or immunological treatment. These treatments are challenging due to short exposure time or dilution by urine production. We have developed a biodegradable device that gradually releases cisplatin into the urinary tract, to eliminate cancer cells. The device is lodged in the renal pelvis, as an alternative strategy for injecting substances into the bladder. METHODS: Using PLGA-produced poly-lactic-glycolic-acid nanofibres, we were able to trap within the polymer matrix the cisplatin that would be released into the urine stream with the gradual breakdown of the device. We tested our ability to build a device that would sit in the renal pelvis, the amount of cisplatin that could be loaded on the device, the rate of release of cisplatin at different concentrations, and the response of different TCC cell lines to exposure to cisplatin released from the disintegrated device. RESULTS: The device was built in the shape of KORIGAMI for maximizing its’ surface area. Cisplatin was imprinted in various concentrations (1.17-2.34% w / w). In a bioreactor that simulates artificial urine interaction with the device, a cisplatin release rate of 26% of its contents within the first 6 hours and a release of 94% of the contents after a week at a flow rate of 30 ml / hour was demonstrated. The stent completely disintegrated within 4 weeks. Two TCC cell-lines were tested (T24 & NTB9) and apoptosis was demonstrated within a few hours at all concentrations tested. CONCLUSIONS: We attempted to build a nanofiber model as a reservoir for active substances trapped in a polymer, that decomposes under urinary flow conditions. We developed a stent that releases at a predetermined rate, a drug for a period of about several weeks. We started with cisplatin, the most effective chemotherapy in the treatment of bladder cancer. We anticipate that the location of the device in the renal pelvis it will reduce bladder irritation of existing stents. We are planned to implant the device in the renal pelvis of a swine model. We believe that a device that allows for long-term controlled release will provide a significant advantage over existing bladder cancer treatments that require multiple instillations. Source of Funding: KAMIN Grant, Israel Ministry of Science © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e911 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gilad Amiel More articles by this author Alexandra Bukchin-Tihomirov More articles by this author Ahmed Kabha More articles by this author Azik Hoffman More articles by this author Eyal Zussman More articles by this author Expand All Advertisement PDF DownloadLoading ...

High loading of trimethylglycine promotes aqueous solubility of poorly water-soluble cisplatin

Trimethylglycine (TMG) is a cheap, natural, and highly biocompatible compound. Therefore, it has been used in the fields of food and life sciences, but the application of solid TMG is limited to utilisation as an “additive”. In the present study, we focussed on the high solubility of TMG in water, derived from the aprotic zwitterionic structure, and proposed TMG as the chemical accounting for a major portion of the aqueous solution (e.g., 50 wt%). High loading of TMG shifted the properties of water and enabled the dissolution of poorly water-soluble cisplatin, an anticancer agent, at high concentration (solubility of cisplatin: 0.15 wt% in water vs 1.7 wt% in TMG aqueous solution). For hepatic arterial infusion, this can reduce the amount of cisplatin administered from 40 to 4 mL. It enables simple injection using a syringe, without the need for catheters and automatic pumps, leading to critical alleviation of the risk to patients. Furthermore, we produced a dry powder from a cisplatin-containing TMG aqueous solution via freeze-drying. Powders can be conveniently stored and transported. Furthermore, cisplatin is often used as a mixture with other drugs, and cisplatin aqueous solutions are not preferred as they dilute the other drugs.

Glutathione-responsive hydrogel and molecularly imprinted polymer nanospheres: New aspect on cisplatin delivery

In this study, Glutathione-responsive hydrogel, MIPs and their related NIPs were synthesized by precipitation polymerization method. Effect of HEMA/MAA and EGDMA/TRIM as functional monomer and crosslinking agents on size, morphology, drug loading and releasing were studied. BACy as a disulfide cross-linking agent was used in praparation of Glutathione-responsive hydrogel. The size and morphology of MIPs and NIPs were examined by FE-SEM technique. Results showed EGDMA-synthesized MIPs had the smaller size than TRIM-prepared MIPs. In order to measure the capacity of MIPs or NIPs for binding to cisplatin, static and dynamic adsorption experiments were done. The amounts of cisplatin were measured by HPLC method. The binding capacity of drug by MIPs is observed to be higher than NIPs. The EGDMA-synthesized polymers showed a high percentage of adsorption. NIPs were saturated much sooner than MIPs. In vitro release assays of cisplatin were done in PBS at pH=7.4 and temperature of 37°C. The results showed that increase of the amount of GSH cause to increase release of the drug from hydrogel.

A Label-free DNA-based Fluorescent Sensor for Cisplatin Detection

Cisplatin, an anti-cancer drug, has been used extensively and has become a “gold standard” treatment for various types of cancer. However, overdosage has serious side effects. Thus, this drug should not be over-administered. The current study presents a label-free but sensitive fluorescent sensor for cisplatin detection based on G-quadruplex (GQ) and fluorescent dye Thioflavin T (ThT). In the absence of cisplatin, ThT binds to GQ, resulting in high fluorescence emission. In the presence of cisplatin, the GQ structure is destroyed after cisplatin–DNA adduct formation, leaving no binding scaffold for ThT, thereby causing a decrease in ThT fluorescence. The amount of cisplatin can be quantified from the changes in ThT fluorescence intensity. Parameters that affect the sensor performance were tested, including pH and concentration of potassium phosphate buffer, type of GQ, number of quartets, loop length, incubation time, and temperature. The sensor provided a linear dynamic range of 10–500 nM with limit of quantification at 10 nM. The sensor was successfully applied to detect cisplatin in urine samples from patients undergoing treatment with high accuracy (recoveries between 74 ± 14 and 112.8 ± 5.0%) and precision (relative standard deviations between 1.15% and 2.12%).

Efficacy of four cleaning solutions for the decontamination of selected cytotoxic drugs on the different surfaces of an automated compounding system

The automated aseptic preparation of ready-to-administer antineoplastic drug solutions with robotic systems reduces the risk of occupational exposure. However, the surfaces in the preparation area of the robot are to be cleaned by wiping with an appropriate cleaning solution. The aim of the study was to evaluate the cleaning efficacy of four cleaning solutions on four surface materials installed in the APOTECAchemo robot. Predefined amounts of cisplatin (Cis), 5-fluorouracil (5-FU), and cyclophosphamide (CP) were intentionally spread on test plates made of stainless steel, aluminium, polyoxymethylene, and polycarbonate just as installed in the robotic system APOTECAchemo. After drying, the plates were cleaned with 0.2% ethanolic NaOH, 0.23% isopropanolic sodium dodecylsulfate (SDS-2P), 0.5% sodium hypochlorite (NaOCl), and 0.1% benzalkonium chloride (BZK) solutions following a standardized wiping protocol. Residual contamination was recovered with wipe tests, Pt was quantified by voltammetry, and 5-FU and CP was quantified by gas chromatography-tandem mass spectrometry (GC-MSMS). The mean residual contamination after cleaning and the cleaning efficacy (CE) rates were calculated and aggregated on different levels. The CE rates varied between 81.5% and 100% and lay in the majority of cases above 90%. The lowest CE rates were registered for Pt contamination. Especially on aluminium surfaces the residual contamination was high. The overall CE rates of the three different drugs and four different surface types amounted to 98.3% for NaOCl, 97.9% for SDS-2P, 96.9% for ethanolic NaOH, and 96.5% for BZK. The tested cleaning solutions proved to be higher than 90% in most cases, but none of them was able to eliminate 100% of the intentional surface contamination of three antineoplastic drugs on the test plates. The cleaning efficacy varied according to the different surface types and antineoplastic drug. Results could be used in the daily clinical practice to develop and implement effective cleaning procedures.

Impact of Neoadjuvant Chemotherapy on the Administration of Concurrent Chemoradiation for Locally Advanced Nasopharyngeal Carcinoma

ObjectivesThe standard of care for locally advanced nasopharyngeal carcinoma (NPC) is concurrent cisplatin chemoradiotherapy. Neoadjuvant chemotherapy can be administered to downsize tumors before concurrent treatment to optimize radiation volumes. Our hypothesis was that the use of cisplatin in the neoadjuvant phase could limit the amount of cisplatin that patients could tolerate in the concurrent phase of treatment.MethodsThis is a retrospective analysis of Canadian NPC patients who received neoadjuvant chemotherapy with the intention to downsize locally advanced tumors prior to concurrent cisplatin plus radiation. Baseline demographic and treatment data were obtained from institutional databases and chart review; all data were analyzed with SPSS (SPSS Inc. Released 2005. SPSS for Windows, Version 14.0. Chicago: SPSS Inc.) Overall survival (OS), disease-specific survival (DSS), and local/regional relapse-free survival (LRRFS) were analyzed using Kaplan-Meier survival functions. Univariate and multivariate models were used to determine factors associated with the total dose of concurrent chemotherapy.ResultsForty-six patients were identified as receiving neoadjuvant chemotherapy before concurrent chemoradiotherapy. In the univariate and multivariate analyses of patients who received concurrent chemotherapy, receiving over 200 mg/m2 concurrent cisplatin with radiation was associated with a higher neoadjuvant dose of chemotherapy received. The median follow-up time was 2.6 years (range, 0.17 years to 10.6 years). At three years, the OS was 83%, DSS was 86%, and LRRFS was 74%.ConclusionsNPC patients have been treated with neoadjuvant chemotherapy at this center with favorable outcomes. Most patients could tolerate concurrent chemotherapy after radiotherapy. Receiving higher doses of concurrent chemotherapy was associated with also having higher doses of neoadjuvant cisplatin. This suggests that neoadjuvant cisplatin is not a limiting factor in the delivery of full-dose concurrent chemotherapy.

Abstract 1121: Redox-mediated inactivation of MGMT DNA repair through thiolation or nitrosylation of the active site cysteine145 sensitizes human gliomas to alkylating agents in vitro and in xenograft models

Abstract O6-Methylguanine-DNA methyltransferase (MGMT) is a simple and unique antimutagenic DNA repair protein that plays a crucial role in conferring tumor resistance to various alkylating agents in brain tumor therapy. MGMT repairs O6-alkylguanines by transferring the alkyl groups to its active site cysteine (Cys145) in a stoichiometric/suicidal reaction thereby restoring the original nucleotide sequence in a single step. Although O6-benzylguanine (BG), a pseudosubstrate inhibitor, currently in clinical trials, potently inhibits MGMT and improves the efficacy of alkylating agents, unacceptable levels of bone marrow toxicity has been a great concern. For developing new MGMT inhibitors, we exploited the reactive nature of the active site Cys145 (pKa 4.5) and its susceptibility for thiolation and nitrosylation, both of which inactivate the MGMT. A new strategy involving inhibition of S-nitosoglutathione reductase (GSNOR) was also explored to increase the steady-state levels of nitrosylated MGMT. First, we designed a redox perturbing glutathione mimetic, a homoglutathione disulfide (hGTX) that has been stabilized with the addition of small amounts of cisplatin (1000:10). Spermine NONOate and S-nitroso penicillamine (SNAP) served as the protein-nitrosylating agents. N6022, a specific and reversible inhibitor of GSNOR was used with nitrosylators in some assays. Both the hGTX and spermine NONOate at 100-400 µM concentrations for 24 h inhibited the repair activity of MGMT >75% in HT29, SF188, and T98G cells. The NONOate strongly curtailed the DNA repair activity of recombinant MGMT showing a direct interaction. The inactivated MGMT protein was promptly eliminated from glioma cells. MGMT disappeared from cells much faster in spermine NONOate +N6022 treated cells, suggesting an extended steady-state of nitrosylated MGMT and its subsequent degradation. MGMT depletion was accompanied by 4-6 fold increases in cell killing by BCNU or temozolomide (TMZ). Binding of the biotinylated-O6-benzylguanine to the MGMT protein was prevented by hGTX and spermine NONOate treatments indicating the Cys145 was indeed the site of modification. In the nude mice bearing T98G or HT29-luc2 xenografts, combinations of hGTX and TMZ or spermine NONOate +TMZ produced markedly synergistic tumor regressions compared with TMZ alone. A reduction of MGMT protein as by immunoblotting and diminished MGMT activity levels in tumor tissues was verified. Apoptosis regulatory proteins were upregulated in excised tumors and H&E staining confirmed no discernible host organ toxicities. Our studies highlight the options for redox-driven therapeutic strategies for MGMT [supported by CPRIT grants RP130266 and RP170207 to KSS]. Citation Format: Debasish Basak, Kalkunte S. Srivenugopal. Redox-mediated inactivation of MGMT DNA repair through thiolation or nitrosylation of the active site cysteine145 sensitizes human gliomas to alkylating agents in vitro and in xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1121. doi:10.1158/1538-7445.AM2017-1121

Aldehyde dehydrogenase 2*2 knock-in mice show increased reactive oxygen species production in response to cisplatin treatment

BackgroundThe aldehyde dehydrogenase (ALDH) enzyme family metabolizes and detoxifies both exogenous and endogenous aldehydes. Since chemotherapeutic agents, such as cisplatin, generate cytotoxic aldehydes and oxidative stress, and chemoresistant cancer cells express high levels of ALDH enzymes, we hypothesized that different ALDH expression within cells may show different chemosensitivity. ALDH2 has the lowest Km for acetaldehyde among ALDH isozymes and detoxifies acetaldehydes in addition to other reactive aldehydes, such as 4-hydroxy-nonenal, malondialdehyde and acrolein produced from lipid peroxidation by reactive oxygen species (ROS). Thus, cells with an ALDH2 variant may sensitize them to these ROS-inducing chemotherapy drugs.MethodsHere, we used wild type C57BL/6 mice and ALDH2*2 knock-in mutant mice and compared the basal level of ROS in different tissues. Then, we treated the mice with cisplatin, isolated cells from organs and fractionated them into lysates containing mitochondrial and cytosolic fractions, treated with cisplatin again in vitro, and compared the level of ROS generated.ResultsWe show that overall ROS production increases with cisplatin treatment in cells with ALDH2 mutation. The treatment of cisplatin in the wild type mice did not change the level of ROS compared to PBS treated controls. In contrast, ALDH2*2 knock-in mutant mice showed a significantly increased level of ROS compared to wild type mice in tongue, lung, kidney and brain tissues without any treatment. ALDH2*2 mutant mice showed 20% of the ALDH2 activity in the kidney compared to wild type mice. Treatment of ALDH2*2 mutant mice with cisplatin showed increased ROS levels in the mitochondrial fraction of kidney. In the cytosolic fraction, treatment of mutant mice with cisplatin increased ROS levels in lung and brain compared to PBS treated controls. Furthermore, ALDH2*2 mutant mice treated with cisplatin showed increased cytotoxicity in the kidney cells compared to PBS treated mutant controls.ConclusionsThese data indicate that deficiency in ALDH2 activity may contribute to increased cisplatin sensitivity and cytotoxicity by producing more ROS by the treatment. Based on these data, the amount of cisplatin used in patients may need to be adjusted based on their ALDH2 variant profile.

Abstract 1216: Thiol-modification induced reactivation of mutant p53 protein in human cancers using a nontoxic stabilized homoglutathione disulfide

Abstract Loss of functional p53 gene is a frequent and most critical event in the genesis and progression of half of all human cancers. Conversion of the non-DNA binding and misfolded-p53 mutant proteins to their wild-type conformation using small molecules to induce apoptosis is currently an intense area of research. Although a few compounds with this property have been described, their abilities to alter the p53 structure in animal models have not been documented and they appear toxic as well. Human cancers harbor elevated levels of oxidative stress and are vulnerable to even slight redox oscillations. To exploit this susceptibility, we have designed a glutathione mimic, namely, homoglutathione disulfide that has been stabilized with the addition of small amounts of cisplatin (1000:10). This compound perturbs the cellular redox, alters kinase activities and induces protein-glutathionylation. Since the human p53 contains several redox-sensitive cysteines in the DNA-binding domain, we explored the possible reactivation of mutant p53 (R273H) present in HT29 and SW620 colon cancer cells by the stabilized hGSSG.Pt (hGTX). Electrophoretic mobility shift assays demonstrated a concentration and time-dependent restoration of DNA-binding for the mutant p53 in HT29 cells by hGTX. DNA affinity immunoblotting, immunoprecipitation and direct western blotting using specific antibodies against the mutant (Mab240) or wild type p53 (Mab1620) or pan p53 (MabDo1) confirmed a progressive generation of wild type p53 and a gradual decline of mutant p53 levels in HT29 cells. The half-life of the altered p53 remained similar to the endogenous mutant form. Treatment of HT29 nuclear extracts with hGTX also induced the binding of mutant p53 with DNA, indicating that thiolation of specific cysteines mediates the reactivation. RT-PCR and western analyses showed significant upregulation of many p53 target genes (2-5 fold). Furthermore, when nude mice bearing the HT29 xenografts were administered HGTX (100 mg/kg daily for 7 days), marked p53 reactivation with concomitant reduction of mutant p53 was observed in tumor tissues. Treatment of hGTX also modulated redox sensitive DNA repair systems like MGMT and Topoisomerase-I. A highly significant amplification of cytotoxicity was induced by anticancer drugs in association with hGTX. We conclude that the alteration of redox milieu following hGTX modifies the reactive thiols in the p53 protein to restore the native conformation. These findings acquired with a nontoxic endogenous metabolite suggest therapeutic applications in combination with chemotherapy agents. Molecular modeling and mass spectrometry studies are underway to determine the structural changes and residues altered by hGTX treatment (Supported by grants from CPRIT (RP130266), NIH (R03CA125872) to KSS. Citation Format: Debasish Basak, Kalkunte S. Srivenugopal. Thiol-modification induced reactivation of mutant p53 protein in human cancers using a nontoxic stabilized homoglutathione disulfide. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1216. doi:10.1158/1538-7445.AM2015-1216

Abstract 4576: Development of thermochemotherapy using cisplatin and ferucarbotran (Resovist®) in head and neck cancer

Abstract Background: Radical surgery for patients with advanced head and neck cancer causes dysfunctions as well as decreases quality of life. To overcome this issue, we developed a new combination therapy of cisplatin and inductive hyperthermia using ferucarbotran (Resovist®). Ferucarbotran, which is made of superparamagnetic iron oxide, generates heat when exposed to an alternating magnetic fields (AMF). Herein, we explored whether ferucarbotran could be used as a heat source for hyperthermia upon exposure to AMF in the presence of cisplatin. Our aim is to evaluate the simultaneous therapeutic efficacy of chemotherapy and inductive hyperthermia for head and neck cancer. Materials and Methods: OSC-19 and HSC-3, human oral cancer cell lines, were used in this study. Cell proliferation was assessed by methyl thiazolyl tetrazorium (MTT) assay. The intracellular level of reactive oxygen species (ROS) was measured using fluorescent dye 2′, 7′-dichlorodihydrofluorescein diacetate. Apoptotic cells were stained with Annexin V, allophycocyanin conjugate and 7-amino-actinomycin D, and measured by fluorescence activated cell sorting (FACS), to evaluate early and late apoptosis. Thermal images and temperature were obtained by thermograpy and thermometer. Alternating magnetic fields were generated by a transistor-driven vertical coil at a frequency of 308 KHz and electric current (EC) 250 A. Results: Ferucarbotran generated heat in a dose- and time-dependent manner when exposed to an AMF, suggesting that ferucarbotran could be used as a heat source for hyperthermia. As we expected, Cisplatin suppressed proliferation of OSC-19 and HSC-3 cells in a dose-dependent manner, not only furucarbtran. First, we performed MTT assay and ROS generation assay to evaluate whether hyperthermia effect enhanced anti-cancer effect in the presence of cisplatin. Simply incubation at 42 °C for one hour enhanced the anti-cancer effect and ROS generation in the presence of cisplatin. Cisplatin induced apoptosis of OSC-19 and HSC-3 cells in a dose-dependent manner. Ferucarbotran further promoted cisplatin-induced apoptosis compared to cisplatin alone, when exposed to an AMF for an hour. Thus, the combination of cisplatin with ferucarbotran /AMF was more effective than cisplatin alone, suggesting that we could reduce the amount of cisplatin in clinical usage. Conclusion: Our findings suggest that combination therapy of cisplatin and ferucarbotran in an AMF may be used to develop a new combination therapy for head and neck cancer. Citation Format: Itaru Sato, Masanari Umemura, Kenji Mitsudo, Xianfeng Feng, Hideyuki Nakashima, Mitomu Kioi, Akiyoshi Miyajima, Haruki Eguchi, Iwai Tohnai, Yoshihiro Ishikawa. Development of thermochemotherapy using cisplatin and ferucarbotran (Resovist®) in head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4576. doi:10.1158/1538-7445.AM2014-4576

Abstract 4585: Crocin-supplemented cisplatin is highly effective in killing breast cancer cells than cisplatin alone

Abstract Breast cancer afflicts women of all ages. Limited treatment options that are currently available include use of hormone antagonists and various platinum-based chemical agents (Cisplatin, Carboplatin and Oxaliplatin). Cisplatin, is a widely used chemotherapeutic drug that is often prescribed for the treatment of hormone resistant breast tumors. Through inter- and intra-strand DNA cross linking, Cisplatin induces apoptosis in cancer cells. Although, it is very effective in its action, the use of Cisplatin is accompanied with high overall toxicity due to its indiscriminate killing of normal cells along with cancer cells; therefore, it has to be used at less than the effective dose. Crocin, is a phytochemical that efficiently kills cancer cells without affecting the normal cells even at higher doses. Due to this property, it is currently being tested as an anti-cancer drug either alone or in combination with various novel and traditional anticancer agents to supplement and even use the otherwise toxic anticancer drugs at a lower dose. In this study, we supplemented Cisplatin with Crocin to reduce the amount of Cisplatin that could kill breast cancer cells. MCF-7 and MDA MB-231 breast cancer cells in culture were initially treated with Cisplatin and Crocin alone for 24, 48 and 72 hours using a spectrum of concentrations for each drug (Cisplatin: 1 to 100ug/ml; Crocin: 0.25 to 10mM) to assess the cell death by MTT assay. The concentration that induced fifty percent killing of the cancer cells was designated as IC50. FACS based Annexin V assay was used to confirm the results obtained from MTT assays. Using isobologram analysis we tested various combinatorial doses of Cisplatin and Crocin that were lower than IC50 to determine the extent of synergism displayed by the drug combinations. The formula used to ascertain the synergy index was: q= I(A+B)/ (IA+IB - IA*IB), where I(A + B) represented the inhibition rate of the Crocin + Cisplatin combination; IA and IB were the inhibition rate of Crocin and Cisplatin individually and respectively. If the drug combination showed q= 0.85 to 1.15, it was considered as simple addictive effect, while a value of q= 1.15 to 2.0, indicated that the drugs were showing a degree of synergism. q= >2.0 indicated significant synergistic effect between the two agents. Experimental data were statistically analyzed to ascertain significant p-value. In our experiments, we could reduce the effective dose of Cisplatin by supplementing it with Crocin, and with this combination, we could achieve enhanced killing of breast cancer cells that was until now only possible with higher doses of Cisplatin, when used alone. These results suggest that cancer patients can benefit from this study as plant based anti-cancer compounds can be used to reduce toxicity from higher doses of traditional anti-cancer drugs, but without compromising their effectiveness. Citation Format: Sachin Gupta, Bhawna Jhamb, Sanjay Katiyar. Crocin-supplemented cisplatin is highly effective in killing breast cancer cells than cisplatin alone. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4585. doi:10.1158/1538-7445.AM2014-4585

Assessment of cisplatin concentration and depth of penetration in human lung tissue after hyperthermic exposure.

The effects of cisplatin on the lung parenchyma during hyperthermic intrathoracic chemotherapy perfusion have not been analysed in detail. The objective of this study was to evaluate both the concentration and depth of the penetration of cisplatin in human lung tissue after hyperthermic exposure under ex vivo conditions. This experimental study was approved by the local ethics committee. Twelve patients underwent pulmonary wedge resections after elective thoracic lobectomies were performed (resected lobe), and the lung tissue (approximately 1-2 cm(3)) was incubated (in vitro) with cisplatin (0.05 mg/ml; 60 min, 42°C). Subsequent tissue beds (depth, 0.5 mm; median weight, 70-92 mg) were prepared from the outside to the middle, and the amount of cisplatin per tissue weight was analysed using atomic absorption spectrometry. Afterwards, the penetration of cisplatin depth was calculated and related to the different concentrations per tissue. Cisplatin penetrated into the human lung tissue after ex vivo hyperthermic exposure. The median amount of platinum [nmol cisplatin/g lung tissue] decreased significantly (P ≤ 0.05) depending on the penetration depth: 32 nmol/g (1 mm), 20 nmol/g (2 mm) and 6.8 nmol/g (4 mm). The calculated median concentrations of cisplatin (µg/ml) were 2.4 µg/ml (1 mm), 1.4 µg/ml (2 mm) and 0.5 µg/ml (4 mm), respectively. Under ex vivo hyperthermic conditions, cisplatin diffused into human lung tissue. The median penetration depth of the cisplatin was approximately 3-4 mm. The penetration of cisplatin into lung tissue may affect the local therapy of residual tumour cells on the lung surface using hyperthermic intrathoracic chemotherapy perfusion in patients with malignant pleural tumours.

Abstract 843: Influx of thiosemicarbazone NSC73306 is mediated by the cisplatin and copper transporter SLC31A1 (CTR1)

Abstract Multidrug resistance (MDR) mediated via the drug efflux pump P-glycoprotein (P-gp/ABCB1) overexpression is a major obstacle to the success of chemotherapy to combat many cancers. Clinically, P-gp expression correlates with poor clinical response to chemotherapy in patients, and its presence on the blood-brain barrier (BBB) can reduce the efficacy of otherwise promising agents targeted to the brain. Attempts to reverse drug resistance by inhibiting P-gp have not been successful, and we have been investigating alternative strategies for targeting multidrug-resistant (MDR) cancer cells. One such strategy is the use of NSC73306, a thiosemicarbazone compound that is selectively toxic towards P-gp-expressing cells. Here, we investigate the cellular uptake of NSC73306, and examine its interaction with P-gp and copper transporter 1 (SLC31A1, CTR1). In porcine kidney epithelial LLC-PK1 cells, cellular accumulation of NSC73306 is saturable, and overexpression of human P-gp in LLC-PK1 cells sensitizes them to NSC73306. Accumulation of [3H]NSC73306 in LLC-PK1 cells is inhibited by cyclosporin A (IC50 = 500 μM), verapamil (IC50= 700 μM) and cisplatin (IC50 = 77 μM). Among these compounds, we tested the effect of cisplatin on NSC73306. Selective toxicity of NSC73306 to P-gp-expressing LLC-PK1 cells was reversed by cisplatin in a dose-dependent manner (up to 40 μM). In contrast, the amount of cisplatin required to kill 50% of LLC-PK1 cells was not affected by increasing the amount of NSC73306 (up to 2μM). Since cisplatin could inhibit NSC73306 uptake, we hypothesize that these two compounds share a common uptake pathway, but cisplatin has additional routes of entry into cells. Cells transiently expressing CTR1 showed significantly higher [3H]NSC73306 accumulation. In contrast, CTR1 knockdown cells had lower [3H]NSC73306 uptake. Although cisplatin is a transport substrate of CTR1, we found that CTR1 protein was overexpressed in cisplatin-resistant KB-CP20 and BEL7404-CP20 cell lines. Compared to their parental cisplatin-sensitive cells, even though these resistance cells show decreased cisplatin accumulation, suggesting that CTR1 is not rate-limited for cisplatin uptake. These cell lines also showed higher [3H]NSC73306 uptake compared to their parental cells. In contrast, MDR cell lines overexpressing P-gp had unchanged CTR1 protein levels. Incubation with NSC73306 for 24 hours down-regulated CTR1 in a dose-dependent manner. These results suggest that NSC73306 is a transport substrate of CTR1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 843. doi:1538-7445.AM2012-843

Abstract 827: Development of stabilized homoglutathione disulfide as a novel nontoxic redox perturbing agent for increased anticancer efficacy

Abstract Human cancers harbor significantly higher levels of oxidative and nitrosative stresses than corresponding normal tissues due to increased metabolic load, accelerated rates of mitochondrial electron transfer, DNA damage, production of cytokines and constitutive expression of inducible nitric oxide synthase. A selective exacerbation of these stresses in tumors remains a viable anticancer strategy; however, this has not been exploited. Increased levels of glutathione disulfide reflects the redox imbalance resulting from various pathological stresses and leads to a plethora of adaptive signaling and cellular defense strategies for terminating apoptotic signals. Consistent with this theme, a glutathione disulfide mimetic termed ‘glutoxim’ (GSSG stabilized with minute amounts of cisplatin; NOV-002 in USA) has gone through extensive clinical trials in Russia, and has been shown to greatly sensitize tumors to anticancer therapies and offer benefits in the treatment of many other diseases. With the goal of obtaining a better, rational, and more powerful redox-perturbing compound, we used a glutathione analog called homoglutathione (HGSH) found in leguminous plants. HGSH differs from GSH in having a terminal beta-alanine in place of glycine (α-glutamyl-cysteinyl-α-alanine as compared to α-glutamyl-cysteinyl-glycine). Synthetic HGSH was mixed with cisplatin at 1000:1 and 1000:10 molar ratios and disulfide formation was induced using H2O2. We obtained total conversion of the thiol to HGSSG as verified by TLC. Stabilized HGSSG was approx. 3.5-fold less efficient as a substrate for glutathione reductase than glutoxim. Exposure of human cancer cell lines to stabilized homoglutathione (100 µM, 1-6 h) led to high levels of bulk protein glutathionylation, a reversible posttranslational modification that gauges the intracellular redox and transduces into functional responses. The new agent increased p53 and its target the p21cip1 protein in a time-dependent fashion in HCT116 cells. Overall phosphorylation of cellular proteins and phosphorylation of the stress kinase JNK1 were also markedly increased after short incubations with stabilized HGSSG. Further, p53 glutathionylation as determined with a specific antibody and the expression of the multifunctional DNA repair/redox modulatory protein APE1were markedly enhanced by this agent. The increased expression of APE1 appears to reflect the involvement of the cytoprotective transcription factor NRF2 in redox modulation. Data on the potentiation of anticancer drugs by stabilized HGSSG and its chemoprotective effects in animal studies will be presented. In summary, the ability of the new GSSG mimetic to alter extracellular and intracellular redox signaling pathways may help to markedly improve/ modulate the various anticancer regimens (supported by RO3 CA125872 and Association for Research of Childhood Cancer). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 827. doi:1538-7445.AM2012-827

GC Determination of Cisplatin in Serum and Urine of Cancer Patients after Chemotherapy as Platinum (II) Pyrrolidinedithiocarbamate Chelate

A gas chromatographic procedure has been developed for determination of cisplatin from pharmaceutical preparation, serum and urine after chemotherapy of cancer patients as platinum(II) pyrrolidinedithiocarbamate chelate. The elution was carried from the column DB-1701 (30 m × 0.32 mm i.d.) coupled with FID detection. Cu(II), Ni(II), Co(III), Mn(II), Fe(III), Zn(II) and VO(II) when present together with Pt(II) separated completely and did not affect the determination of platinum. The linear calibration curve for platinum (II) was within 1–30 μg mL−1 with a detection limit of 300 ng mL−1. The amount of cisplatin detected from serum and urine was 250–325 and 20–116 ng mL−1 with relative standard deviations (RSD) of 0.8–1.2%, and 0.9–1.2%. The % recovery of Pt from serum and urine by standard addition was 98 and 98.2% with RSD 1.4 and 1.1%.

Transscleral permeability and intraocular concentrations of cisplatin from a collagen matrix

This study determined the in vitro permeability of cisplatin through isolated human sclera delivered by a collagen matrix vehicle. Short-term and long-term levels of cisplatin were also measured in the rabbit eye after a subconjunctival injection. Cisplatin in either a collagen matrix or a control balanced salt solution (BSS) vehicle was applied to human sclera mounted in a perfusion chamber. The amount of cisplatin that diffused across the sclera was measured hourly for 24 hours using atomic absorption spectrometry. Studies of conjunctival injections of cisplatin in rabbits included determination of intraocular cisplatin levels. Cisplatin had a peak in vitro scleral permeability constant of 8.3 ± 1.2 × 10−6 and 20.1 ± 1.8 × 10−6 cm/s, delivered in collagen matrix and BSS, respectively (mean ± SD). At the end of the in vitro experiments, 35.9 ± 4.6% of the cisplatin remained in the collagen matrix, while 0.8 ± 0.2% remained in the BSS vehicle. Subconjunctival injections of cisplatin in the collagen matrix achieved 3.3 ± 0.1 μg/mg in the vitreous at 1.5 hours and 0.1 ± 0.1 μg/ml at 2 weeks. The collagen matrix preparation also achieved a cisplatin concentration of 73.5 ± 23.9 μg/mg in the choroid and retina at 1.5 hours and 3.2 ± 1.3 μg/mg at 2 weeks. Compared to BSS, the collagen matrix vehicle provided a more controlled release of cisplatin, and after subconjunctival injections in rabbits, attained higher drug levels in several ocular tissues.—Hans E. Grossniklaus

Transscleral permeability and intraocular concentrations of cisplatin from a collagen matrix

This study determined the in vitro permeability of cisplatin through isolated human sclera as delivered by a collagen matrix vehicle. Short-term and long-term intraocular levels of cisplatin were also measured in the rabbit eye after a subconjunctival injection. Cisplatin in either a collagen matrix vehicle or a control balanced salt solution (BSS) vehicle was applied to human sclera mounted in a specially designed in vitro perfusion chamber. The amount of cisplatin that diffused across the sclera was measured in hourly samples for 24 h using atomic absorption spectrometry. In vivo studies were also performed in Dutch Belted rabbits given subconjunctival injections of cisplatin in collagen matrix or in BSS. Eyes were enucleated at 1.5 h and 2 weeks after injection, frozen, and dissected to determine the intraocular cisplatin concentrations. Cisplatin had a peak in vitro scleral permeability constant of 8.3+/-1.2 x 10(-6) and 20.1+/-1.8 x 10(-6) cm/s, delivered in collagen matrix and in BSS, respectively (mean+/-S.D.). At the end of the in vitro experiments, 35.9+/-4.6% of the cisplatin remained in the collagen matrix, while 0.8+/-0.2% remained in the BSS vehicle. Subconjunctival injection of cisplatin in the collagen matrix vehicle achieved 3.3+/-0.1 microg/ml in the vitreous humor at 1.5 h and 0.1+/-0.1 microg/ml at 2 weeks. This vehicle also achieved a cisplatin concentration of 73.5+/-23.9 microg/mg in the choroid and retina at 1.5 h and 3.2+/-1.3 microg/mg at 2 weeks. Compared to BSS, the collagen matrix vehicle provided a more controlled release of cisplatin, and after subconjunctival injection into rabbits, attained higher drug levels in several ocular tissues.

Transscleral permeability and intraocular concentrations of cisplatin from a collagen matrix

This study determined the in vitro permeability of cisplatin through isolated human sclera as delivered by a collagen matrix vehicle. Short-term and long-term intraocular levels of cisplatin were also measured in the rabbit eye after a subconjunctival injection. Cisplatin in either a collagen matrix vehicle or a control balanced salt solution (BSS) vehicle was applied to human sclera mounted in a specially designed in vitro perfusion chamber. The amount of cisplatin that diffused across the sclera was measured in hourly samples for 24 h using atomic absorption spectrometry. In vivo studies were also performed in Dutch Belted rabbits given subconjunctival injections of cisplatin in collagen matrix or in BSS. Eyes were enucleated at 1.5 h and 2 weeks after injection, frozen, and dissected to determine the intraocular cisplatin concentrations. Cisplatin had a peak in vitro scleral permeability constant of 8.3±1.2×10 −6 and 20.1±1.8×10 −6 cm/s, delivered in collagen matrix and in BSS, respectively (mean±S.D.). At the end of the in vitro experiments, 35.9±4.6% of the cisplatin remained in the collagen matrix, while 0.8±0.2% remained in the BSS vehicle. Subconjunctival injection of cisplatin in the collagen matrix vehicle achieved 3.3±0.1 μg/ml in the vitreous humor at 1.5 h and 0.1±0.1 μg/ml at 2 weeks. This vehicle also achieved a cisplatin concentration of 73.5±23.9 μg/mg in the choroid and retina at 1.5 h and 3.2±1.3 μg/mg at 2 weeks. Compared to BSS, the collagen matrix vehicle provided a more controlled release of cisplatin, and after subconjunctival injection into rabbits, attained higher drug levels in several ocular tissues.

Polymer chemotherapy for head and neck cancer.

To study a new method of delivery of chemotherapy for the treatment of squamous cell carcinomas (SCCs) of the head and neck, to evaluate the pharmacokinetics of four anticancer agents (cisplatin, fluorouracil [5-FU], methotrexate [MTX], and paclitaxel) loaded into the biodegradable polymer, polyanhydride polymer poly(FAD:SA), and to evaluate the effectiveness and toxicity of the drug-polymer combination against human SCCs, both in vitro and in vivo. Poly(FAD:SA) was loaded with different chemotherapeutic drugs and its in vitro and in vivo drug release and tissue penetration characteristics were studied. The biocompatibility and toxicity of the polymer-drug combination were determined. The effectiveness of the drug-polymer was evaluated against three different human SCCs (larynx O11, pharynx FADU, and floor of mouth UM- SCC1) cultured in vitro and in nude mice carrying human SCC xenografts. The in vitro drug release pharmacokinetics of the drugs were performed using atomic absorption spectrometry for cisplatin and high-pressure liquid chromatography for the 5-FU, MTX, and paclitaxel studies. In vitro tumor cytotoxicity was assessed by growth assay. In vivo cytotoxicity was assessed by growth rate inhibition in a nude mouse model. All four chemotherapy drugs demonstrated a continuous release that followed first-order kinetics from the polymer. More than 95% of the MTX and 5-FU, 70% of the cisplatin, and 20% of the paclitaxel was released within the 10 days of the assay. Tumor cytotoxicity revealed that the polymer was very effective against the human SCCs O11, FADU, and UM- SCC1 in vitro. When a small amount of polymer (1-2 g) was added to the cell culture and left for 7 days, 96.6% of the UM-SCC1 cells, 86.9% of the FADU cells, and 94.6% of the O11 cells were killed. When the culture medium was then changed every 2 days to remove the effect of nutrient depletion or chemicals released by the degrading polymer, 74% of the UM-SCC1 cells, 94.5% of the FADU cells, and 66.1% of the O11 cells were killed at 7 days. The tumor animal model was the nude mouse carrying human floor of mouth SCC xenografts. Different amounts of cisplatin were incorporated into the polymers (5% and 7% drug/polymer at a weight/weight [wt/wt] load). Thirty-five days after implantation of the polymer in nude mice, the mean treated tumor size was 65.5% of controls in the 5% group and 31.8% in the 7% group. Seventy days after implantation the mean treated tumor size was 41.4% of controls in the 5% group and 38.1% in the 7% group, indicating a statistically significant delay of tumor growth compared with controls or with intraperitoneally injected cisplatin. The blank polymer was well tolerated by the mouse and had no effect on tumor growth. The study results indicate that polymer chemotherapy is effective against a variety of SCCs of the head and neck, both in vitro and in vivo, and may become a useful therapeutic option for head and neck cancer.