A Drug of Such Damned Nature.1 Challenges and ... - ACS Publications

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Perspective

A drug of such damned nature . Challenges and opportunities in translational platinum drug research. Dorian M Cheff, and Matthew D. Hall J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01351 • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 16, 2017

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Journal of Medicinal Chemistry

A drug of such damned nature.1 Challenges and opportunities in translational platinum drug research.

Dorian M. Cheff and Matthew D. Hall* NCATS Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, Maryland 20850, United States

ACS Paragon Plus Environment

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Abstract The platinum-based anti-cancer agents cisplatin, carboplatin and oxaliplatin, represent a spectacular translational science achievement. The basic research observations that led to the discovery of Pt complexes as DNA-binding agents that elicit cell arrest, the preclinical tumor regression studies, and the inorganic medicinal chemistry that led to clinical implementation of effective platinum complexes in the clinic, have fueled multidisciplinary research into platinum-based drugs. While the successes are clear and the research activity continues, a significant window of time has passed since a new Pt drug has been approved for clinical use. Here we assess the current Pt drug landscape, challenges for future Pt development, and discuss opportunities for improving our understanding of Pt drugs that utilize contemporary translational science tools such as chemical biology and real-time imaging. The underexplored spaces may reveal new opportunities for Pt drug development.


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Introduction Platinum stands out among chemotherapeutic classes for its spectacular successes and utility in combination therapy, but also for its inconvenience to the patient and the resistance that can arise. The first report related to the discovery of cisplatin was published over 50 years ago (1965) (see Hoeschele2 for a detailed description of the early experiments). However, as is well reported, it was likely first synthesized by Michele Peyrone around 1840,3 and was critical to Nobel prize-winner Alfred Werner’s work on inorganic isomerism.4

Cisplatin became the first of three platinum-based anticancer agents registered for use with the FDA (1978), followed by carboplatin (1989) and oxaliplatin (2002) (structures shown in Figure 1). Other Pt-based drugs have entered clinical trials, and some are approved for use in specific countries (discussed below). In parallel with this clinical effort, significant basic research has been conducted on Pt-based complexes with a number of aims, including: to understand their mechanism(s) of action; to synthesize and test new Pt complexes as anticancer drug candidates; to characterize the ways in which cells become resistant to Pt drugs; to explore whether other (non-Pt) metal complexes can offer useful biological activity; to study the (bio)chemistry of Pt complexes in the context of reactions with biomolecules; and to characterize the cellular pharmacology and fate of Pt drugs.

This perspective aims to serve as a reflection on the ghosts of platinum research past, present, and future. It is written to focus attention and inspire discussion on the strengths


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of the field, and the future of Pt research. It was inspired partly by Richard Callaghan’s honest reflections on the relevance of drug transporters in cancer resistance5 entitled ‘Providing a molecular mechanism for P-glycoprotein; why would I bother?’. The corresponding author has spent 15 years studying Pt complexes, over that time transitioning from inorganic medicinal chemistry and biological spectroscopy, to cell biology and mechanisms of resistance to Pt drugs.6-9 The discussion here is written from the perspective of one who has been immersed in the field of Pt drugs, but who, through the natural peregrinations of research, has evolved a view of the field from afar that might offer an external perspective on the strengths and weaknesses of the field. It is also an attempt to be candid. The difference is vast between the honest (and colloquial) posterside conversation at conferences on the state and direction of a field, and the sober sanitized nature of published perspectives that are written to ensure no future reviewer will be offended. In that context, this Perspective aims to be a useful conversation piece.

Like other ‘micro-fields’ of study, the topic of ‘metals-in-medicine’ (or inorganic medicinal chemistry) has been subject to the vicissitudes of biomedical fashions and fads. With each new technology that emerges to improve cancer treatment, the hand-wringing over the relevance of ‘shotgun’ therapeutics begins.10 It seems, anecdotally, that the typical expectation is that the need for Pt drugs will decline as a result of the new (‘better’) technology, followed by the realization that the technology is not the complete solution that everyone anticipated, and finally the clinical demonstration that the new technology works very well in combination with a Pt drug!


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O H 3N

Cl

H 3N

Pt H 3N

O

H2 N

H 3N

O O

Cisplatin

O

O

O

Pt

Pt Cl

O

Carboplatin

N H2

Oxaliplatin

Figure 1. Structures of cisplatin, carboplatin, and oxaliplatin. In each case the cis nonleaving amines are at the left, and the leaving groups (chloride, bidentate dicarboxylate, or oxalate) are at the right.

The health impact and economic benefit of Pt drugs has been tremendous.11 While these authors do not believe Pt drugs will leave the clinic for many generations to come, the expectation seems to be that ‘shotgun’ therapeutics should be phased out, and this is understandable given the side-effects experienced by patients, and the superiority of targeted drugs against some malignancies in the genomic age.12 From a basic and translational research point of view, new biomedical discoveries with therapeutic implications should be welcomed as a way to empower Pt research and effectiveness in the clinic. A colleague once said that the major referee critique against their rejected grant application was that with the arrival of gene therapy, there would no longer be a need for chemotherapy!

Despite the time that has passed since the discovery of cisplatin, the number of publications per year continues to increase for all three clinical Pt complexes. There are more than 60,000 publications indexed in Pubmed for ‘cisplatin’, and more in 2015 than any other year (>3,200). The same is true for carboplatin (more than 14,000 total, >790 in


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2015) and oxaliplatin (over 8,000 total, >850 in 2015) (depicted in Figure 2A). It is notable that oxaliplatin surpassed carboplatin in publications in 2015 for the first time. Comparison with other DNA-targeting chemotherapeutic agents developed in the same period (Figure 2B, chlorambucil, mitomycin, mitoxantrone, bleomycin) emphasizes the scientific and medical impact of the ‘platins’. In each case, an initial surge in publications per year occurred, followed by a plateau. In fact, the only ‘shotgun’ chemotherapeutics that appear to come close are doxorubicin (Adriamycin) and the ‘blockbuster’ drug paclitaxel (Taxol), which show similar research attention (Figure 2C). Even a modern ‘blockbuster’ targeted drug such as the Bcr-Abl inhibitor imatinib (Glivec) shows static research output, while the antibody therapeutic trastuzumab (Herceptin), which targets HER2/neu receptor, does display continued growth in research output. The comparisons in publications made here are reflective only of scientific interest, they are not a measure of clinical impact, but among the >80,000 indexed papers on platinum drugs (and likely many thousands more on other Pt experimental therapeutics and compounds) there are a core of innovative papers that set the tone for topics of interest. This includes topics such as structure activity relationships,13 Pt-DNA structures,14 structure-rule-breakers15 such as active trans complexes16, and highly active non-Pt complexes such as osmium,17 to name a few examples. While citation tracking of published papers that pre-exist the internet are not complete, the 10 most highly cited research reports (excluding literature reviews, using Google Scholar18) describing the three clinical Pt drugs all appear to be clinical reports (not shown).


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A.

Publications per year

4000 3000 2000 1000 0 1970

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Year Cisplatin

Carboplatin

Oxaliplatin

B. 600

Publications per year

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Year Chlorambucil

Mitomycin

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Bleomycin

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C.

Figure 2. A. Publications indexed in Pubmed referring to the drugs, cisplatin, carboplatin, or oxaliplatin in the title or abstract, displayed by year. B. As a comparison, publications describing the FDA-registered DNA-damaging agents chlorambucil, mitomycin, mitaxantrone and bleomycin. C. Publications describing ‘blockbuster’ oncology therapeutics paclitaxel, doxorubicin, imatinib and trastuzumab.

Given the remarkable clinical importance of Pt drugs, it is critical to keep in mind the contrasting negative perception of them. Certainly the lay-person’s view of the drug being a toxic ‘heavy metal’ is technically accurate, perhaps the worst press being Lance Armstrong’s inaccurate statement that “for testicular cancer, they treat you with plutonium, one of the harshest chemicals there is,”19 (though the cure itself is testament to the drug’s effectiveness). Armstrong also stated in his autobiography that they gave him “…a cocktail of three different drugs, bleomycin, etoposide, and cisplatin, and they were so toxic that the nurses wore radioactive protection when handling them”20 (emphasis


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added) – either confusing the radiotherapy arm of treatment, or the personal protective equipment (PPE) worn by nurses to minimize chronic workplace exposure to cytotoxic (and possibly mutagenic) drugs.

The historical clinical literature is peppered with the perspective that cisplatin would not be approved by the FDA were it discovered today. Cisplatin’s clinical usefulness was ultimately rescued by pre-hydration to offset nephrotoxicity and anti-emetics to manage nausea,21 after much experimentation. The challenge of irreversible ototoxicity in patients, which is not a dose-limiting toxicity but results life-changing effects such as deafness and tinnitus, remains unresolved.22 It is interesting to consider just how close cisplatin came to not being registered in spite of its activity.23 The engrossing and lengthy transcript of the Witness Seminar held by the Wellcome Trust Center for the History of Medicine in 200623 contains multiple references by participants reflecting on the promising efficacy of cisplatin against malignancies such as ovarian cancer in early trials in England (circa 1971/2), but also the clinical challenges associated with toxicity. These challenges were a significant topic of discussion at the 1973 Oxford meeting,24 perhaps best captured by Hill who summarized pessimistically that ‘New and better analogs are awaited with great interest,’25 and this consensus led to the desire for less toxic analogs. The Johnson Matthey work on analogs led to the development of carboplatin (initially coded JM-826), but other options were pursued. While not mentioned in the official proceedings publication that followed the 1973 meeting, nor in the Witness Seminar, the popular science writer June Goodfield attended the 1973 meeting, and described the inprogress Pt story in a chapter of her book ‘Cancer Under Siege’27 based on interviews she


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conducted with researchers at the meeting. The Pt chapter title “I want the platinum blues!” is a direct reference to Barnett Rosenberg’s stated belief during the 1973 meeting that the platinum blue complexes,28 with their lesser nephrotoxicity than cisplatin, would be the ultimate clinical Pt drug!

Nevertheless, cisplatin and carboplatin prevailed. Any reader who has not observed a patient suffering from the specific effects of platinum chemotherapy need to look no further than a social media platform such Twitter29 to read posts by patients. Alongside the aforementioned cisplatin side-effects, oxaliplatin produces peripheral neuropathy that results in cold hypersensitivity, such as shooting pains if a patient’s skin comes into contact with a metal surface,30 as well as peripheral neurotoxicity, often causing paresthesias and numbness. The drugs certainly stay with patients—it has been shown that platinum is detectable in the urine of subjects 8 years following their last Pt chemotherapy round.31 In the veterinary setting, cisplatin also presents benefits and challenges. While cisplatin is a single-agent curative for equine sarcoids (administered directly into the tumor site via needle as an oil suspension)32, 33, all veterinary trainees also learn that ‘cisplatin splats cats’, a reference to the species-specific pulmonary edema and death that precludes its use in felines.34

Intrinsic and acquired resistance to Pt drugs also occurs in patients. Drug resistance is normally studied by culturing a cell line in increasing concentrations of a given drug to force the acquisition of a resistance phenotype. It is recognized that 2-dimensional tissue culture is not the ideal cell model, but it is certainly the easiest model to study). Another


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weakness in this strategy is that single-agent resistance in cell lines is very different from the combination chemotherapies a tumor is normally exposed to, and therefore develops resistance to. For example, patients diagnosed with ovarian cancer usually undergo surgical resection (debulking), followed by chemotherapy composed of a platinum (cisplatin or carboplatin) and a taxane (paclitaxel or docetaxel) drug, repeated every 21 or 28 days.35, 36 A recent Nature supplement on tackling drug resistance in ovarian cancer singled out platinum as the cause of the resistance problem.37 Despite the fact that the front-line treatment for high-grade serous ovarian carcinoma is a taxane and a platinum, the article’s accompanying video narration states “most die because their cancer develops resistance to the most common form of treatment – platinum based chemotherapy” (i.e., the taxane is not to blame). The article is unfairly titled ”The Problem with Platinum” despite being about ovarian cancer, and looks forward to ‘new approaches (that) promise to break through the platinum barrier.’38 The article does not explain that platinum chemotherapy extends the life of suffers by years, and that it is front-line because no replacement chemotherapy regimen has been demonstrated to be superior. This example reinforces the negative image of platinum chemotherapy among those who peer-review manuscripts on new Pt agents, review Pt grant applications, and read Pt publications. Yet Pt drugs are an essential part of many chemotherapeutic regimens. When asked why they studied cisplatin resistance, a world-known clinician shrugged and said simply, “because it will still be in the clinic in 50 years, so we need to continue to try and understand it.”

The critical path for researchers in experimental therapeutics is to translate discoveries towards the clinic.39 As such, the design, synthesis, and study of experimental Pt


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complexes are, by definition, translational. The critical question is: how can Pt drugs be relevant to new discoveries in our understanding of cancer, and how can Pt drugs be used in concert with new therapeutic strategies? It is incumbent on metals-in-medicine experts to ask these questions, because there are unique aspects to Pt drugs, and their chemistry, which limit those outside the field to appropriately design experiments using Pt complexes—for this reason, collaboration is key. When the corresponding author recently published a study on the effect of DMSO on the cancer cell line cytotoxicity and mechanism of cell death of Pt drugs, and the extent to which DMSO is used as the solvent for Pt drugs in the literature,40 he was met with some very positive41 and some very negative feedback (personal communication!). The fact that Pt complexes react with DMSO was already well known within the metals-in-medicine community,42 which is dwarfed in size by the oncology community using the compound in experimental studies. The finding that over a third of the oncology and experimental therapeutic literature used DMSO as a solvent for Pt drugs was distressing as the implications for the extant literature were potentially profound,40 and it was advised to not publish that observation as part of the study. However, alerting the wider oncology community that works with Pt drugs is critical (and others have sought to make researchers aware in other ways).

Key challenges for the field Below, we assess recent trends in the platinum medicine field, and discuss key challenges for metals-in-medicine generally. The relevant pre-existing work is cited in each case, and it is hoped that this will stimulate interest and experimentation in new fields, and entice experts in related fields into entering the waters. We chose four ‘recent trends’ to


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give a sense of where we see current major developments, and discuss ‘key challenges’ that we believe deserve further investigation (we do not suggest they are novel, just underexplored). There is obviously a skew to these based on our interest in oncology experimental therapeutics and translational science, and we have attempted to discuss the most difficult questions. We are hopeful that these topics will stimulate thought and discussion on the topic, and that they highlight much that lies fallow.

1. Seeing is believing: microdose imaging of clinical and experimental Pt drugs in humans. Image-based insight into drug tumor distribution is a powerful tool during pre-clinical drug development, and for image-guided personalization of therapeutic regimens.43 For example, [18F]paclitaxel (FPAC) has been used for positron emission tomography (PET) imaging to show that uptake levels in breast tumors correlated with clinical response.44 Such tools are under-developed for clinical and investigational Pt-based drugs, and we believe implementation will aid in answering questions related to tumor drug levels, clearance rates, off-target organ residence times, and side effects (perhaps including sites such as the inner ear that present a toxicity challenge). As it stands, the information that exists relating to patient tumor-drug localization has relied on direct measurement of bulk Pt levels in post-surgical tissue from non-small cell lung cancer,45 which demonstrated a correlation between Pt levels and survival for 44 patients, and more recently in a smaller study of muscle-invasive bladder cancer.46 A limitation of these valuable studies is that patients had received Pt chemotherapy over a month prior to tissue resection. While they are single time-point studies, the obvious advantage of directly measuring Pt levels is that


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the moiety measured is the one that exerts activity, and the challenge is that the proportion of total Pt responsible for cell-killing activity is not known but presumably a very small proportion of total Pt in the cell. Spectroscopies such as X-ray fluorescence spectroscopy (XFS, also known as synchrotron resonance induced X-ray fluorescence, SRIXE), and NanoSIMS (Nano-scale surface ionization mass spectrometry), can provide spatial information on Pt localization, and one imagines a wealth of post-chemotherapy samples are in pathology banks and available that may be amenable to such a study, but the challenge again remains that the limitation is that clinical samples are ex vivo samples from a single time-point.

Radiolabeled Pt complexes have been generated for laboratory studies, and could be used for animal biodistribution47 and time-course studies. Cisplatin has no carbon, and [3H] labeling is not feasible because protons on Pt ammine ligands exchange rapidly in water. Carboplatin has been labeled on the ‘leaving’ cyclobutyldicarboxylate ligand, 48, 49 limiting its utility as one is essentially tracing the non-pharmacologic leaving group rather than the bioactive moiety, particularly over longer time-points. Oxaliplatin’s 1,2cyclohexylamine non-leaving ligand has been [14C] labeled, and this seems the most appropriate Pt radioligand to study for this reason.50 In a similar way, satraplatin and miriplatin contain non-leaving amines useful for [14C] or [3H] labeling, but neither appears to be reported.

An emerging theme in drug development is the concept of Phase 0 studies that precede the usual Phase I dose escalation studies. Phase 0 studies utilize a non-pharmacologic


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‘microdose’ that allows insight into the human pharmacokinetic profile.51 Ideally, the investigational agent is labeled with [14C] for detection by accelerator mass spectrometry (AMS), or possibly another radioisotope with a long half-life, but less-sensitive mass spectrometry-based detection tools are also utilized. No report currently exists for Pt microdose studies in humans, though it seems achievable for complexes amenable to [14C] labeling (see above), and may be a potential method for comparing a small number of candidate Pt drugs. The development of a pharmacodynamic microdose study that aimed at assessing Pt-DNA adducts in subjects by isolating peripheral blood mononucleocyte cells (PBMCs) as a predictive biomarker was reported in 2013,52 but results have not been disclosed as of this writing.

What is possible for temporal imaging strategies in human subjects with platinum drugs, then? There are four accessible radioisotopes of Pt: 191Pt (t1/2 = 3 days), 193mPt (t1/2 = 4.3 days), 195mPt (t1/2 = 4 days), and 197Pt (t1/2 = 17 hours)53 from which radiolabeled Pt complexes could be generated. By using radiolabeled Pt, the structure of the drug itself is unmodified. All radioisotopes produce gamma emission, and would be amenable to either gamma camera scintigraphy (producing 2D images) or single-photon emission computed tomography (SPECT) imaging (producing 3D images). Studies reporting [191Pt]cisplatin (1985)54 and [193mPt]cisplatin (1973)55 preparation have received followup; [191Pt]cisplatin appears to have been intended partly as a radiotherapeutic pharmacologic form of cisplatin that did not progress into humans,56 and partly for imaging (see below), and the [193mPt]cisplatin was not used for imaging. Multiple reports


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of preparations of [195mPt]cisplatin exist from 1973 onwards,55, 57-60 and performance assessment showed that the minimum detection limit for a 2 cm tumor was 8 ppm.61

All but one of the handful of imaging studies have utilized [195mPt]cisplatin, and specific examples are shown in Figure 3. The first report with [195mPt]cisplatin displayed a gamma camera image at very low resolution in a 35-year-old woman with metastatic carcinoma of the cervix55 (Fig. 3A), and a similar strategy was independently reported the following year.62 A decade later, Owens et al. produced [191Pt]-labeled cisplatin, carboplatin, and the platinum(IV) clinical candidate, iproplatin, and gamma camera images were used to assess liver and kidney levels in patients with a number of different malignancies, but tumor accumulation itself could not be discerned.63

The first report to utilize imaging for clinical insight came from Wolf and co-workers (1989), who used scintigraphic imaging to assess [195mPt]cisplatin levels in brain tumors of patients receiving intra-venous (i.v.) versus intra-arterial (i.a.)64 administration (intraarterial administration of chemotherapy via the carotid to maximize delivery of drug to brain tumors was of great clinical interest in the 1980s). Scans were collected each minute to create time-activity curves, and tumor signal (ipsilateral) was compared with the non-tumor brain hemisphere (contralateral), revealing lower tumor or brain uptake of cisplatin administered i.v., and 3-10 times increased cisplatin levels when administered i.a. (Fig 3B). Brain tumor could be clearly distinguished because of the very low surrounding brain Pt levels. Areberg et al. imaged [191Pt]cisplatin distribution in 14 patients, and in 5 cases were able to distinguish tumor uptake in patients with a range of


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malignancies including oral, lung and testis65 (Fig 3C). Critically, tumor location was confirmed by separate computed tomography (CT) scans, and tumor Pt concentrations were derived from quantitated signal and compared against Pt pharmacokinetics. More recently, a study of [195mPt]cisplatin scintigraphy distribution in five healthy volunteers has been reported (along with associated PK), with the highest organ exposure in kidneys (Fig 3D).66

PET allows higher-resolution imaging than SPECT, but also has a number of restrictions associated with it, including the limited number of radioisotopes able to produce positrons upon radioactive decay. While Pt radioisotopes do not appear to be amenable to PET imaging, [11C] and [18F] are the most regularly utilized radioisotopes in PET imaging, though not yet reported for Pt drugs. However, multiple syntheses of [13N]cisplatin have been reported,67-69 the first in 1985. The challenge for clinical imaging is the short half-life of the 13N radioisotope (t1/2 = 10 mins), shorter even than [11C] (t1/2 = 20 min) and [18F] (t1/2 = 1.8 h). Only one report exists using [13N]cisplatin for PET imaging (compared with three reports on its preparation!).70 Ginos et al. assessed biodistribution, blood clearance and stability first in rats, and could not detect displacement of the [13N] ammine ligand from cisplatin in deproteinized blood samples. PET studies were performed in two patients with glioblastoma following both i.v. and i.a. (carotid artery) injection, in conjunction with regular chemotherapy administration, producing brain:tumor concentrations of 30:1, with increasing tumor accumulation over 15 minutes (Fig 3E).70 A.

B.


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C.

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D.

E.

Figure 3. Examples of images acquired from human subjects treated with radiolabeled cisplatin. A. 1973. 35-year old woman with metastatic carcinoma of the cervix imaged


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following injection with [195mPt]cisplatin.55 The strong activity arises from high concentrations in the kidneys. This research was originally published in JNM. Lange, R.C., Spencer, R.P., Harder H.C. The Antitumor Agent cis-Pt(NH3)2Cl2: Distribution Studies and Dose Calculations for 193mPt and 195mPt. J Nucl Med. 1973;14:191-195. © by the Society of Nuclear Medicine and Molecular Imaging, Inc. B. Images of a patient with astrocytoma (brain tumor) injected with [195mPt]cisplatin either i.v. (left) or i.a. (right).64 Low brain signal compared to surrounding tissue can be seen at left. The high signal (dark) at right corresponds to the tumor region. C. Static anterior images over the head, neck and thorax region of a patient with metastatic lung cancer immediately after injection with [191Pt]cisplatin, with several tumors arrowed.65 D. Anterior (frontal) view of healthy subject 5 hours following injection of [195mPt]cisplatin.66 E. 1987. Composite 13 minute images of [13N]cisplatin PET scans following i.v. administration, with the drug being infused during the data collection.70 This research was originally published in JNM. Ginos, J.Z., Cooper, A.J.L., Dhawan, V., Lai, J.C.K., Strother, S.C., Alcock, N., Rottenberg, D.A. [13N]Cisplatin PET to Assess Pharmacokinetics of Intra-Arterial Versus Intravenous Chemotherapy for Malignant Brain Tumors. J Nucl Med. 1987;28:18441852. © by the Society of Nuclear Medicine and Molecular Imaging, Inc.

The images collated in Figure 3 demonstrate the evolution of radiolabeled cisplatin over time. Three of the four patient studies (i.e. subjects with tumors) were published in the 1980s, and most of the studies are proofs-of-concept rather than clinical studies. Given that both scintigraphy/SPECT (Pt radioisotope) and PET imaging ([13N]) with cisplatin have been demonstrated as useful—why have they not been explored, both in animal


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models and humans? There are a litany of clinical challenges for Pt drugs, including understanding the acquisition of resistance, virtually no knowledge at all of tumor drug concentrations, and the opportunities for using imaging for differentiating candidate Pt drugs. Producing a fluorinated analog of a Pt drug such as oxaliplatin (non-leaving amine ligand) is an unexplored possibility, provided the analog retains pharmacologic activity (and therefore relevance). Medical imaging technology has progressed significantly in the past 30 years, and identification of tumor sites can be achieved with [18F]fluorodeoxyglucose (FDG) and MRI/CT to facilitate interpretation of drug localization. Coordination between chemists, biologists and clinicians will be needed for any clinical imaging program, and could ultimately produce a clinical tool for identifying patient populations who would benefit from Pt-containing chemotherapeutic regimens.

2. Time for a new drug Cisplatin and carboplatin are workhorses of the clinic, and oxaliplatin has added a new spectrum of activity. Oxaliplatin was approved in the United States in 2002, and it is stated in the literature that it was first approved for clinical use in 1996 in France71 (reflected in Table 1,72 though we have been unable to identify a primary source for this date). Europe-wide approval for oxaliplatin occurred in 1999.73 Aside from these three approved drugs, a range of Pt complexes have entered clinical trials in the United States and Europe (reviewed extensively in 200774 and 201072), the most notable being the Johnson Matthey products cis,trans,cis-[PtCl2(OH)2(isopropylamine)2], (iproplatin, JM975), cis,trans-[PtCl2(acetato)2(NH3)(cyclohexylamine)]


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(satraplatin, JM21676), and cis-[PtCl2(NH3)(2-methylpyridine)], (picoplatin, JM473, AMD473, ZD047377), and the multinuclear complex [{trans-PtCl(NH3)2}2-mu-(transPt(NH3)2(H2N(CH2)6-NH2)2)]4+ (BBR3464) developed by the Farrell lab.78 While cisplatin, carboplatin and oxaliplatin are essentially approved worldwide, there are four drugs approved for clinical use in specific nations (Table 1 and Figure 4). Carboplatin was developed with the aim of producing a complex with cisplatin’s activity, but without its toxicity. It is worth considering how the clinical activity of the more recent Pt drugs was identified.

Oxaliplatin (originally called l-OHP) was one of many Pt complexes containing 1,2diaminocyclohexane ligands synthesized based on the promising activity of these complexes.79 It was identified as a highly soluble analog that was active against L1210 murine leukemia cells in mice (the standard for front-line testing at the time) and was active against cisplatin-resistant L1210/DDP cells along with other mouse tumors.80 A study of oxaliplatin’s cytotoxicity against the NCI60 panel of cell lines identified oxaliplatin as highly active against colorectal cancer cell lines,71 and this led to clinical trials with 5-fluorouracil that produced improved responses and led to regulatory approval.71 Despite challenges associated with peripheral neuropathy, oxaliplatin continues to enter clinical trials for efficacy against other agents, including rectal, pancreatic, and stomach cancers.81

Nedaplatin (Figure 4, originally termed 254-S72) was approved for use in Japan in 1995, against a range of malignancies including head & neck cancers.82 It has the same non-


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leaving cis-ammine ligands as cisplatin and carboplatin, and a glycolato ligand as the leaving group. It was developed by Shionogi Pharmaceutical Company, Japan, based on a desire to develop a drug with lesser side-effects than cisplatin. Nedaplatin was one of a small series of Pt glycolato complexes synthesized with various amine ligands, all of which were shown to be highly soluble.83 Nedaplatin was found to be less toxic than cisplatin, and active against a range of animal xenografts,84 and progressed to clinical trials. While it is generally thought of as a second-line platinum drug,82 a recent Japanese clinical trial comparing nedaplatin plus docetaxel with cisplatin plus docetaxel for advanced or relapsed squamous cell lung cancer found that survival was significantly longer, and side effects lessened, using nedaplatin instead of cisplatin.85 This suggests a greater role in the clinic may lie ahead for nedaplatin, and certainly more trials comparing nedaplatin with first-line drugs such as cisplatin and carboplatin.86

Heptaplatin (Figure 4, originally termed SKI 2053R) was approved in Korea in 1999 for the treatment of gastrointestinal malignancies. 87 The compound was part of a series of over forty [2-substituted-4,5-bis(aminomethyl)-1,3-dioxolane]platinum(II) complexes with bidentate carboxylate leaving groups88—the name heptaplatin evidently arose from the seven-membered ring formed between the diamine non-leaving ligand and Pt. The in vivo activity against L1210 tumors was determined, and the most active complexes were tested against stomach cancer cell lines (for which there are limited treatment options), and found to be superior to cisplatin and carboplatin. Heptaplatin was selected as the lead among other analogs based on its aqueous solubility and stability, low nephrotoxicity, and activity against stomach cancer cell lines.89 Clinical trials in Korea led to approval


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for use of the agent against advanced gastric cancers, in combination with 5fluorouracil.90

Lobaplatin (Figure 4, originally termed D-19466) was originally developed in Germany by ASTA Pharma, has entered clinical trials in multiple nations,91 and was approved for use in China in 2003. It emerged from a series of Pt analogs containing a 1,2bis(aminomethyl)cyclobutane ligand synthesized at ASTA Pharma, with bidentate lactate as the leaving group.92 It was selected for progression to clinical development based on aqueous solubility and stability, superior cytotoxicity to cisplatin against a number of cancer cell lines including melanoma and hepatoma lines, and an absence of nephrotoxicity in mice at a dose that was efficacious against P388 and L1210 tumors (and was more active than both cisplatin and carboplatin),93 and entered clinical trials. The drug passed through several hands in Germany before being licensed for development in China. It is approved in China for the treatment of metastatic breast and small-cell lung cancer, and acute myelogenous leukemia,94 and has been recommended for combination with paclitaxel as second-line treatment for esophageal cancer.95

Miriplatin (Figure 4) was recently approved in Japan, and is structurally distinct in that it features long alkyl chains on its carboxylate leaving groups (myristates) to confer lipophilicity.96 The complex was specifically created for treatment of hepatocellular carcinoma (HCC). It has been shown that the excipient ethiodized oil (Lipiodol), composed of iodine in fatty acid esters derived from poppy seed, is selectively retained in HCC tumors when administered directly via the hepatic artery (a therapeutic strategy


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termed transarterial chemoembolization, TACE).97 TACE can act as a tumor drugdelivery strategy, and clinical trials with cisplatin suspended in ethiodized oil have been performed, but suspension of drugs insoluble in the oil presents challenges.98 To address this, Sasaki and co-workers prepared a series of ‘liposoluble’ cyclohexane-1, 2-diamine platinum(II) complexes with long-chain carboxylate (C10 – C24) leaving groups.99 The criteria for lead selection were: ‘suspensibility’ in ethiodized oil; Pt release from the suspension to aqueous saline; and in vivo antitumor activity against L1210 murine leukemia cells, and miriplatin, with myristate (tetradecanoate) leaving groups, was selected for preclinical and clinical trials (summarized in96) and was ultimately approved. Dosage is 70 mg of drug suspended in 3.5 mL of ethiodized oil, administered to the patient once a day through a catheter inserted into the hepatic artery.

H 3N

O

O

O

NH 2 O Pt NH 2 O

Pt H 3N

O

O Heptaplatin

Nedaplatin

O CH3

H2 N

NH 2 O Pt NH 2 O

O

(CH)12

O

(CH)12

Pt O

N H2

CH3 O

Lobaplatin

Miriplatin


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Figure 4. Structures of drugs registered for use in specific countries. In each case the cis non-leaving amines are at the left, and the leaving groups are at the right.

Table 1. Drugs registered for clinical use (structures shown in Figure 1). Drug Cisplatin Carboplatin Oxaliplatin Nedaplatin Lobaplatin Heptaplatin Miriplatin

Registered Worldwide Worldwide Worldwide Japan China Korea Japan

Year 1978 1989 1996 1995 2003 1999 2009

Ref 72 100 72 72 72 101 96

The common requirements in the discovery stage for the newer agents described above were solubility, stability and a reduction in side effects compared with cisplatin. None of these four agents (nedaplatin, heptaplatin, miriplatin, lobaplatin) have been reported to display superior activity to cisplatin/carboplatin, though they have been demonstrated to be comparable. This probably accounts for the fact that these drugs have received little clinical attention in the United States or Europe, and presumably commercial factors are also at play. These newer drugs can in a sense be considered ‘me-too’ drugs102 that were developed based on the known structure-activity rules (SAR) for the ‘big three’ Pt drugs.15 That is, a cis arrangement of two non-leaving amine ligands, and two ‘leaving’ ligands. Oxaliplatin can be excused from the ‘me too’ accusation based on its different scope of activity against colorectal cancer. Miriplatin was specifically developed for a formulation that localized to HCC tumors in the liver, though the point stands that structurally it complies with the SAR of cisplatin. The reality is that in each case these later-generation complexes exist to replace another Pt drug, rather than to introduce a


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new spectrum of activity, which is a common motivator for me-too drug development. The challenge is the need for the newer ‘me-too’ Pt agent to demonstrate superiority over the clinical standard-of-care Pt drug, replacing one Pt drug with another – carboplatin succeeded in this, satraplatin did not.

There are three strands for the future development of Pt-based drugs: first, the creation of me-too drugs; second, the continued identification of Pt drug combinations with other agents against specific cancers that provides improved patient benefit; and third, the generation of new Pt compounds that push the boundaries of Pt SAR for testing and identification of novel activity against malignancies poorly responsive to current Pt agents.

We believe the greatest hope for Pt drug development lies in creating and identifying compounds with a new spectrum of activity—be it through phenotypic profiling of cancer cell line sensitivity, or medicinal chemistry driven towards identifying a highly active complex against a malignancy of choice. A major challenge for clinical chemotherapy is drug resistance, and this is the case for most patients enrolled in Phase I/II clinical trials being treated with an experimental drug. It seems unlikely that the solution to clinical resistance to a platinum drug will lie with a new Pt drug.6 This does not mean that there is little prospect for new Pt drugs to enter the clinic, but while testing new Pt compounds in cytotoxicity assays against cisplatin-resistant cell lines may inform on a possible alternative cell-killing mechanism to cisplatin, activity against cisplatin-refractory tumors is unlikely to be the basis of a clinical trial.


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However, one can envisage a Pt drug with a very similar activity profile to cisplatin that could result in replacement of cisplatin/carboplatin as front line drugs. One scenario could be a Pt drug that does not cause ototoxicity in patients. Ototoxicity in various forms (deafness, tinnitus) is not a dose-limiting toxicity as it does not prevent the patient from receiving the drug, but cisplatin can cause immediate and permanent alterations to a patient’s hearing, and the same is true for carboplatin.103 The replacement of cisplatin with carboplatin in clinical regimens has been careful (why change something that works?), and requires side-by-side clinical trials. For example, in the case of nasopharyngeal cancer, most patients receive cisplatin as the front-line agent, despite the fact that individual trials have shown no difference in treatment efficacy,104 and that carboplatin was better tolerated. Ototoxicity is not avoided though, and while the mechanism has not been definitively determined, a mouse model exists for cisplatininduced ototoxicity that could be a useful basis for counter-screening for Pt drugs that do not induce ototoxicity.105

So what are the questions we should be asking of new agents? New clinical candidates that offer genuinely new activity are needed, including activity against intractable malignancies such as glioblastoma and other brain tumors, melanoma, and pancreatic cancer. Brain tumors are an interesting case in point. In the previous section it was noted that brain signal was extremely low during gamma imaging of radiolabeled cisplatin; this appears to be because clinical platinum drugs are inefficient at crossing the blood-brain barrier (BBB). The brain:plasma ratio for cisplatin and carboplatin is between 1:5 and


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1:10,106 whereas peripheral tissues demonstrate Pt levels closer to parity with plasma levels. A Pt development program driven by brain tumor cell accumulation (rather than cell line killing) could result in an agent with an improved activity profile against brain tumors. This is just one example, there are of course many such scenarios where pushing beyond simple phenotypic monolayer cell line killing could open up new avenues for Pt drug discovery.

In that context, what pre-clinical Pt candidates appear to be in the pipeline? Information on Pt candidates progressing towards clinical trials is often limited, but Investigational New Drug approval for a clinical trial (in the US, or equivalent procedures elsewhere) is usually accompanied by a press release. There are also websites indexing clinical trials for various regions that can give insight into open trials with new platinum agents. One challenge is in identifying the structure of investigational agent and in some cases similar structures can be inferred from literature published by founders of a given pharmaceutical company.

The ‘phosphaplatin’ complex, PT-112 (1), is being developed by Phosplatin Therapeutics (USA).107 It is currently listed as being in Phase I clinical trials in the United States to evaluate its safety, tolerability, pharmacokinetics and preliminary clinical effects.108 Several academic publications by the lead author Rathindra Bose (who passed away in 2015) describe the ‘phosphaplatin’ class of complexes (Figure 5). The initial synthesized series including both Pt(II) and Pt(IV) complexes with bidentate pyrophosphate complexes, containing cis ammine (NH3), ethane-1,2-diamine and 1,2-


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diaminocyclohexane (dach) non-leaving groups.109 The complexes comply with Pt SAR rules, but the pyrophosphato ligand confers high aqueous solubility, and does not hydrolyze over weeks in aqueous solution.110 The dach-containing complex termed ‘pyrodach-2’ demonstrated greater cytotoxicity than cisplatin against cisplatin-resistant cells, in spite of lower cellular accumulation.110 A recent paper further assessed the dach complexes and revealed a lack of in vitro DNA interaction, little role for DNA repair genes in sensitivity, and differential gene expression changes in treated ovarian cancer cells, and the investigators have concluded that an alternative mechanism of action to cisplatin is at play.111 The same paper revealed that the R,R enantiomer of pyrodach-2, ‘RRD2’ was approved as an Investigational New Drug, hence it is the agent designated 1 by Phosplatin Therapeutics. Whether 1 truly acts by a mechanism divorced from DNA binding remains to be proven; the reduced cytotoxicity compared with cisplatin, and the high tolerated doses in mice (>60 mg/kg i.v.)112 is consistent with increased stability just like carboplatin and oxaliplatin, both of which contain bidentate leaving groups. Along with trials in the US (drug to be administered intravenously), Phosplatin Therapeutics have also granted a regional license for development and commercialization of 1 in China and nearby countries.113


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O

HO

ONa

H2 N

O

2

O

P

O

H 3N

O

P

O Pt

O

Pt N H

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O ONa

H 3N

HO

O O

O

PT-112

Dicycloplatin

O

O

N H N

O Cl

N H

O

Pt N H

O

O

Cl H N N O

O Kowol

Figure 5. Structures of pre-clinical Pt complexes and related structures. 1 (R,R-trans-1,2cyclohexanediamine dihydrogen pyrophosphate)platinum(II)) in development by Phosplatin Therapeutics. Dicycloplatin in development by Sopo-Xingda Pharmaceutical, containing carboplatin (structure highlighted in red, also shown in Figure 1) hydrogenbonded to 1,1-cyclobutane dicarboxylic acid (CBDCA). The ‘Kowol’ complex is not preclinical, but is an exemplar from the literature of a Pt(IV) complex possessing axial ligands with pendant maleimide.

The Pt-containing therapeutic BTP-114114 (2) (structure not disclosed) is in development by Placon Therapeutics (USA, scientific co-founder Stephen Lippard115), which recently spun out from Blend Therapeutics (USA) to focus on Pt drugs.116 2 was recently approved as an Investigational New Drug by the FDA.114 2 has been disclosed to be a pro-drug of cisplatin with a pendant maleimide group, which covalently binds to serum albumin, providing an extended plasma half-life.117 Maleimides react with thiols, and


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human albumin contains a single thiol at residue 34,9 that has been demonstrated to be accessible to maleimide conjugation.118 Based on the disclosure information, it seems likely that 2 is a Pt(IV) complex with axial ligands containing functionalized maleimide groups. Pt(IV) complexes are thought of as ‘pro-drugs’ that release their axial ligands upon reduction to yield the square planar active Pt(II) complex.8 2 may be similar to a concept reported by Kowol and co-workers in 2013,119 where pendant maleimides were coupled to the axial ligands of Pt(IV) complexes (Figure 5). In the case of the Kowol complexes, the Pt(IV) complexes reacted with albumin rapidly (halftime ~1h)119 and showed tumor regression activity. Lippard and co-workers have published a series of asymmetric Pt(IV) complexes designed to non-covalently associate with albumin via their pendant fatty acids (Figure 6b),120 but no information on an analogous covalent complex have been disclosed. Generally speaking, the albumin-binding pro-drug strategy may in some ways parallel abraxane, the albumin-bound formulation of paclitaxel,121 which was developed to circumvent toxicities associated with paclitaxel and its excipient. Molecules such as Evans Blue are avid albumin binders and used on that basis to assess BBB integrity (or similarly tethered to Pt(IV)).122 Along with prolonged pharmacokinetics,117 2 may also be anticipated to benefit from the leaky tumor effect that can allow for high macromolecule accumulation in tumors.123


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Figure 6. Schematic of a likely mechanism of action of a compound similar to 2, based on disclosed description of mechanism.117 A Pt(IV) complex containing cisplatin in the equatorial plane, with a pendant maleimide on an axial ligand (left) conjugates with Cys34 of human albumin in the blood stream. Following prolonged circulation, the Pt(IV) complex is ultimately reduced, yielding cisplain, which can then exert its anticancer activity.

Dicycloplatin is effectively a reformulation of carboplatin, wherein the excipient contains an extra molar equivalent of carboplatin’s leaving group, CBDCA that is believed to hydrogen bond with carboplatin and facilitate its solubilization.124 Developed by Sopo-Xingda Pharmaceutical (China), it has greater solubility and stability in solution than carboplatin, and equivalent activity to carboplatin125 A number of clinical trials have been conducted in China, the most recent reported being a Phase II study comparing paclitaxel plus carboplatin versus paclitaxel plus dicycloplatin in patients with advanced non-small-cell lung cancer,126 with both combinations showing similar response and survival rates, concluding that a Phase III trial of dicycloplatin is warranted to confirm efficacy.


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Along with new small molecules, there has been a significant emphasis on liposomal reformulations of platinum complexes.127, 128 While slightly outside the intended scope of this article, there are some interesting points related to the Pt drug being delivered, and the pro-drugs generated as part of the formulations. The archetypal success story in nanoscale drug formulation is that of pegylated liposomal doxorubicin (marketed as Doxil).129 The weak base doxorubicin is hyper-accumulated within the acidic lumen of the pegylated liposomes, creating a high ‘payload’ per liposome. To date, a physico-chemical property of cisplatin and its counterparts has not been identified that would allow noncovalent trapping at high levels (though there is no reason why a complex that can be acid-trapped cannot be created for liposomal loading). However, several liposomal preparations encapsulating unmodified cisplatin have been reported,130 the most advanced being lipoplatin, a liposomal formulation of encapsulated cisplatin with a loading level of 1:10130 that shows prolonged circulation and diminished toxicities. Lipoplatin has now progressed through Phase I, II and III trials, and it was granted orphan drug status by the EMEA in 2007 for treating pancreatic cancer.131 While clinical trials appear to be ongoing, it is not yet approved for full use in any market. Other liposomal formulations encapsulating unmodified Pt agents are also in pre-clinical development.

Beyond lipoplatin, a panoply of liposomal and other nano-scale drug carriers containing Pt drugs are reported in the literature, some of which are progressing towards clinical trials. These involve a covalent attachment of the Pt complex to the nanocarrier to facilitate high Pt drug payload per nanoparticle. This attachment is conferred either via


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the axial ligand of a Pt(IV) pro-drug containing cisplatin, carboplatin or oxaliplatin that is released upon reduction,132 or via coordination of carboxylate leaving ligands (Figure 7). What is intriguing is that, unlike the liposomal formulations of intact Pt drugs, both conjugation strategies involve creating a new chemical entity (NCE) that presumably requires full safety assessment. For either strategy, when the Pt detaches from the nanoparticle it produces a version of the active drug; for Pt(IV) attachment the intact drug is presumably yielded; for carboxylate-tethered Pt(II) drugs, the result is presumably a diaquated complex that is highly reactive133 (this aspect of Pt release does not appear to have been explored in detail).

Tethering strategies have led to Pt nanocarrier formulations that have entered clinical trials. For example, Nanocarrier Co. (Japan) have developed several micellar nanoparticles formed from block co-polymers containing a polyamino acid core that coordinates to Pt(II) via carboxylate ligands, using both cis-diammine (cisplatin analog, termed Nanoplatin, {[Pt(1,2-diaminocyclohexane)]}-loaded polymeric micelles or NC6004) and dach (oxaliplatin analog, termed DACH-Platin, or NC 4016), schematically shown in Figure 7.134 Both forms are currently in clinical trials against a range of malignancies in Japan and the US.135, 136 An alternative leaving group is included in the oxaliplatin analog ProLindac (AP5346) (3),137 where the Pt is loaded onto a hydroxypropylmethacrylamide (HPMA) biocompatible polymer via an O,N chelate (Figure 7). Like DACH-Platin, (3) releases a hydrolyzed form of oxaliplatin. (3) entered Phase I and II clinical trials, but appears to have been discontinued.138


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Figure 7. Schematic demonstrating the chelation of Pt-dach in DACH-Platin and (3). Release from the constructs presumably produces the hydrolyzed form of oxalipatin.

While it is clear that structure-activity rules are being adhered to, for both new small molecules and nanocarrier formulations, there is clearly scope of exploration of new chemotypes and new strategies for improving the activity over cisplatin, and reducing the reactivity and toxicities compared with cisplatin both by modifying pharmacokinetics and targeting the Pt payload to tumors. The literature on drug delivery systems incorporating cisplatin and other active Pt complexes is expanding rapidly and it is likely more will enter clinical trials in the near future.127 Understanding Pt drug mechanism and activity, and utilizing the various drug delivery mechanisms available to create new therapeutic


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opportunities clearly leads the way for the next Pt therapeutic to be approved for use in the clinic.

3. A role for Pt in antibody-drug conjugates? The emergence of biologics including antibody-directed therapeutics is changing the chemotherapeutic landscape. A cornerstone of this technology are antibody-drug conjugates (ADCs), where a highly specific antibody is directed to a tumor-specific target, delivering a small molecule cytotoxin.139 There are three components to ADCs. First, a monoclonal antibody directed towards a cancer-specific cell-surface target. Second, to the antibody is tethered a linker that connects with the cytotoxic ‘payload’, that can be cleaved to release the cytotoxin. Third, a highly cytotoxic small molecule is conjugated to the linker. It is critical that the synthetic modification to conjugate the small molecule does not interfere with the activity of the molecule. A number of ADCs are in development. For example, trastuzumab emtansine (marketed as Kadcyla) possesses an antibody that targets HER2-positive cells, and delivers a maytansine-like molecule called DM1, a highly cytotoxic molecule that acts by binding to tubulin.140 A critical need for ADCs is that the conjugated cytotoxin is highly potent, as each ADC delivers a few cytotoxin molecules to the cell at most.139 In fact, molecules usually incorporated into ADCs are often natural product derived molecules targeting microtubules (such as maytansinoid or Monomethyl auristatin) that are too toxic for systemic administration. ADCs allow such toxins to be administered as they are inactive till released where they are localized at the tumor site, improving the therapeutic window for these agents.


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Given the emerging clinical importance of ADCs and other antibody- and immunedirected therapeutic approaches, it is unsurprising that combination chemotherapies with Pt drugs are being explored as part of clinical trials.141 However, to date there is little exploration of the possibility of conjugating Pt drugs to antibodies in order to create ADCs. It is possible that that Pt drugs do not provide adequate cytotoxicity, but the improvement in therapeutic window and reduction in side effects such as ototoxicity may confer an advantage.

The main study published describes a trastuzumab-Pt(II) conjugate to target HER2positive cells.142 In this work, an oxaliplatin-like complex was conjugated to trastuzumab via a dipeptide linker that could be cleaved by the cellular protease cathepsin, rigidified to ensure that the Pt complex cannot reach around and coordinate with the antibody. This is important as it has been shown that cisplatin can coordinate with trastuzumab, though it does not affect trastuzumab’s antibody-binding region.143 A Pt:antibody ratio of 6:1 was reported, and the conjugation of Pt(II) did not affect the binding of trastuzumab to HER2 on breast cancer cells. The trastuzumab-Pt construct resulted in almost four-fold higher cellular Pt levels compared with an equimolar dose of oxaliplatin, though it did not result in increased cytotoxicity. There are other examples of Pt-antibody constructs, for example for utilization in mass cytometry, but these were not created with linkers, nor for therapeutic use.144 Waalboer et al. report the use of Pt(II) complexes as bifunctional ADC linkers (as the linker, not as the active drug) connecting a model fluorophore with trastuzumab as proof-of-concept.145


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Away from antibodies, an extensive literature exists on the general concept of targeting cancer cells by appending biomolecules to the axial ligands of Pt(IV) complexes.146 By way of example, axial coordination of peptides that bind to integrins have been reported with the intention of targeting cellular uptake to brain tumor cells.147, 148 While promising, cellular targeting guided by small molecules is less likely to provide the exquisite specificity of antibodies directed against validated oncology targets. Pt ADC-related studies are in their infancy, but given that the therapeutic antibodies, and the chemistry needed to conjugate bioactive small molecules, are already in place, translational studies will answer the question of whether there is a role for Pt here. As with the natural product-derived agents currently used, a survey back through the literature to find the most toxic Pt complexes (without great concern for therapeutic window) may mean a ‘new’ Pt chemotype will become a clinical candidate.

4. Chemical biology for cellular fate and mechanisms of action of cisplatin. The verdant field of chemical biology is underpinned by the use of chemical tools to gain insight into biological processes, including understanding of the mechanism of action of small molecule therapeutics. For example, target identification for a drug can be achieved with modified molecules containing a ‘click-able’ pendant group that can be used for proteomic identification of protein targets of a small molecule, or fluorescent labeling to monitor cellular fate. For Pt drugs, the development of chemical biology is occurring at a time when new questions are being asked of the cellular targets of Pt drugs.149 For


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example, it is clear that Pt drugs interact with a range of protein targets, and understanding their mechanistic relevance (or lack of it) will require significant effort.

Tracking Pt drugs in cells, and their interaction with biomolecules, is not straightforward. As mentioned earlier, few labeling strategies are available for native Pt drugs (i.e., without modification of their chemical structure). This has led to the development of Pt complexes with fluorophores, usually tethered to an amine ligand.150 Examples include complexes conjugated to the fluorophores carboxyfluorescein diacetate (CFDA),151 N-(7nitro-2,1,3-benzoxadiazol-4-yl)ethane-1,2-diamine (NBD),152 and BODIPY.153, 154 These complexes have been used to track the localization of the complexes in a range of cell lines. An obvious limitation to adding a fluorophore to a small inorganic complex is that it may significantly alter the physicochemical properties and biological activity of the Pt complex, and is limited by the assumption that the Pt-fluorophore connection is intact (otherwise it is free fluorophore being imaged).154

To address these limitations, and to allow ‘label-free’ study of Pt drug fate, a number of alternative strategies have been pursued. For example, New and colleagues reported a fluorescent sensor that was used to ‘post-label’ monofunctional Pt species in fixed cells to image its localization.155 Pt complexes have been reported that incorporate minimal structural changes to add a reactive handle for direct post-labeling of the complex. Bierbach and colleagues reported a Pt-acridine with a pendant azide group, and following cell treatment and fixation ‘click’ chemistry was used to append a alkyne-functionalized Alexafluor 488 dye.156


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Cl

N N3

Pt NH 3

Cl

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N N3

Pt NH 3

Reporter

Picazoplatin (Picoplatin)

H2 N

Cl Pt Cl

N3 N H2

Target

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Target


N N

Pt NH 3

N N

Reporter

Figure 8. Scheme showing the structure of picazoplatin (with the structure of the clinical candidate picoplatin shown in red), and its reaction with biomolecules. Subsequently ‘click’ chemistry can be used to conjugate an alkyne-functionalized reporter of choice (blue) to the bound picazoplatin via its pendant azide. A Pt complex containing a 2-azido1,3-propanediamine non-leaving bidentate ligand is also shown at bottom left.

In this context, a series of studies by the DeRose group have reported modified Pt drugs with functionalizations that allow chemical biological post-labeling experiments to be performed, such as protein target identification, DNA and RNA binding studies, and incell detection.157 By incorporating an azide or alkyne handle for Click chemistry, relatively small changes to the host Pt complex can be made. The initial study reported an analog of picoplatin with an azide group appended to the 2-methylpyridine ligand


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(termed picazoplatin, Figure 8), that could be post-labeled with a fluorophore after reaction with DNA or RNA in vitro.158 RNA extracted from yeast cells (S. cerevisiae) treated with picazoplatin were post-labeled with Alexa Fluor 488 to demonstrate that tRNA is a cellular substrate for Pt drugs.159 A Pt complex containing a 2-azido-1,3propanediamine non-leaving bidentate ligand (Figure 8) was shown by post-labeling to bind with bovine serum albumin as a model target protein.160 Confocal images of a similar Pt complex post-labeled in HeLa cells demonstrated the potential for cell-based studies.161 The chemical biology tools now exist to interrogate the cellular targets of Pt drugs, both nucleic acids and protein, and to extend into detailed in vivo studies.

Conclusions Platinum drugs play a critical role in the clinic, and will continue to do so for many years to come. While Pt drugs have been extensively studied, new fields of biology pave the way for new discoveries and understanding of Pt drugs. The development of new Pt drugs that enter clinical trials is a realistic and achievable goal, but requires boldness and sensitivity to the challenges that current Pt drugs can present to patients in the clinic: efficacy that is tempered by side-effects.


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Corresponding Author *Matthew Hall, NIH/NCATS, 9800 Medical Center Drive, Rockville, Maryland, 20850 Telephone: (301) 217-5727; Fax: (301) 217-5736; E-mail: [email protected]

Biographies: Dorian Cheff received her Bachelor of Science degree in Neuroscience from the University of Michigan in 2015. She is now at the National Center for Advancing Translational Science, NIH as a post-baccalaureate Intramural Research Training Award (IRTA) Fellow working on assay development and high-throughput drug screening for potential oncology-focused small molecule therapeutics. Her current research interests include efforts to screen Pt compound libraries.

Matthew Hall received his B.Sc. (Hons) in Science and his Ph.D. in Chemistry from the University of Sydney, Australia, under Professor T. W. Hambley, working on understanding the mechanism of action of platinum(IV) complexes. Following a 1-year postdoctoral fellowship with Professor Val C. Culotta at the Johns Hopkins School of Public Health, he joined the Laboratory of Cell Biology, National Cancer Institute (NCI), National Institutes of Health (NIH), under Dr. Michael Gottesman, studying (in part) the mechanisms of cisplatin resistance and multidrug resistance. He joined NCATS in 2015, where his group engages in assay development and high-throughput screening for earlystage small molecule probe discovery, with an emphasis on challenges in oncology drug discovery, including challenges associated with Pt drug research.


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Abbreviations Used ADC, antibody-drug conjugate; AMS, accelerator mass spectrometry; BBB, blood-brain barrier; CBDCA, 1,1-cyclobutanedicarboxylic acid; CFDA, carboxyfluorescein diacetate; CT, computed tomography; dach, 1,2-diaminocyclohexane; FPAC, [18F]paclitaxel; HCC, hepatocellular carcinoma; HPMA, hydroxyprpylmethacrylamide; I.A, intra-arterial; I.V., intravenous; NanoSIMS, nano-scale surface ionization mass spectrometry; NBD, N-(7nitro-2,1,3-benzoxadiazol-4-yl)ethane-1,1-diamine; NCE, new chemical entity; PBMC, peripheral blood mononucleocyte; PET, positron emission tomography; SAR, structureactivity relationships; SPECT, single photon emission computed tomography; TACE, trans-arterial chemoembolization; XFS/SRIXE, x-ray fluorescence spetroscopy

Acknowledgements The authors wish to thank Kyle Brimacombe for his work in designing our figures. This research was supported [in part] by the Intramural Research Program of the NIH, National Center for Advancing Translational Sciences.

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