Say No to DMSO: Dimethylsulfoxide Inactivates Cisplatin, Carboplatin, and Other Platinum Complexes

Published OnlineFirst May 8, 2014; DOI: 10.1158/0008-5472.CAN-14-0247

Cancer Therapeutics, Targets, and Chemical Biology Research

Matthew D. Hall1, Katherine A. Telma1, Ki-Eun Chang1, Tobie D. Lee1, James P. Madigan1, John R. Lloyd2, Ian S. Goldlust3, James D. Hoeschele4, and Michael M. Gottesman1

Abstract The platinum drugs cisplatin, carboplatin, and oxaliplatin are highly utilized in the clinic and as a consequence are extensively studied in the laboratory setting. In this study, we examined the literature and found a significant number of studies (11%–34%) in prominent cancer journals utilizing cisplatin dissolved in DMSO. However, dissolving cisplatin in DMSO for laboratory-based studies results in ligand displacement and changes to the structure of the complex. We examined the effect of DMSO on platinum complexes, including cisplatin, carboplatin, and oxaliplatin, finding that DMSO reacted with the complexes, inhibited their cytotoxicity and their ability to initiate cell death. These results render a substantial portion of the literature on cisplatin uninterpretable. Raising awareness of this significant issue in the cancer biology community is critical, and we make recommendations on appropriate solvation of platinum drugs for research. Cancer Res; 74(14); 3913–22. 2014 AACR.

Introduction (8), some of which have entered clinical trials (e.g., picoplatin, satraplatinand BBR3464; ref. 9). Cisplatin (cis-[PtCl2(NH3)2]), carboplatin ([Pt(O,O'-cdbca) A critical aspect of the activity of platinum drugs in vitro and (NH3)2], cbdca ¼ cyclobutane-1,1-dicarboxylate), and oxaliplatin ([Pt(R,R-cyclohexane-1,2-diamine)(O,O'-ethanedioato)]) are in vivo is their interaction with the solvent environment. For used in combination with other agents to treat a range of example, platinum drugs are activated via replacement of malignancies including testicular, ovarian, head and neck, leaving groups with water inside the cell, a process termed bladder, esophageal, and small cell lung cancer (1–4). With aquation (10, 11). For cisplatin, this is the loss of a chloride the exception of testicular cancer, acquired or intrinsic resis- ligand and its replacement with water, which is a more reactive tance generally occurs and tumors are not eliminated by leaving group. For this reason, cisplatin is formulated for treatment (3–5). Cisplatin exerts its toxicity by DNA binding clinical use in saline solution with a high chloride concentra- and downstream apoptotic signaling, and resistance is con- tion (154 mmol/L) to prevent drug aquation before adminis- ferred by reducing apoptotic signaling, upregulating DNA tration, stabilizing the drug and preventing side reactions damage repair mechanisms, altering cell-cycle checkpoints, before cell entry. A limiting factor for platinum drugs is and disrupting assembly of the cytoskeleton (4, 6). The pleio- their relatively low solubility, and clinical cisplatin is formu- tropic mechanisms underlying platinum resistance are well lated at a concentration of 1 mg/mL (3.3 mmol/L). In labora- described, but their clinical significance is uncertain (4, 6). tory and drug screening settings, stock solutions of organic- These factors have prompted continued experimentation with based drugs are predominately prepared in the solvent DMSO ¼ platinum drugs to understand their mechanisms of action (O S(CH3)2), which is viewed as a virtual "universal solvent" alone and in combination with other therapeutics (7), as well able to solubilize most small molecules at high concentrations as the synthesis and testing of new platinum complexes with (up to 100 mmol/L, for example; ref. 12). DMSO contains a the prospect of uncovering compounds with promising activity nucleophilic sulfur, which allows it to coordinate with platinum complexes, displacing ligands and changing the structure of the complexes (13–16). This renders platinum complexes Authors' Affiliations: 1Laboratory of Cell Biology, Center for Cancer unstable in DMSO. Massart and colleagues first reported that 2 Research, National Cancer Institute; Advanced Mass Spectrometry Facil- DMSO reduced cisplatin's cytotoxicity toward cultured thyr- ity, National Institute of Diabetes & Digestive & Kidney Diseases, NIH, Bethesda; 3Division of Preclinical Innovation, National Institutes of Health ocytes (17), and Dernel and colleagues reported that a polymer- Chemical Genomics Center, National Center for Advancing Translational based drug delivery system limited activity of cisplatin against Sciences, NIH, Rockville, Maryland; and 4Department of Chemistry, East- ern Michigan University, Ypsilanti, Michigan stage IIb appendicular osteosarcoma in dogs (18). Little information exists on the effect of DMSO on other platinum drugs Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). and complexes. Yet, as discussed in this article, cisplatin and other platinum Corresponding Author: Michael M. Gottesman, Laboratory of Cell Biol- ogy, National Cancer Institute, NIH, 37 Convent Drive, Rm. 2108, Bethesda, complexes are regularly dissolved in DMSO for biologic experi- MD 20892. Phone: 301-496-1921; Fax: 301-402-0450; E-mail: ments, both in vitro and in vivo in experimental models, and [email protected] DMSO solutions of cisplatin have been utilized in the clinical doi: 10.1158/0008-5472.CAN-14-0247 veterinary setting (19). This use may be due to the lack of a 2014 American Association for Cancer Research. comprehensive understanding of the effect of DMSO on the

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activity of platinum complexes in the cancer biology commu- mL (1.6 mmol/L) in our laboratory, as described previously (20, nity. Irrespective of the reason, the implications for published 21). KB lines were originally generated in the laboratory of M.M. studies on cisplatin's mechanism using DMSO solutions are Gottesman. DLD-1 cells were provided by the National Cancer profound. Institute (part of the NCI-60 collection). All cell lines were We sought to examine the range of solvent systems utilized thawed immediately before experimentation, and cell lines are for platinum drugs in peer-reviewed research, and the effects of characterized by NCI using short tandem repeat profiling. The DMSO on platinum drug activity. A number of journals were cisplatin stock solution used for culturing CP.5 cells was assessed to gauge the solvent types used in studies of cisplatin. prepared in PBS. The cisplatin-resistant cells were main- We then assessed the impact of DMSO versus clinical formula- tained in the presence of cisplatin, which was removed from tions of a number of platinum complexes on the cytotoxicity, growth medium 3 days before all experiments. All cell lines cellular recognition of DNA damage, and cell death signaling. were grown as monolayer cultures at 37 Cin5%CO2,using Mass spectrometry was used to directly assess the interaction either DMEM (KB cells) or RPMI (DLD1 cells) with 4.5 g/L of DMSO and clinical formulations with each platinum glucose (both from Invitrogen), supplemented with L-gluta- complex. mine, penicillin, streptomycin, and 10% FBS (BioWhittaker). Resistance of CP.5 cells to cisplatin was confirmed on a Materials and Methods regular (at least monthly) basis, using cell viability assays as Literature review described herein. Five journals that regularly publish studies on small-mole- cule therapeutics and their mechanism were examined: Cancer Cytotoxicity and cell growth Research (http://cancerres.aacrjournals.org), Molecular Cancer Cell survival was measured by the MTT (Invitrogen) assay. Therapeutics (http://mct.aacrjournals.org), Molecular Pharma- Cells were seeded at a density of 5,000 cells per well in 96-well fi cology (http://molpharm.aspetjournals.org), Journal of Phar- plates and incubated at 37 C in humidi ed 5% CO2 for 24 macology and Experimental Therapeutics (JPET; http://jpet. hours. Serially diluted (1:3, RPMI or DMEM used as diluent, aspetjournals.org), and the Public Library of Science (PLoS; chloride concentration 120 mmol/L) compound was added fi http://www.plos.org) journals. In each case, the word "cisplat- to give the intended nal concentrations. Solvent tolerance fi in" was entered as a search term on the respective journal testing up to 0.5% under identical conditions con rmed website search engine, restricted to the term appearing in the growth of all cell lines was unaffected. Cells were then incu- title or abstract of articles. Manuscripts were then individually bated an additional 72 hours, and the MTT assay was per- reviewed to identify and assess only those papers reporting in formed according to the manufacturer's instructions (Molec- vitro data. These papers were then assessed for the solvent or ular Probes). Absorbance values were determined at 570 nm on solution used for dissolving cisplatin, and these were deter- a Spectra Max 250 spectrophotometer (Molecular Devices). All mined and recorded; similarly, it was noted if the solvent MTT assays were performed in triplicate. The IC50 values were fi system was not disclosed. In the majority of cases, if the de ned as the drug concentrations required to reduce cellular solvent used was not mentioned in the Materials and Methods proliferation to 50% of the untreated control well. We used section, it was not explicitly disclosed anywhere in the man- Prism 6 (GraphPad Software) software for graphs and statis- fi uscript in a manner that allowed unambiguous determination tics. All data are expressed as mean SD. For bright- eld 5 of the experimental strategy used—this was recorded as "Not imaging, DLD-1 cells were seeded at a density of 3 10 cells fi reported." Thirty-five manuscripts were assessed for each per well in 6-well plates and incubated at 37 C in humidi ed journal, with the exception of the JPET, where only 28 relevant 5% CO2 for 24 hours. Drug was added directly to each well and articles were identified. cells were incubated for another 72 hours. Media were aspi- rated and cells were rinsed with PBS before imaging. Materials Cisplatin, carboplatin, DMSO, dimethylformamide (DMF), H2AX immunofluorescence staining dimethylacetamide (DMA), formamide, acetamide, and cre- Fixed DLD-1 cells were stainedwithPhospho-Histone mophor EL were purchased from Sigma-Aldrich. Oxaliplatin H2A.X (Se139; 20E3) Rabbit mAb (Alexa Fluor 488 Conjugate; was purchased from LC Laboratories. Satraplatin was pur- all components from Cell Signaling Technology) following chased from Sequoia Research Products. [PtCl2(en)] and trans- the manufacturer's instructions and as previously described 4 platin were purchased from Alpha Aesar. Clinical formulations (22). Briefly, cells were seeded at 5 10 cells per chamber in of cisplatin, carboplatin, and oxaliplatin were kindly provided 8-chamber coverslips and incubated for 24 hours. Cells were by Dr. Tito Fojo, Clinical Center, National Cancer Institute then incubated with Pt complexes for 24 hours before being (Bethesda, MD). Compounds were assessed in various solvent washed with PBS, fixed in 4% paraformaldehyde, then systems as described below. blocked with blocking buffer (PBS/5% normal horse serum/0.3% Trition X-100) for one hour. Diluted antibody Cell lines and cell culture (1:50 in PBS/1% BSA/0.3% Triton X-100) was then applied, This study used the DLD1 human colorectal carcinoma cells, and cells were incubated overnight at 4 C. Cells were then parental human cervical carcinoma cell line KB-3-1 (a subline washed and stained with 40, 6-diamidino-2-phenylindole of HeLa), and its cisplatin-resistant subline KB-CP.5. KB-CP.5 nuclear DNA stain, and imaged on a Zeiss LSM 710 NLO cells were originally selected in a single step in 0.5 mg cisplatin/ confocal microscope.

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DMSO Inactivates Platinum Drugs

Preparation of cell lysates, quantification of protein, and Results Western blot analysis Solvent utilization in in vitro studies of cisplatin fl After 48-hour drug treatments, both oating and adherent Five journals that regularly publish studies on small-mole- DLD-1 colorectal cancer cells were washed with PBS and cule therapeutics and their mechanism were examined for lysed together in NP-40 lysis buffer [50 mmol/l Tris/HCl, pH reports pertaining to the activity and mechanism of cisplatin: 8.0, 150 mmol/L NaCl, 1% NP-40, and supplemented with Cancer Research, Molecular Cancer Therapeutics, Molecular complete protease inhibitor cocktail tablets (all from Sigma- Pharmacology, JPET, and the PLoS journals. Thirty-five papers Aldrich) and PhosStop phosphatase inhibitor tablets reporting in vitro data were assessed for each journal (28 for (Roche)] and centrifuged to remove insoluble material. 2X JPET), and the solvent used for dissolving cisplatin in each case Laemmli sample buffer (Sigma-Aldrich) was added to equiv- was determined and recorded. Solvent systems utilized were alent amounts of cellular lysates (40 mg), which were then simple salt buffer, cell culture media, clinical formulation resolved by SDS-PAGE on 4% to12% Bis-Tris gels (Invitro- (often indicated as sourced from a research hospital's in-house gen) and transferred onto Immobilon PVDF membrane pharmacy), DMF, DMSO, saline, PBS, and water (Table 1). (Millipore). Membranes were blocked in 5% (w/v) nonfat Where the solvent used was not identified in the Materials dried skimmed milk powder in TBS-Tween 20 (TBST; 20 and Methods, this was recorded as "Not reported," with a very mmol/L Tris/HCl, pH 7.6, 137 mmol/L NaCl, and 0.2% Tween small number of exceptions where the solvent was categori- 20; Sigma-Aldrich; blocking buffer) and probed with appro- cally identified elsewhere in the manuscript. Manuscripts priate primary antibodies overnight followed by anti-mouse assessed are listed in Supplementary Tables S1–S5, with the or anti-rabbit IgG horseradish peroxidase (HRP)-conjugated solvent used assigned for each. Although only in vitro data were secondary antibody (Cell Signaling Technology) for one assessed, it should be noted that cisplatin dissolved in DMSO hour. Membranes were washed in TBST and incubated in has also been used for in vivo studies (23). either Western Blotting Luminol Reagent (Santa Cruz Bio- As shown in Table 1, for each journal the majority of papers technology) or Immobilon Western Chemiluminescent HRP did not report the solvent used (26%–50% of papers). Following Substrate (Millipore) and the signal developed on HyBlot ES this, a DMSO solution or clinical formulation was the second fi fi lm (Denville Scienti c). All primary antibodies were from most used solvent. DMSO was the most heavily utilized in-lab Cell Signaling Technology: PARP (#9542), cleaved caspase-3 solvent. For example, DMSO was used in 34% of Cancer (#9664), phospho-H2A.X (#9718), and b-actin (#3700). Research and 20% of Molecular Cancer Therapeutics articles that were examined. Mass spectrometry In the clinic, cisplatin is typically provided as a lyophilized Accurate mass data were obtained on a Waters Premiere powder in a vial containing 50 mg cisplatin, 450 mg NaCl, and LCT time-of-flight mass spectrometer operated in the positive 500 mg mannitol. When dissolved in 50 mL of water, this results ion W-mode at 10 K resolution. The high-performance liquid in a 1 mg/mL solution (3.3 mmol/L) of cisplatin dissolved in chromatography solvent pump was operated at 200 mL/minute 150 mmol/L saline. The saline prevents aquation of the com- and the solvent composition was 50:40:10 water:methanol: plex in solution before administration to the patient (24). acetonitrile. All solvents were LC/MS grade and were pur- Clinical formulation was used for in vitro experiments in chased from Sigma-Aldrich. Samples were flow injected via a 11% to 23% of papers. However, in some studies, cisplatin was sample loop and ionized in the electrospray ion source. The dissolved in PBS (chloride concentration 140 mmol/L—phos- electrospray capillary was operated at 3.5 kV and at 350 C. The phate has been reported to not appreciably alter cisplatin desolvation gas was purified nitrogen. The MS data were aquation; ref. 25) or saline (chloride concentration 154 analyzed using Waters MassLynx 4.1 software. mmol/L). When these chloride-containing solutions are

Table 1. Reported solvent or formulation of cisplatin used for in vitro studies in selected peer-reviewed scientiﬁc journals, stated as percentage of total (with number of reports in parentheses)

Cancer Molecular Cancer Molecular Formulation Research Therapeutics PLoS Pharmacology JPET Not reported 43 (15) 37 (13) 43 (15) 26 (9) 50 (14) DMSO 34 (12) 20 (7) 17 (6) 11 (4) 14 (4) Water — 3 (1) 11 (4) 11 (4) 7 (2) Buffer —— —3 (1) — DMF — 3 (1) 3 (1) 3 (1) — Cell media —— 3 (1) 6 (2) — Clinical 14 (5) 23 (8) 17 (6) 14 (5) 11 (3) Saline 3 (1) 11 (4) — 20 (7) 14 (4) PBS 3 (1) 3 (1) 3 (1) 6 (2) 4 (1)

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considered along with clinical formulation, their use was and solutions of each drug in DMSO (at 20 mmol/L stock predominant, accounting for 20% to 40% of reports (Cancer solution). Satraplatin is only available as capsules for oral use, Research, 20%; Molecular Cancer Therapeutics, 37%; PLoS, 20%; and aqueous solubility is poor. As such, a 4:1 water:DMSO Molecular Pharmacology, 40%; JPET, 29%). solution was prepared to enable assessment of the effect of Other solvents were used in only a small number of cases. It water on activity. No clinical formulation exists for transplatin is possible that DMF (on one occasion) was used as an or [PtCl2(en)], but given that both contain chlorido leaving alternative organic solvent as it is able to solubilize cisplatin groups (like cisplatin), both complexes were dissolved in saline. and does not contain a sulfur group. Similarly, water can The responses of DLD-1 and KB-3-1 parental cells, and the solubilize cisplatin. However, in the absence of chloride, cis- cisplatin-resistant subline of KB-3-1, termed KB-CP.5, were platin and other complexes with chlorido leaving groups evaluated to examine whether sensitivity to each platinum become aquated, producing a mixture of species with agent, and cross-resistance in cisplatin-resistant cells, were increased reactivity and altered cytotoxicity (26, 27). affected by DMSO (Table 2; Fig. 2). The CP.5 cells demonstrated cross-resistance to all Pt complexes (>10-fold) with the excep- In vitro assessment of effect of DMSO on platinum tion of transplatin. The clinical formulations of cisplatin, complexes carboplatin, and oxaliplatin showed equivalent cytotoxicity to To assess the effect of DMSO on the activity of platinum their respective saline/aqueous solutions. drugs and complexes, the activity of the drugs cisplatin, DMSO had a signiﬁcant effect on the cytotoxicity of all carboplatin, oxaliplatin, and satraplatin, and the experimental complexes with monodentate (singly coordinated) ligands, complexes transplatin (trans-[PtCl2(NH3)2]) and [PtCl2(en)] diminishing the cytotoxicity of cisplatin, carboplatin, and (structures shown in Fig. 1) were assessed. Satraplatin is a [PtCl2(en)], and to a lesser effect, transplatin. These effects stable, lipophilic platinum(IV) complex designed to be orally could be easily observed by bright-ﬁeld microscopy (for cis- available (28), and although it entered clinical trials against platin, carboplatin, and oxaliplatin; Fig. 2A–C), and by dose– adult and pediatric malignancies (most prominently prostate response cell killing (Fig. 2D–F). The strongest effects were on cancer in combination with prednisone), it has not been cisplatin, with a greater than 40-fold loss of cytotoxicity against approved for use by the FDA (29). Transplatin is the "inactive" both DLD-1 and KB-3-1 cells (Table 2), and [PtCl2(en)], with a geometric isomer of cisplatin (30), and [PtCl2(en)] is regularly greater than 30-fold loss of cytotoxicity, both complexes con- used in experimental papers, and like cisplatin contains two tain the "'classic" cis-dichlorido leaving group arrangement. For cis-chlorido leaving groups (8). example, cisplatin dissolved in saline demonstrated an IC50 of The activity of clinical formulations of the drugs cisplatin 3.8 0.6 mmol/L, whereas its IC50 when dissolved in DMSO was (3.3 mmol/L in 0.9% saline with 10 mg/mL mannitol), carbo- 178 9.2 mmol/L. Carboplatin demonstrated reduced cyto- platin (27 mmol/L in 5% glucose solution; ref. 31), and oxali- toxicity compared with cisplatin (34.2 3.4 mmol/L against platin (12.6 mmol/L in 5% glucose solution; 32) was compared KB-3-1 cells), consistent with its diminished reactivity (32), but with analogous solvent preparations to the clinical formulation DMSO reduced its cytotoxicity further (3- to 10-fold, 243 11.2 (i.e., saline for cisplatin, water for carboplatin, and oxaliplatin), mmol/L against KB-3-1 cells). In contrast, DMSO seemed to

Figure 1. Structures of the platinum complexes used in this study.

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DMSO Inactivates Platinum Drugs

Table 2. Cytotoxicity (IC50, mmol/L) of platinum complexes against parental (DLD-1 and KB-3-1) and cisplatin-resistant (KB-CP.5) cell lines

IC50 (mmol/L)

DLD-1 KB 3-1 KB CP.5 Cisplatin Clinical 3.3 0.5 1.1 1.0 33.2 8.5 Saline 3.8 0.6 1.9 0.4 34.3 1.6 DMSO 178 9.2 48.1 3.7 376 283 Saline þ DMSO (384 mmol/L) 3.4 0.3 1.1 0.2 ND Carboplatin Clinical 68.8 22.6 41.4 10.8 460.7 54.4 Aqueous 78.4 5.1 34.2 3.4 1,014 10.4 DMSO 573 38.0 243 11.2 118 74.3 Oxaliplatin Clinical 1.5 0.2 1.14 0.05 ND Aqueous 3.9 0.6 3.0 0.5 74.3 34.1 DMSO 1.6 0.2 0.7 0.3 84.0 72.7 Satraplatin Water: DMSO (4:1) 3.8 0.4 2.4 0.3 37.3 3.2 DMSO 2.9 0.6 2.5 0.3 8.6 1.6 Transplatin Saline 335 165 >500 716 287 DMSO 587 31.8 433 30.6 134 80.3

[PtCl2(en)] Saline 2.7 1.9 11.4 1.5 569 113 DMSO 87.1 36.3 135 7.1 256 92.2

have a slight potentiating effect on oxaliplatin, with both platin cytotoxicity (e.g., IC50 for the clinical formulation against KB-3-1 (aqueous ¼ 3.0 0.5 mmol/L, DMSO ¼ 0.7 0.3 DLD-1 ¼ 1.5 0.2 mmol/L, in DMSO ¼ 1.6 0.2 mmol/L). mmol/L) and DLD-1 (aqueous ¼ 3.9 0.6 mmol/L, DMSO ¼ Although DMSO disrupted the activity of cisplatin, cisplatin- 1.6 0.2 mmol/L) cells being slightly more sensitive to the resistant cells retained cross-resistance toward the DMSO- DMSO-formulated compound. DMSO had no effect on satra- formulated drug. CP.5 cells demonstrated 10-fold resistance to

Figure 2. Comparison of the effect of DMSO on the biologic activity of cisplatin, carboplatin, and oxaliplatin. Bright-ﬁeld images (A–C), and dose–response curves (D–F) of DLD-1 cells exposed to platinum drugs (cisplatin, carboplatin, and oxaliplatin) in different solvents (water/saline, clinical formulation, DMSO).

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¼ ¼ cisplatin (CP.5 IC50 34.3 1.6 mmol/L vs. KB 3-1 IC50 1.9 dissolution in DMSO, by replacement of a Cl with DMSO (m/z 1.6 mmol/L), and cross-resistance to aqueous carboplatin (32- ¼ 343), and replacement of a Cl and an NH3 ligand by two fold), oxaliplatin (25-fold), and satraplatin (4-fold). CP.5 cells DMSO molecules (m/z ¼ 404; refs. 38, 39). We also observed a demonstrated 8-fold resistance to cisplatin formulated in higher molecular weight species (m/z ¼ 665.9) corresponding DMSO, and cross-resistance to oxaliplatin and satraplatin was to a bridged form of DMSO-substituted cisplatin, mNH2-[Pt þ maintained. Both carboplatin and transplatin dissolved in (NH3)(Cl)(DMSO)]2 (Supplementary Table S7) not previously DMSO seem to have greater potency against CP.5 cells than reported. These are shown schematically in Supplementary when formulated in aqueous media (water and saline, respec- Fig. S3. Carboplatin, transplatin, and [PtCl2(en)] produced tively). By way of example, the IC50 of transplatin dissolved in reaction products with DMSO, all involving replacement of a DMSO was very high (433 30.6 mmol/L) but lower against Cl ligand (Supplementary Table S7). Carboplatin and oxali- CP.5 cells (134 80.3 mmol/L). This collateral sensitivity platin also produced self-association multimers, as previously suggests that reaction with DMSO produces a complex (vide reported (32). Mass spectra of satraplatin in DMSO did not infra) with greater activity than cisplatin. The poor cytotoxicity produce any peaks corresponding to interaction with DMSO, and in vivo activity of transplatin are generally ascribed to its consistent with the inertness of platinum(IV) complexes (40). greater reactivity, but it is known that trans Pt complexes with Oxaliplatin did not produce any observable peaks correspond- bulkier ligands have reduced reactivity, and with this improved ing to reaction with DMSO. stability comes biologic activity (33). To assess the effect of DMSO on the DNA damage and cell The effect of DMSO on aqueous cisplatin killing produced by Pt drugs (9), we examined phosphorylation The evidence thus far indicates that most Pt complexes of the histone H2A family member H2A.X. H2A.X becomes (including cisplatin and carboplatin) are deactivated upon phosphorylated at serine 139 (gH2AX) in response to double- dissolution in DMSO stock solutions, and aqueous-based stranded breaks elicited by DNA damage, recruiting repair solutions are essential for biologic studies. However, cisplatin factors to sites of damage, and is also phosphorylated as a is usually used in combination in the clinic, and synergy of Pt result of apoptosis induced by DNA damage (34). In this way, drugs with experimental and established therapeutics are H2A.X has been shown to be phosphorylated in cells exposed to regularly assessed (41). As organic-based therapeutics are cisplatin, carboplatin, and oxaliplatin (35, 36). Cells were usually dissolved in DMSO, we wondered whether cisplatin treated with cisplatin, carboplatin, and oxaliplatin formulated in aqueous solution or growth media would be deactivated by a in aqueous solution (saline or water) or DMSO, and gH2A.X foci small component of DMSO if it is introduced into the same were evaluated by immunofluorescence microscopy after 24 solution. hours (Fig. 3A). Cells treated with cisplatin (5 mmol/L) in saline We first examined whether DMSO would inhibit cisplatin's or carboplatin (5 mmol/L) in water clearly elicited a DNA cytotoxicity. To examine this, cisplatin dissolved in saline was damage response, whereas cells treated with the compounds diluted into growth medium, followed by 30 mL/mL DMSO (3%, dissolved in DMSO resulted in a striking diminution of gH2A.X effective concentration 384 mmol/L in 154 mmol/L saline), staining. Despite being approximately equitoxic with cisplatin, followed by serial dilution and dosing to cells. DMSO did not oxaliplatin resulted only in low-level staining. Satraplatin in have any effect on cisplatin's cytoxicity on KB-3-1 or DLD-1 water and DMSO showed equivalent gH2A.X staining, whereas cells (e.g., DLD-1 cisplatin saline IC50 ¼ 3.3 0.5 mmol/L, ¼ transplatin and [PtCl2(en)] dissolved in DMSO elicited weaker cisplatin saline and DMSO IC50 3.4 0.3 mmol/L; Table 2). gH2A.X staining than their saline-dissolved comparison (not Despite the lack of biologic effect, mass spectrometry of saline shown). solutions of cisplatin and cisplatin in the presence of 3% DMSO Immunoblot analysis of gH2A.X supported immunofluores- revealed a peak corresponding to the replacement of one cence microscopy staining (Fig. 3B). To directly study whether chloride ligand with DMSO (Table 3). These data suggest that DMSO affected cell death, we incubated DLD-1 cells with DMSO introduced into combination studies with cisplatin cisplatin (5 mmol/L), carboplatin (50 mmol/L), and oxaliplatin does not affect its activity. However, researchers should be (5 mmol/L) in either DMSO or water, and whole-cell lysates aware that DMSO does interact with cisplatin even in dilute were collected after 48 hours for Western analysis (Fig. 3B). environments. Induction of cleaved PARP and cleaved caspase-3 was caused by cisplatin, carboplatin, and oxaliplatin, consistent with their Assessment of alternative solvent systems for platinum role in platinum-mediated apoptosis (37). These events were complexes prevented by formulation of the three drugs in DMSO. One limitation of aqueous formulations of cisplatin is its maximal solubility of just more than 3 mmol/L, whereas Modification of platinum complexes by DMSO and other DMSO solutions can be prepared of more than 10 mmol/L. solvents We examined whether other organic solvents had a similar To assess the nature of the interactions between cisplatin effect on cisplatin. Stock solutions of cisplatin were prepared and DMSO, we compared electrospray ionization mass spectra in amenable solvents: DMF, DMA, formamide, acetamide, in positive mode of all six complexes in aqueous and DMSO cremophor EL (all 10 mmol/L stock solutions), and water solutions (Supplementary Table S7, only peaks related to (3.3 mmol/L; Table 3). Cremophor EL and acetonitrile did DMSO adducts are shown). Previous chemical analysis has not fully dissolve cisplatin, but were tested as fine ultrasoni- demonstrated that cisplatin produces multiple species upon cated suspensions. None of the solvents profoundly affected

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A Cisplatin Carboplatin Oxaliplatin Aqueous

5 μmol/L, saline 50 μmol/L, water 5 μmol/L, water

Figure 3. Comparison of the effect of DMSO on cisplatin, carboplatin, and oxaliplatin-mediated DNA

damage and cell death. A, cellular DMSO DNA damage recognition caused by platinum drugs observed via confocal images (63) of DLD-1 cells exposed to platinum drugs as indicated and stained with an 5 μmol/L, DMSO 50 μmol/L, DMSO 5 μmol/L, DMSO antibody that recognizes phosphorylated (g) H2A.X. B, immunoblot analysis of protein B lysate from DLD-1 cells treated as described for A and probed for expression of total PARP, cleaved PARP, cleaved caspase-3, and phosphor (g) H2A.X, with b-actin probed as a control for protein Control Cisplatin Cisplatin(DMSO) (saline)Oxaliplatin Oxaliplatin (DMSO)Carboplatin (water) Carboplatin (DMSO) (water) loading. Total PARP Cleaved PARP

Cleaved caspase-3

Phospho-H2A.X

β-Actin

cisplatin's cytotoxicity against DLD-1 cells, and although not poorly soluble drugs such as paclitaxel (42). Although 3% solvent statistically signiﬁcant, DMF resulted in the greatest decrease is a high concentration, the IC50 concentration for cisplatin in cytotoxicity of cisplatin (IC50 ¼ 5.0 1.3 mmol/L compared (1–3 mmol/L) in our toxicity assays occurs at a drug dilution with 3.8 0.6 mmol/L for cisplatin in saline). that is in the presence of 0.2% to 0.05% solvent, a concentration However, each organic solvent alone (at a high concentration at which no toxicity toward DLD-1 cells was observed. of 3% in media, consistent with the lowest dilution of 10 mmol/L cisplatin in media required to perform a dose–response) dem- Discussion onstrated cytotoxicity toward DLD-1 cells (Table 3). The excep- We have demonstrated here the profound effects of DMSO tion was acetonitrile, with the limitation that acetonitrile was on platinum drugs and complexes that contain monodentate one of the two solvents unable to fully dissolve cisplatin. The ligands. Drug cytotoxicity is diminished, as evidenced by cell most toxic solvent was cremophor EL, a polyethoxylated castor growth and DNA damage response. The replacement of ligands oil used as the excipient for intravenous administration of on the platinum complexes could be observed by mass

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Table 3. Cytotoxicity (IC50, mmol/L) of cisplatin, dissolved or suspended in solvents, against DLD-1 cells

Cisplatin IC50 (mmol/L) Stock (mmol/L) Soluble 3% solvent viability (%) Control 100 Clinical 3.3 0.5 3.3 Y — Saline 3.8 0.6 3.3 Y 96 10 DMSO 177.6 9.2 20 Y 90 5 DMF 5.0 1.3 10 Y 79 5 DMA 3.5 0.8 10 Y 24 3 Formamide 3.7 1.8 10 Y 57 2 Cremophor EL 2.5 0.2 10 N 0.7 0.1 Acetonitrile 3.4 0.3 10 N 103 6 Water 4.0 0.5 3.3 Y 106 7

spectrometry. This study was prompted by a recognition of the whether DMSO could act as an antidote for cisplatin-induced high rate of use of DMSO with cisplatin in the literature, and toxicities, based on previously reported protective effects of partly by limitations encountered in developing a high- DMSO (17). This work was initiated in the context of the desire throughput screen using cisplatin—drugs are transferred by to utilize known thiols as reactive "protective agents" to temper pins and surface tension has been optimized to transfer a fixed the side effects of cisplatin (48). Fischer and colleagues later volume of DMSO. Using an alternative solvent would not be demonstrated that although the neurotoxic side effects of possible without significant optimization. A significant number cisplatin are tempered by DMSO, loss of cisplatin's cytotoxicity of reports in all five journals examined utilized DMSO-dissolved offset any potential benefit (38). cisplatin, raising questions about how to interpret the results of It is not clear whether the cytotoxicity observed in DMSO- a large number of previous studies. Our findings also reinforce formulated cisplatin is due to the mixture of chemical entities the current focus on the challenge of reproducing scientific present in solution, or the unreacted portion of cisplatin literature (43). remaining in equilibrium. Given varying cross-reaction in These findings extend to most platinum complexes. Thou- solutions utilized in laboratories, it would be expected to be sands of platinum complexes (and other metal-based com- difficult to replicate results with saline, PBS, or clinical for- plexes) have been synthesized and tested in vitro in an effort to mulations of platinum complexes. For example, a crystallo- identify complexes with greater potency than cisplatin, a graphic study of cisplatin and carboplatin binding to histidines different mechanism of action from cisplatin, and/or an ability of hen egg-white lysozyme found one platinum bound to His15 to overcome cross-resistance in cisplatin-resistant cells (8, 6). when cisplatin was dissolved in aqueous medium. When Given the general lack of information about how to formulate dissolved in DMSO, two platinum atoms bound to His15, experimental agents and the likelihood that complexes will leading to the conclusion that DMSO could facilitate plati- dissolve in DMSO, most compounds for testing against cell num-protein binding (49). Uribe and colleagues reported lines are also regularly dissolved in DMSO. For example, of the increased sensory hair cell death in zebrafish cotreated with 20 most recent papers reporting new chemical entities based cisplatin and DMSO but not other solvents, which was ascribed on platinum in the Journal of Medicinal Chemistry, seven (35%) to the cell permeabilizing effects of DMSO, and warned that used DMF to dissolve complexes for biologic testing, five (25%) DMSO could produce false-positive effects in other drug did not state the solvent used, and three (15%) used DMSO screens due to DMSO's biologic activity (50). (Supplementary Table S6). Alternative solvents such as DMF The effects of DMSO on platinum drug activity in vitro can be have been reported not to cause cytotoxicity below 0.5% (44), profound. We believe the practice of dissolving platinum drugs and solutions of cisplatin dissolved in DMF retain activity (45). in DMSO must cease, and if the solvent is to be utilized, new However, although DMF is recognized as cytotoxic toward cells platinum agents must demonstrate a lack of interaction with (46), there seems to be no comprehensive assessment of DMF's DMSO. For experimentation, cisplatin should be prepared in a utility as a drug (or platinum complex) solvent. It is critical saline-based solvent (3 mmol/L), and carboplatin (27 mmol/L) then, that an understanding of drug interaction with solvent, and oxaliplatin (12.6 mmol/L) in water. When available, clinical and solubility in solvent, be ascertained for experimental formulation can substitute. It is the hope of the authors that metal-based therapeutics. this report will raise awareness of this significant issue in the The problem of cisplatin deactivation by DMSO was first cancer biology community, introduce caution when interpret- reported over 20 years ago, and the underlying chemistry had ing results of published studies utilizing DMSO, inform future been generally well defined (13, 16). Jones and colleagues experimental design, and guide editorial guidelines for mech- demonstrated that coadministration of cisplatin with DMSO anistic and experimental therapeutic research. in Sprague–Dawley rats reduced nephrotoxicity (47), and Massart and colleagues observed that DMSO inhibited cisplat- Disclosure of Potential Conflicts of Interest in cytotoxicity toward cultured thyrocytes while investigating No potential conflicts of interest were disclosed.

3920 Cancer Res; 74(14) July 15, 2014 Cancer Research

DMSO Inactivates Platinum Drugs

Authors' Contributions Grant Support Conception and design: M.D. Hall, M.M. Gottesman This work was supported by the Intramural Research Program of the NIH, Development of methodology: M.D. Hall, K.A. Telma National Cancer Institute. K.-E. Chang is an NIH Medical Research Scholars Acquisition of data (provided animals, acquired and managed patients, Program scholar, a public-private partnership supported jointly by the NIH and provided facilities, etc.): M.D. Hall, K.-E. Chang, T.D. Lee, J.P. Madigan, J.R. generous contributions to the Foundation for the NIH from Pﬁzer Inc, The Doris Lloyd, I.S. Goldlust Duke Charitable Foundation, The Alexandria Real Estate Equities, Inc., Mr. and Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Mrs. Joel S. Marcus, and the Howard Hughes Medical Institute, as well as other computational analysis): M.D. Hall, K.A. Telma, T.D. Lee, J.P. Madigan private donors. Writing, review, and/or revision of the manuscript: M.D. Hall, K.-E. Chang, I. The costs of publication of this article were defrayed in part by the S. Goldlust, J.D. Hoeschele, M.M. Gottesman payment of page charges. This article must therefore be hereby marked Administrative, technical, or material support (i.e., reporting or orga- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this nizing data, constructing databases): M.D. Hall fact. Study supervision: M.D. Hall

Acknowledgments Received February 21, 2014; revised April 16, 2014; accepted April 16, 2014; The authors thank George Leiman for editorial assistance and Dr. Craig published OnlineFirst May 8, 2014. Thomas for helpful insights.

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3922 Cancer Res; 74(14) July 15, 2014 Cancer Research

Say No to DMSO: Dimethylsulfoxide Inactivates Cisplatin, Carboplatin, and Other Platinum Complexes

Matthew D. Hall, Katherine A. Telma, Ki-Eun Chang, et al.

Cancer Res 2014;74:3913-3922. Published OnlineFirst May 8, 2014.

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