ANTICANCER RESEARCH 34: 455-464 (2014)

Review Dicycloplatin, a Novel Analog in : Synthesis of Chinese Pre-clinical and Clinical Profile and Emerging Mechanistic Studies

JING JIE YU1, XUQING YANG2, QINHUA SONG2, MICHAEL D. MUELLER1 and SCOT C. REMICK1

1Mary Babb Randolph Cancer Center and Molecular Medicine Core Facility, Department of Biochemistry, School of Medicine and Department of Basic Pharmaceutical Sciences, School of Pharmacy, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, U.S.A.; 2Sopo-Xingda Pharmaceutical, Inc., Beijing/Jiang-Su, P.R. China

Abstract. Dicycloplatin (DCP) has better solubility and Dicycloplatin is a novel platinum analog developed in China. stability than both and . Pre-clinical and It was approved by the State Food and Drug Administration phase I studies demonstrated significant antitumor activity and (SFDA) of China in 2012. This compound is in the midst of fewer adverse events than carboplatin. Phase II clinical trials pre-clinical and early-phase clinical evaluation in China. in advanced non-small cell lung cancer found efficacy and Herein we review findings that have appeared in the Chinese safety of DCP-plus- comparable to carboplatin-plus- medical literature and report our mechanistic studies. paclitaxel but better tolerability. This article summarizes and reviews pre-clinical and clinical data for dicycloplatin from DCP: Chemical Structure and Properties the Chinese medical literature. We also report on new mechanistic findings in our laboratory in West Virginia, USA. DCP is a platinum-based compound synthesized from Patient blood samples were collected for DCP-prototype platinum powder through 10 steps. The host part of DCP is determination by liquid chromatography mass spectrometry carboplatin and the guest part is 1,1-cyclobutane (LC-MS/MS). Molecular studies of ovarian cancer cells dicarboxylic acid (CBDCA); they are linked by hydrogen treated with DCP or cisplatin were carried out for gene- bonds (Figure 1). DCP is a hydrogen-bond supramolecule; signature profiling using immunoblotting. Pharmacokinetic carboplatin is a covalent-bond molecule. This may explain mass-spectrometry showed different spectrum profiles of DCP why DCP seems to have a more stable chemical structure, and carboplatin in plasma. Plasma concentration of DCP good water solubility, and a better safety profile than prototype was 17.1 μg/ml 2h after administration, with a peak carboplatin (1). The crystal structure of DCP has been concentration of 26.9 μg/ml at 0.5 h. Immunoblotting showed determined. In aqueous solution, using electrospray DCP-induced activation of DNA damage pathways, including ionization mass spectrometry (ESI-MS), a model of the double-phosphorylated checkpoint kinase 2 (CHK2) and structure of DCP was soluble and stable (1). No chemical breast cancer 1 (BRCA1) and triple-phosphorylated p53, decomposition of DCP in aqueous solution was observed compared to controls. Cisplatin produced a similar profile, after several years of storage at room temperature. with increased p53 protein. DCP and cisplatin activate DNA- damage response through similar pathways. DCP may be Pre-clinical Pharmacology more soluble and stable, and better-tolerated. Early in vitro cytotoxicity studies of DCP in China demonstrated significant antitumor activity against a variety of human cancer cell lines, including BCG-823, BEL-7402 Correspondence to: Jing Jie Yu, MBRCC, West Virginia University, and EJ with an IC range of 25-30 μM (2-5). Studies of 2312 HSC, P.O. Box 9300, Morgantown, WV 26506-9300, U.S.A. 50 DCP-induced in PC3 cells and Tel: +1 3042938661, Fax: +1 3042934667, e-mail: [email protected] lung cancer NCI/H446 cells showed that DCP can induce Key Words: Dicycloplatin, SFDA approval, platinum compound, cell-cycle arrest, inhibit proliferation and lead to apoptosis cancer chemotherapy, review. (6-8). In a study of platinum DNA damage caused by

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Figure 1. The chemical structure of dicycloplatin and a computer- Figure 2. In vivo comparison of therapeutic efficacy of dicycloplatin generated structural model of the dicycloplatin supramolecule in (DCP) and carboplatin in A549 NSCLC xenografts. a: DCP and aqueous solution. Illustrative representation of the data originating from carboplatin in treatment of A549 NSCLC xenografts. b: Images of mice Yang et al. (1). treated with DCP compared to controls. Data provided by Sopo-Xingda Pharmaceutical, Inc. cisplatin, carboplatin and DCP, researchers found the Sprague-Dawley (SD) rats significantly less than cisplatin, breaking potency on pBR322 plasmid DNA to be with similar episodes of bone-marrow suppression as those cisplatin>DCP>carboplatin (9). found with carboplatin (12, 13). In vivo evaluation of DCP DCP has also shown in vivo anti-tumor effects against the toxicity suggests that DCP causes the least damage to major experimental A549 human non-small cell lung cancer target organs of rats, and there exist the usual platinum- (NSCLC) tumor xenograft model, with 67-90% inhibition of associated problems with pregnancy (14, 15). These reports tumor growth compared to 51-63% by carboplatin (Figure 2) provided a generally favorable safety profile for DCP, (5, 10, unpublished data). In summary, DCP shows supporting the possibility for its clinical evaluation. significant antitumor activity. These data provide a favorable pre-clinical profile for DCP. Pre-clinical

Pre-clinical Toxicology Pharmacokinetic studies of DCP in comparison to cisplatin and carboplatin in rats suggest that DCP and carboplatin have Acute toxicity was investigated by single intravenous and faster renal than cisplatin, and transfer proteins may intraperitoneal administrations of different doses. The results be involved in excretion of DCP and its metabolites (16). indicate that the toxicity profile of DCP [Lethal dosage Substrates of drug transporters, such as probeneside and (LD50)=210 mg/kg] is higher than that of carboplatin (164 verapamil, can reduce the renal excretion of platinum if used mg/kg) and cisplatin (14.27 mg/kg) (5, 11). Studies of in combination with DCP. P-glycoprotein [P-gp] organic cytotoxicity and hematological toxicity among DCP, cisplatin anion transporter (OAT) or organic anion-transporting and carboplatin found that DCP induced renal toxicity in polypeptide (OATP) may be involved in the excretion of DCP

456 Yu et al: Dicycloplatin in Cancer Chemotherapy (Review)

Table I. Plasma pharmacokinetic parameters in rats and dogs following dicycloplatin administration.

Preclinical pharmacokinetic parameters of dicycloplatin

Dose Cmax AUCinf T1/2α T1/2β Vc Cl (mkg) (μg/mL) (μg×h/mL) (h) (h) (L/kg) (L/kg/h)

10 54 38.8 0.27 11.9 0.17 0.26 20 130 80.4 0.24 10.8 0.16 0.25 40 255.3 176.6 0.25 11.6 0.16 0.22 5 20 41.7 0.56 18.1 0.28 0.123 10 37.4 81.1 0.72 22.8 0.326 0.14 20 81.6 146.2 0.58 21.6 0.318 0.154

Note: Rats data (first 3 lines); Dogs data (last 3 lines). Cmax: maximum plasma concentration. AUCinf: area under the curve from time 0 to infinite; T ½ α: early distribution half-lives; T ½ β: late elimination half-lives; Vc: volume of distribution; Cl: clearance. Data provided by Sopo-Xingda Pharmaceutical, Inc.

(17). A pharmacokinetic study of DCP in dogs reported that the maximum plasma concentration (Cmax), the areas under concentration (AUC) and dosage appeared linearly-correlated, and the summarized plasma pharmacokinetic parameters in rats and dogs following DCP treatment are shown in Table I. Zhao et al. reported findings in rats and dogs that plasma concentration of platinum originating from DCP decreased rapidly within the first 4h and declined slowly long-term (Figure 3a,b). This study also showed distribution of DCP with the highest concentration in plasma and some in intestine and prostate (Figure 3c) (18).

Early-Phase I Clinical Trial

In the early clinical phase I and phase II trials, all patients signed informed consent according to Chinese regulatory guidelines (19). A phase I clinical and pharmacokinetic study to determine efficacy and safety was conducted at State Key Laboratory of Oncology in Southern China (20). DCP was administered to 29 patients with cancer at escalating doses from 50 mg/m2 to 650 mg/m2. Pharmacokinetic analysis was performed for 26 patients to determine the total and ultrafiltered platinum concentrations in plasma. The results demonstrated a linear pharmacokinetics characterized by distribution half-life of Figure 3. Pharmacokinetics of platinum originating from dicycloplatin in rats and dogs. a: Concentration–time profiles of platinum originating 1.49±0.39 h and low steady-state apparent volume of from dicycloplatin in plasma following intravenous administration in male 2 distribution (177.88±44.92 l/m ). The maximum tolerated dose rats (dicycloplatin, 10 mg/kg) (mean±SD; n=6). b: Concentration–time of DCP was 550 mg/m2. The terminal plasma half-life of total profiles of platinum originating from dicycloplatin in plasma following platinum ranged from 41.86 to 77.20 h without significant intravenous administration in male dogs (dicycloplatin, 5 mg/kg) dose dependency; however, the half-life of free platinum (mean±SD; n=6). c: Tissue distribution of DCP in rats. Mean concentrations of platinum in plasma and tissues at 6, 30 and 120 min ranged from 42.34 to 61.07 h. In conclusion, DCP displayed after intravenous administration of dicycloplatin at a dose of 60 mg/kg 2 a favorable safety profile at doses between 50 mg/m and (mean±SD, n=5). Illustrative representation of the data originating from 550 mg/m2 and first efficacy signals were observed. Dose- Zhao et al. (18).

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Figure 4. Dicycloplatin in the treatment of lung cancer with brain metastasis. Data provided by Sopo-Xingda Pharmaceutical, Inc.

Figure 5. Dicycloplatin may pass the blood-brain barrier demonstrated by chemosensitivity assay in brain tissue (data provided by Sopo-Xingda Pharmaceutical, Inc) and by theoretical calculation.

limiting toxicities were , anemia and emesis. using DCP plus paclitaxel, compared to carboplatin plus The recommended starting dose for subsequent phase II paclitaxel, was approved by Chinese FDA (19, 22, 23). These studies is 450 mg/m2 once every three weeks (20). multi-center trials involved 15 hospitals nationwide and enrolled 138 patients who met the criteria (22). Patients were Phase II Clinical Trials randomly assigned to the experimental group (DCP-plus- paclitaxel) or control group (carboplatin-plus-paclitaxel). The first phase II clinical trial of DCP followed 39 patients Unfortunately, there is no overall report; separate with small cell lung cancer was conducted in 2003. Liu and institutional articles were published by Cancer Center of Sun colleagues reported an overall improved effect and tolerance Yat-Sen University (32 patients) in 2009 (19) and Jiang-Su for the novel platinum drug (21). Tumor Hospital (20 patients) in 2011 (23). In both reports, the In Jan 2007, randomized multicenter phase II clinical trials two arms were balanced with regard to gender, age, ECOG among chemotherapy-naïve patients with advanced NSCLC (Eastern Cooperative Oncology Group) performance status,

458 Yu et al: Dicycloplatin in Cancer Chemotherapy (Review)

Figure 6. Mass spectrometry for dicycloplatin and carboplatin. a: dicycloplatin spectrum. b: carboplatin spectrum. disease stage and histological classification. The experimental group was given DCP at 450 mg/m2 plus paclitaxel at 175 mg/m2 q3w; the control group received carboplatin AUC=5 plus paclitaxel at 175 mg/m2 q3w. Response rate and adverse reactions were evaluated according to the RECIST criteria with survival follow-up. Results from the Sun Yat-Sen University showed a 1-year survival rate of 54.8% in the DCP arm versus 20.1% in the control arm (p=0.028) (19). At the Jiang-Su Tumor Hospital, the median progression-free survival time was 4.1 months in the control arm and 4.9 months in the DCP arm (p<0.05); there was no significant difference in the response rate, median overall survival time and 1-year survival rate between the two arms. However, the 3-year survival rate was 22.2% in the DCP group, versus 11.1% in the control group (23). Adverse reactions in these trials included myelosuppression and gastrointestinal reactions. Lympho- cytopenia and bone-marrow suppression were more frequent in the control group. No significant difference was found for other adverse reactions between the two groups. There was one report that the adverse reactions caused by DCP resulted in pancytopenia (24). Researchers concluded that efficacy and safety of the DCP-plus-paclitaxel regimen were comparable to Figure 7. Mass spectrometry demonstrates a high level of prototype of DCP in plasma 2-h after administration. Thirty minutes after DCP those of carboplatin plus paclitaxel, with better tolerance of administration, peak plasma concentration of DCP is 26.9 μg/ml. Two DCP-plus-paclitaxel among chemotherapy-naïve patients with hours after administration, 17.1 μg/ml of prototype DCP was measured advanced NSCLC (19, 22, 23). in patient plasma, compared to 5.01 μg/ml of carboplatin.

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Figure 8. DNA-damage response induced by cisplatin (CDDP) and dicycloplatin (DCP) in ovarian cancer A2780 cells. Cells were treated with cisplatin or dicycloplatin for 1 h. Cells were washed and re-incubated in drug-free media for the indicated times. Proteins were extracted, separated by polyacrylamide gel electrophoresis (PAGE), transferred onto PVDF membrane, and probed with the indicated antibodies.

A Case Report that Hints DCP Can Cross shown in Figure 5 (right panel), DCP sensitivity in glioma the Blood–Brain Barrier (BBB) tissues is much higher compared to other chemotherapeutic agents (data provided by Sopo-Xingda Pharmaceutical, Inc.). A case report was provided by Sopo-Xingda Pharmaceutical, Worth noting here, Zhao et al. reported that a visible Inc. In brief, a 42-year-old male with lung cancer (upper apical distribution of platinum originating from DCP was seen in region of right lung) was diagnosed in June 2006 at Beijing brains of rats (18). Theoretically, a larger polar surface area Hospital based on Positron Emission Tomography/Computerized (PSA) hints at greater solubility and a higher calculated logP Tomography (PET/CT). The patient showed significantly (ClogP) value predicts a better permeability than a smaller increased metabolic activity of the mass and high SUV PSA and logP (25-27). Therefore, we calculated the PSA and (standardized uptake value) at delayed imaging. Later, the cancer logP for cisplatin, carboplatin and DCP. As shown in Figure spread to lymph nodes, mediastina and meninges. An encephalic 5 (left panel), DCP has the largest PSA and the largest tumor was also found. A regimen of large-dose DCP (1200 mg ClogP. Thus, DCP seems to have better solubility and by i.v. q3w) and oral Iressa was begun as adjuvant treatment to permeability based on these characteristics, with greater support cranial radiotherapy at PLA General Hospital in Beijing. likelyhood of crossing the BBB. Thus, DCP has the greater This regimen continued for four months with five high-dose likelihood of crossing the BBB. treatments of DCP and whole-brain irradiation to 38 Gy in 19 Still, mechanistic questions remain. What is the effect- daily fractions, followed by a boost to the lesion area with driving part of the compound? Is DCP hydrolyzed to additional 22 Gy delivered in 11 daily fractions. During carboplatin inside the body? What is its mechanism of treatment, the patient developed slight leucopenia and action? To address these questions, we conducted molecular thrombocytopenia which were resolved by symptomatic mechanism studies in our laboratory in light of the clinical treatment. The patient was discharged on 2/1/2008 with normal pharmacokinetic studies of DCP in China. Our findings are white blood cell count, platelet count, biochemistry, and blood presented in the following section. coagulation tests. Magnetic resonance imaging (MRI) on 1/25/2008 showed no brain lesion (Figure 4). Patients and Methods That successful treatment suggested the possibility that DCP crosses the BBB. In vitro data of histoculture drug Researchers at the Cancer Center of Sun Yat-Sen University, Guangzhou, response assay [MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5- China analyzed plasma level of DCP prototype in four patients. Patients diphenyltetrazolium bromide] in various tumor specimens gave informed consent according to Chinese regulatory guidelines (19). demonstrated DCP chemosensitivity in brain tissue. As Patients received 450 mg/m2 of DCP by i.v. within 1-h. Blood samples

460 Yu et al: Dicycloplatin in Cancer Chemotherapy (Review) were collected 30-min prior to DCP treatment and at 30-min, 1-h, TBS and 0.1% Tween 20). Membranes were then incubated with 1.5-h and 2-h afterwards. The whole blood samples were mixed and primary antibodies overnight at 4˚C. The primary antibodies applied immediately centrifuged for 2 min at 1,000 × g to yield plasma. were against ataxia telangiectasia mutated (ATM), p-p53 Aliquot of 0.2 ml of plasma samples was added with 0.2 ml ice-cold phosphoserine-15, p53, CHK2 phosphothreonine-68, anti-CHK2, acetonitrile; the mixtures were stored at −80˚C. BRCA1 phosphoserine-1497, BRCA1, BRCA2, r-H2AX (H2A histone family member X), p48 and p21. To demonstrate equal Pharmacokinetic mass spectrometry. Preparation of standard drug loading of each sample, membranes were re-probed using antibody solution: 8.1 mg of DCP (Beijing Sopo-Xingda, China) and 12.5 mg to β-actin (Sigma-Aldrich Corp. St. Louis, MO, USA). The signals of carboplatin were added to 75% and 50% methanol, respectively, of immunoreactive proteins were visualized using horseradish and ultracentrifuged for 3 min, each mixed for 5 min to obtain peroxidase-conjugated sheep anti-mouse or donkey anti-rabbit concentrations of 810 μg/ml for DCP and 1,250 μg/ml for antibodies and enhanced by Chemiluminescence ECL detection carboplatin. Aliquots of DCP and carboplatin were stored at −20˚C system (Amersham International PLC, Buckinghamshire, UK). for instrument operation. Prior to analysis, 0.2 ml of plasma was added to 0.2 ml ice-cold Results acetonitrile, then mixed and centrifuged at 14,500 × g for 10 min; 0.1 ml of supernatant was then diluted with 0.7 ml of ice-cold In China, prototype concentrations of DCP were determined acetonitrile, with 10 μl used for the analysis. AP-4000™ LC- using AB-4000™ LC-MS/MS instrumentation. Carboplatin MS/MS instrumentation was used to determine the prototype level prototype level was also determined. The results of DCP. demonstrated that the spectrum-profiles of DCP and Molecular mechanistic studies at WVU Cancer Center are carboplatin are different, with a peak plasma-concentration summarized below. for DCP molecule at 514.0 and a peak plasma concentration for the carboplatin molecule at 372.0 (Figure 6). DCP Cell culture and drug treatment. Human ovarian cancer A2780 cells were propagated as an adherent monolayer in RPMI-1640 medium prototype levels in blood samples from four patients who supplemented with 10% heat-inactivated fetal bovine serum (FBS), received DCP treatment were also analyzed. In plasma, 2-h 100 μg/ml penicillin and 100 μg/ml streptomycin (Life after DCP administration, 17.1 μg/ml of prototype DCP was Technologies, Grand Island, NY, USA). Cells were grown at 37˚C in still present following a peak concentration of 26.9 μg/ml at a humidified atmosphere consisting of 5% CO2, 95% air and 0.5-h. In comparison, 5.01 μg/ml of carboplatin was seen 2- allowed to grow to 80% confluence. For drug treatment h after drug administration (Figure 7). experiments, cells were plated the day before exposure to drugs. At WVU, for in vitro mechanistic investigations, we Cisplatin (Sigma-Aldrich, St. Louis, MO, USA) was prepared fresh by first dissolving it in phosphate-buffered saline (PBS), treated ovarian cancer cells with cisplatin or DCP, then without Ca++ or Mg++, at a concentration of 1 mg/ml, then diluting allowed cells to grow for various periods of time before it into pre-warmed media to achieve the IC50 dose of 5-day survival isolation for cellular proteins to be probed with antibodies (3 μM). DCP (>99.0% purity; Sopo-Xingda, Beijing, China) was selected from the DNA-damage repair signaling pathways. prepared by first dissolving it in water and then diluting it in pre- Results of immunoblotting showed DCP-induced warmed media to achieve the concentration of 500 mg/ml. Plated activation of the DNA damage response pathways, including cells were allowed to grow for 24-h and treated with cisplatin or phosphorylations of CHK2 at threonine 68, p53 at serine 15, DCP for 1-h. At the end of 1-h exposure to drug, cells were washed twice with PBS, and then incubated with drug-free media for a and BRCA1 at serine 1497. The increases in p-Chk2, p-p53 required period of time. and p-BRCA1 after 1-h DCP exposure occurred in a time- dependent manner. Quantitative analysis of phosphorylated Protein extraction and immunoblotting. Treated and untreated cells CHK2, p53 and BRCA1 expression showed that the amount were extracted with whole cell lysis buffer [20 mM Tris-HCl (pH of DCP-induced phosphorylation of CHK2 and BRCA1 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 doubled at 48-h and tripled for phosphorylated p53 at 24-h, mM sodium pyrophosphate, β-glycerophosphate, 1 mM Na3VO4, 1 compared to controls (Figure 8, right panel). p53 protein mM phenylmethylsulfonyl fluoride (PMSF), 10 μg /ml leupeptin, expression did not seem changed after DCP treatment; 10 μg/ml aprotinin and 5 μg/ml pepstatin] for 30 min before centrifugation (at 16,000 × g for 30 min at 4˚C). Supernatant was however, an increase in p21 expression was seen at 48 h. collected as whole-cell lysate for western blot analysis. Protein Activations induced by cisplatin in the DNA-damage concentrations of extracts were determined by Bio-Rad Protein response pathways were also observed. After 1-h cisplatin Assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum exposure at IC50 dose, we observed increased protein albumin as standard. expressions of ATM and r-H2AX. The peak of CHK2 The whole-cell lysates were separated on 12% sodium dodecyl phosphorylation at Thr-68 was seen at 48 h. At 12 h after sulfate (SDS)-polyacrylamide gels and transferred to PVDF cisplatin treatment, the p53 protein level was high, (polyvinyl difluoride) membrane (Bio-Rad) using standard electrophoresis and electroblotting procedures. Pre-stained accompanied by p53 phosphorylation at serine 15. In molecular weight markers were purchased from Invitrogen addition, cisplatin-mediated activation of p53 resulted in (Carlsbad, CA, USA). To reduce non-specific binding, blots were activation of downstream proteins p48 and p21 (Figure 8, pre-incubated for 1-h in a blocking buffer (5% nonfat dry milk, 1× left panel).

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Discussion Dr. Chu-Biao Xue (Incyte Corp, USA) for help in calculating PSA and logP for platinum compounds; Dr. Su Li (Sun Yat-Sen University, As the structure of DCP is composed of carboplatin and China) for prototype-drug measurement; and Dr. Xiaobing Liang (WVU, USA) for western blot experiments. CBDCA, it is important to consider if DCP is hydrolyzed to carboplatin inside the body. Chinese researchers measured References prototype DCP concentration in patient blood samples after DCP administration, with carboplatin plasma concentration 1 Yang XQ, Jin XL, Song QH, Tang KL, Yang ZY, Zhang XF and as comparison. The results showed that the majority of DCP Tang YQ: Structural studies of dicycloplatin, an antitumor is still present as prototype DCP in plasma 2-h after supramolecule. Sci China Chem 53: 1346-1351, 2010. administration, with a small portion of carboplatin hydrolyzed 2 Yan DM, Tu LL, Peng XY, Li WJ and Shen ZR: In vitro and in from DCP during drug metabolism. Unfortunately, there was vivo antitumour activity of dicycloplatin, a new active platinum no quantified ratio of DCP conversion to carboplatin. Further complex. Chin J Mod Appl Pharm 24: 352-356, 2007. 3 Liu T, Su H, Fang JC, Wang NQ, Yang XQ and Cui WC: Studies evidence, including clinical pharmacokinetics data in more on the cooperative anticancer effects of dicycloplatin and 8 patient samples, is needed. chemotherapeutic drugs in mice transplanted hepatoma 22 or Li and colleagues report that DCP inhibited the sarcoma 180. Chin J Mod Clin Med 2: 1-3, 2003. proliferation of cancer cells and increased the percentage of 4 Yan DM, Tu LL, Zheng XL Qian BC: Apoptosis Induced by apoptosis, including (ROS) (8). Dicycloplatin in Human Ovarian Cell Line A2780. J Oncol 15: Their observations include collapse of mitochondrial 667-670, 2009. membrane potential, release of cytochrome c from the 5 Yang XQ, Guan ZZ, Song QH, Li S, Jin XL, Yang ZY, Cui WC, Wang NQ, Fang JC and Liu CN: Dicycloplatin, a Novel Drug mitochondria to the cytosol, and upregulation of p53, which for Cancer Chemotherapy. Presentation in Beijing, China, 2007. were accompanied by activation of caspase-9, caspase-3, 6 XU MW, ZHANG J, XU CG, WANG W: Apoptosis induced by caspase-8, and poly (ADP-ribose) polymerase cleavage. They dicycloplatin in human androgen independent prostate cancer concluded that DCP induces apoptosis through reactive line PC3. J Mod Urol 17: 541-544, 2012. oxygen species stress-mediated death receptor pathway and 7 Wang C and Yan RF: Assay of the lung cancer cell apoptosis mitochondrial pathway, which is similar to carboplatin. It induced by dicycloplatin. Pract Oncol J 19: 21-22, 2005. would be interesting to confirm if this is the pathway for all 8 Li GQ, Chen XG, Wu XP, Xie JD, Liang YJ, Zhao XQ, Chen platinum drug-induced apoptosis. WQ and Fu LW: Effect of dicycloplatin, a novel platinum chemotherapeutical drug, on inhibiting cell growth and inducing Our studies of DNA-damage response induced by DCP, cell apoptosis. PLoS ONE 7: 1-13, 2012. compared to those induced by cisplatin, demonstrate that 9 Ma HZ, Liu ZX, Wang QJ, Yan CH and liao MY: The breaking several major kinases of the DNA-damage response effects of cisplatin, carboplatin and dicycloplatin on pBR322 pathways are activated by the two platinum drugs. These plasmid DNA. Carcinog Teratogen Mutagen 15: 220-222, 2003 observations suggest that DCP and cisplatin share similar 10 Liu T, Su H, Fang JC, Wang NQ, Yang XQ and Cui WC: A mechanisms in platinum-induced DNA damage and cellular comparison between dicycloplatin and carboplatin anticancer response, indicating DCP is a platinum analog (28-32). effect in animal models. Chin J Basic Clin Med 9: 36-38, 2003. 11 Yan CH, Shi C, Li YF and Liao MY: Comparison of toxicities Meanwhile, of note, we found that DCP does not increase of dicycloplatin with cisplatin and carboplatin. Chin J New p53 protein level in DCP-treated cells. That is different from Drugs 14: 1156-1159, 2005. what we observed in the same cisplatin-treated cell model. 12 Wang C and Yu T: Comparative studies of cytotoxicity and Further investigation is needed to determine if the difference hematologic toxicity induced by cisplatin, carboplatin and between cisplatin and DCP are relevant to clinical outcomes. dicycloplatin in mice. Med J Liaoning 18: 273-274, 2004. In summary, improving therapeutic efficacy and reducing 13 Wang NQ, Fang JC and Liu T: Toxicity studies of new adverse reactions associated with platinum chemotherapy are anticancer drug, dicycloplatin. Proc Chin Pharmacol 18: 30-31, primary goals of cancer research (33, 34). Developing new 2001 (in Chinese). 14 Lin F, Lu L, Yang XQ, Cui WC and Wang FM: The comparison chemotherapeutic agents with higher efficacy and lower study of major target organ hurt induced by dicycloplatin and its toxicity is an important strategy. DCP seems to have unique derivates in rats. Carcinogen Teratogen Mutagen 17: 276-279, 2005. chemotherapeutic properties and may pass the BBB and 15 Lin F, Feng FL, Yang XQ, Cui WC and Wang FM: In vivo prostate lipid membrane. Further investigation of molecular evaluation of dicycloplatin toxicity on pregnancy in rats. Chin J mechanisms of the three platinum agents (cisplatin, carboplatin Toxicol 19: 72-73, 2005. and DCP) is underway. 16 Li XQ, Chen XJ, Ren WC, Zhang H, Gu X and Wang GJ: Effects of probenecid, , digoxin and verapamil on Acknowledgements the renal excretion of dicycloplatin in rats. J China Pharmaceut Univ 37: 447-449, 2006. We acknowledge the Molecular Medicine Core Facility, Mary Babb 17 Li XQ, Xu FL, Chen XJ, Zhang PY, Wang SL and Gu X: The Randolph Cancer Center, West Virginia University and Jiang-Su effects of probeneside and other drugs on the renal excretion of SOPO (Group) CO., Ltd. in China for supporting this study. We thank dicycloplatin in rats. J Qinghai Med Coll 26: 243-246, 2005.

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