MnDPDP: Contrast Agent for Imaging and Protection of Viable Tissue

The semistable chelate manganese (Mn) dipyridoxyl diphosphate (MnDPDP, mangafodipir), previously used as an intravenous (i.v.) contrast agent (Teslascan™, GE Healthcare) for Mn-ion-enhanced MRI (MEMRI), should be reappraised for clinical use but now as a diagnostic drug with cytoprotective properties. Approved for imaging of the liver and pancreas, MnDPDP enhances contrast also in other targets such as the heart, kidney, glandular tissue, and potentially retina and brain. Transmetallation releases paramagnetic Mn2+ for cellular uptake in competition with calcium (Ca2+), and intracellular (IC) macromolecular Mn2+ adducts lower myocardial T1 to midway between native values and values obtained with gadolinium (Gd3+). What is essential is that T1 mapping and, to a lesser degree, T1 weighted imaging enable quantification of viability at a cellular or even molecular level. IC Mn2+ retention for hours provides delayed imaging as another advantage. Examples in humans include quantitative imaging of cardiomyocyte remodeling and of Ca2+ channel activity, capabilities beyond the scope of Gd3+ based or native MRI. In addition, MnDPDP and the metabolite Mn dipyridoxyl diethyl-diamine (MnPLED) act as catalytic antioxidants enabling prevention and treatment of oxidative stress caused by tissue injury and inflammation. Tested applications in humans include protection of normal cells during chemotherapy of cancer and, potentially, of ischemic tissues during reperfusion. Theragnostic use combining therapy with delayed imaging remains to be explored. This review updates MnDPDP and its clinical potential with emphasis on the working mode of an exquisite chelate in the diagnosis of heart disease and in the treatment of oxidative stress.


Background
MRI is an imaging modality which in its native form produces important diagnostic information with purely instrument-based techniques [1]. Diagnostic routine on the other hand commonly relies upon the use of intravenous (i.v.), extracellular (EC) contrast agents containing gadolinium (Gd). At present, new contrast agent free (native) techniques are advancing into clinical practice whereas a strong standing of Gd agents seems reaffirmed after linear and semistable chelates were discarded and by paying attention to kidney function [2].
Still, there is a demand for new contrast enhancing techniques with properties beyond the scope of both native and Gd based MRI. Especially, there is a high need for agents that enable imaging and quantification of tissue viability at a cellular or close to molecular level. In addition to ensuring efficacy and safety, preferred new agents should be able to improve upon the treatment of patients undergoing diagnostic imaging. In retrospect, such an agent, manganese (Mn) dipyridoxyl diphosphate (MnDPDP), has already been available but vanished before its potential was recognized by the imaging community.
Paramagnetic Mn 2+ was the first metal ion studied for contrast enhancement in MRI [3], but fear of cardiotoxicity and rapid progress of Gd agents restrained the development of Mn-ion-enhanced MRI (MEMRI) [4,5]. As a consequence, MnDPDP (Teslascan ™ , GE Healthcare, Oslo, Norway) became the only i.v. Mn agent for human use ( Figure 1) approved for imaging of liver and pancreas [6,7]. After a decade low product earning led to cessation of marketing (USA 2003) or direct market withdrawal (Europe 2011). At that time intracellular (IC) Mn 2+ was recognized as an excellent biomarker of cellular events in various tissues and organs including heart and brain, but mainly in animals [8][9][10][11][12][13][14][15] and only partly in humans [16][17][18][19]. In parallel, human studies of MnDPDP and its key metabolite MnPLED (Mn dipyridoxyl diethyl-diamine) as small molecular catalytic antioxidants controlling reactive oxygen and nitrogen species (ROS, RNS) were in an early phase [20][21][22][23][24][25]. e aims of the present review are twofold: to focus on a multifunctional chelate with highly differing functions and mechanisms (basic properties) and with early examples from human use to indicate its future possibilities in MEMRI and therapy (application in humans).

Basic Properties
e behavior of MnDPDP as chelated prodrug in medical biology represents a blend of disciplines, ranging from physics and chemistry to pharmacokinetics and physiology in health and disease. From traversing these fields, come the basics of MRI and of antioxidant treatment. In spite of an inherent complexity, interactions between multiple factors seem mostly fortuitous.
A prerequisite for diverse functions of MnDPDP and MnPLED is a chelator being able to release and bind biologically active metal ions in a highly hierarchic manner ( Figure 2). Accordingly, with DPDP and PLED the log conditional stability constants [27], a main index of metalchelator affinities, for Mn 2+ are well above those of calcium (Ca 2+ ) and magnesium (Mg 2+ ) but also well below those of zinc (Zn 2+ ) and of copper (Cu 2+ ) and far below those of iron (Fe 3+ ). Accordingly, in tissue compartments MnDPDP and MnPLED undergo successive transmetallation steps  depending mainly on chelator-metal affinities (log values) and only partly on concentrations of Mn 2+ and competing cations [26][27][28][29]. Of prime importance is that Mn 2+ displaces Ca 2+ from binding to physiological ion channels in the cell membrane and to IC storage and release sites.
Zn 2+ , with log value of 19.0 (16.7) in binding to DPDP (PLED) and relative abundance in plasma and interstitium, is a powerful transmetallator of Mn 2+ with 15.1 (12.6) and, in retrospect, also of Gd 3+ in gadodiamide with reported 14.9 [30]. With far higher log values, traces of Fe 2+ may outstrip any other endogenous cation from binding to DPDP and PLED. Accordingly, i.v. administration of MnDPDP in humans caused a transient fall not only in plasma Zn 2+ [29] but also in serum Fe 2+ [6], with bottom reached at 2 hours and returning to baseline at 24 hours. Furthermore, a comparison with clinical chelators reveals that the in vitro log values of FeDPDP (33.5) and FePLED (36.9) [27] are as high as or even higher than those reported for, respectively, deferoxamine (31) and deferitazole (33.4) [31].
Like other metal ions, endogenous Mn 2+ appears bound, mainly to large molecules in plasma and cytosol and in organelles where Mn 2+ attains catalytic functions [32][33][34]. A model role is shown in mitochondrial superoxide dismutase (SOD) containing Mn 2+ -Mn 3+ as redox pair in its catalytic site (MnSOD). Another consequence of macromolecular binding is an increase in the rotational correlation time between Mn 2+ and protons in water, thereby greatly enhancing r 1 of potential Mn 2+ adducts [26].

Biotransformation in Human
Volunteers. According to a thorough review by Toft et al. [29], i.v. administered MnDPDP distributes and releases active metabolites in plasma and interstitium (Figure 3(a)). In one pathway, µmolar Zn 2+ transmetallates 75-80% of Mn 2+ in a clinical dose of MnDPDP (5-10 μmol/kg) for stepwise uptake in target cells. After bolus injection of 5 and 10 μmol/kg, about 20% of Mn 2+ is released within 2 min by µmolar Zn 2+ present in plasma. ereafter about 50% is released in a delayed manner by gradually available Zn 2+ and possibly by millimolar Ca 2+ and Mg 2+ within the interstitial space.
In another pathway, alkaline phosphatase (ALP) in plasma and microcirculation [29] converts water soluble MnDPDP (ZnDPDP) via monophosphate MnDPMP (ZnDPMP) to lipid soluble MnPLED (ZnPLED). Dephosphorylation enables MnPLED to diffuse across cell membranes and even enter organelles like mitochondria. e 20-25% end product MnPLED disappears from plasma over 60-90 min, whereas ZnPLED remains detectable up to 8 hours. Elimination from the body differs between Mn 2+ and its ligands [29]. e liver acts as a Mn 2+ sink with rapid turnover from plasma and hepatobiliary excretion, while the chelating moieties undergo renal elimination. e administered Mn is recovered within 1-2 hours (urine 25%), few days (feces 50-60%), and weeks (feces). 2+ . Mn 2+ is a most potent stalker of Ca 2+ , conductor of both cell function and energy metabolism. Hence a graded Mn 2+ uptake and retention in cardiomyocytes and other excitable cells (Figure 3(b)) mirror the activity of Ca 2+ transporters and IC ligands to which Mn 2+ has a higher affinity [8,9,12,[35][36][37]. Mn 2+ entry into target cells like cardiomyocytes occurs predominantly via voltage dependent L-type Ca 2+ channels that open briefly during depolarization [35]. Transient receptor potential (TRP) operated Ca 2+ (and Na + ) channels [37] and bidirectional Na + /Ca 2+ exchangers (NCXs) [36] may also mediate Mn 2+ influx or retention, probably more in injured than normal cardiomyocytes or in myofibroblasts during repair. Mitochondrial Mn 2+ entry is via a Ca 2+ uniport and exit from mitochondria and cytosol occurs via  NCXs. Neuronal Mn 2+ uptake occurs via N-type Ca 2+ channels but requires prior transport over the blood-brainbarrier (BBB) and diffusion via cerebrospinal fluid [14][15][16]. Divalent metal ion transporters [38] are active in longer term cell exchange of Mn 2+ . e use of Mn 2+ as a Ca 2+ analog to study normal physiology and contrast enhancement in the animal heart and brain have been highlighted in reviews not dealt with here [8,9,[12][13][14][15]. However, repeated notions of MnDPDP being a cardiotoxic agent still deserve comment [4,8]. us the high affinity to Ca 2+ channels may in theory depress cardiovascular function during high dose and rapid i.v. administration of Mn 2+ -releasing agents.

Cell Mn 2+ Uptake and Competition with Ca
is was exemplified by Wolf and Baum with MnCl 2 in anesthetized animals in the early days of MRI [4].

Safety and Brain Accumulation.
MnDPDP has an about 10 times higher safety margin than MnCl 2 reflecting a more gradual release of Mn 2+ [8]. In offspring of rats both agents produced skeletal defects related to Mn only [42]. Mn 2+releasing agents are thus contraindicated in early pregnancy and preferably in patients with pheochromocytoma. In humans, mild transient side effects mediated by nitric oxide (NO) [20,43] like flushing, occasional headache, and mild diarrhoea are observed during high dose infusion or rapid injection of MnDPDP [6].
In the adult human body, the Mn content, 10-20 mg (182-364 μmol) [32,33], is in the order of an imaging dose. Still transient accumulation in most tissues seems to be well tolerated. An important exception is the brain where a transient and limited Mn 2+ uptake may become a safe tool in functional MRI while a persistent Mn elevation in basal ganglia may induce oxidative injuries. Also Parkinson-like symptoms are feared outcomes from long term exposure to Mn metal whether being environmental, following total parenteral nutrition, or being caused by liver failure [33,44,45]. Importantly, with MnDPDP, single doses up to 25 μmol/kg were applied in phase II trials without reported signs of Parkinsonism [6], and based on the success with MEMRI for study of brain physiology in animals [14,15] Reich and Koretsky are exploring the possibility of using MnDPDP to image neuronal activity and neural tracts in patients with multiple sclerosis [46]. However, Sudarshana et al. recently reported [47] that i.v. infusion of a standard imaging dose (5 μmol/kg) of MnDPDP in healthy human volunteers raised signal intensity (SI) in exocrine glands in the head and neck, in the choroid plexus, and in the anterior pituitary gland but not beyond the intact BBB.

MEMRI and Contrast
Enhancement. MR properties of IC Mn 2+ , as the agent that ultimately shortens longitudinal relaxation time (T 1 � 1/R 1 ) but to a lesser degree transversal relaxation time (T 2 � 1/R 2 ) of excited protons, have been studied mostly with use of MnCl 2 as Mn 2+ -delivering agent. Main mechanisms influencing efficacy of Mn 2+ enhancement in a highly excitable tissue like the LV myocardium have been comprehensively analyzed by Seland et al., Hu et al.,. Using relaxography to examine small animal hearts, mostly additive factors related to T 1 behavior, R 1 -Mn relationships, macromolecules, and field dependence were studied.

Monoexponential T 1 Relaxation.
In the rat heart, a high transmembrane water exchange rate (∼10 s −1 ) caused tissue T 1 relaxation, representing the sum of IC and EC water protons, to become monoexponential. Only after an extreme Mn 2+ overload was a second, probably mitochondrial, T 1 peak disclosed.

Correlation between R 1 and Mn Content.
A linear correlation was found between tissue R 1 and Mn content up to about 10 times normal, i.e., from about 45 to about 500 μmol/kg dry wt. is makes R 1 a reliable parameter of Mn 2+ uptake and cell function whereas MEMRI of mitochondria, otherwise an exciting target, becomes less likely without supplementary MR techniques [48,50]. As expected, the about one order of magnitude higher R 1 of bound vs. free Mn 2+ makes MEMRI possible with a low µmolar dose of a Mn 2+ -releasing agent.

Magnetic Dispersion and Resolution: Low vs. High Field
Imaging. A limitation is that magnetic dispersion above 0.2-0.5 Tesla (T) [24] reduced tissue r 1 (s −1 ·mM −1 ) from 40-50 at 0.5 T, to 30-35 at 2.35 T, and to 20-25 at 7 T [48,50]. Conversely, compensating for a reduction in r 1 of Mn 2+ adducts at higher fields, the signal to noise ratio (SNR) in T 1 weighted images (T 1 WI) increases by at least one order of magnitude. Furthermore, the scale for measuring tissue T 1 expanded by about 30% (native gain) and 40% (Mn 2+ -enhanced gain) when raising the field strength from 0.5 T to 7.0 T [26,50].
Taken together, MEMRI with IC Mn 2+ adducts can be applied for both low (0.5-1.5 T) and high field (3.0-7.0 T) imaging. In the heart, a further advantage is that MEMRI may comply with and improve upon recent and impressive achievements in native T 1 -based methods [51,52].

MEMRI vs. Gd-Based MRI.
e efficacy of MEMRI is, as expected, also highly influenced by physiologic and pharmacokinetic factors which differ from Gd based MRI. In theory, IC Mn 2+ uptake requires an active metabolism and function and requires that healthy cells retain Mn 2+ by strong IC binding and slow efflux. Contrary to this, EC Gd agents accumulate briefly within the interstitial, including disrupted IC, water phase. Consequently, when measuring myocardial infarct size (IS) in rats with permanent coronary artery ligation (Figure 4), IC Mn 2+ adducts lower T 1 mainly in viable cardiomyocytes while Gd-complexes do so in dead or severely injured tissue (Bruvold M, Seland JG, Jynge P, unpublished material).

Tissue Protection in Oxidative
Stress. Following a side track from contrast agent research into the field of "oxidative," i.e., combined oxidative-nitrosative, stress and antioxidants [53][54][55], Asplund et al. discovered that MnDPDP and MnPLED dilated arteries [20] by mimicking MnSOD, with the proposed mechanism that suppression of superoxide preserved endothelial derived NO for activation of adenylate cyclase and cyclic GMP thereby relaxing vascular smooth muscle cells [43].
Experimental data indicate that both EC MnDPDP and IC MnPLED can be characterized as small molecular enzyme mimetics endowed with catalytic antioxidant properties ( Figure 5). In acute or subacute conditions of oxidative stress and inflammation, they seemingly act in either of two ways: by supplementing SOD activity in plasma and IC and by binding prooxidant metals like Cu + and Fe 2+ which leak from IC sites [34,[56][57][58][59]. MnDPDP and MnPLED may thereby improve the balance between salient (low-level) and damaging (highlevel) ROS-RNS: by preserving NO and hydrogen peroxide for cell signaling [53,54,59] and by inhibiting release of superoxide, hydroxyl (OH), and peroxynitrite (ONOO − ) [43,55,56,59]. Other secondary mechanisms may include stabilization of lysosomes and mitochondria [60,61]. Altogether, these properties make MnDPDP a promising drug delaying tissue injury and inhibiting inflammatory responses. A further implication of strong chelator binding of Fe 2+ , besides inhibiting oxidative stress in severe inflammation, is an apparent potential to slow replication of rapidly dividing malignant cells [58,62] and microorganisms [63].

MEMRI in Humans
As amply documented in animals and partly confirmed in humans, MEMRI enhances tissue contrast by Mn 2+ uptake and retention in excitable cells in liver, pancreas, kidney cortex and medulla, myocardium, endocrine and exocrine glands, and potentially retina and brain [4, 8-19, 39, 40]. With MnDPDP, preclinical studies were frequent prior to or just after the millenium shift, and readers are referred to comprehensive reviews from that time [8,9,11,13, 16, 39,
In LV myocardium, an optimal dose of MnDPDP (5-10 μmol/kg) lowered T 1 to midway (∼725 ms) between native values (1020 ms) and reported Gd-enhanced values (350-550 ms) [76]. Importantly, delayed MEMRI, highly feasible within 3-4 hours, provides an advantage for exploitation in patient turnover, in screening of viability, and potentially in theragnostic use of MnDPDP. In liver, a stable time window was shorter, 1-2 hours. e high tissue R 1 , however, makes it possible to quantify liver function and viability by a dose far lower than 5-10 μmol/kg. 2+ Uptake. Myocardial Mn 2+ uptake from MnDPDP was monitored by continuous online recording of R 1 in healthy young adults [18]. With the same dose (5 μmol/kg), duration of infusion ( Figure 9) presented different profiles for ΔR 1 and Mn 2+ uptake, biphasic (5 min) or linear (30 min). On the other hand, ΔR 1 over 40 min did not differ between infusion groups (5 min, 0.32 s −1 ; 30 min, 0.35 s −1 ).

Analysis of Mn
When a tracer kinetic model, based on cell influx of Mn 2+ from an assumedly reversible (EC) into a largely irreversible (IC) compartment [79], was applied to the R 1 curves, an unidirectional influx constant for Mn 2+ (K i ) was measured as an index of Ca 2+ channel activity. As revealed in kinetic (Patlak) plots, the resulting K i values (arbitrary units) were identical in the two infusion groups, 5 min (5.73) and 30 min (5.72). An attempt to measure tissue fraction of the Mn 2+ -donating compartment, i.e., the EC volume (ECV), revealed results far from an expected 25% level.
With adjustment of infusion time measurements of K i and possibly of ECV, the latter a hallmark of Gd-based MRI [1,[80][81][82], may become exquisite tools in clinical physiology. It is also attractive to assess myocardial L-type Ca 2+ channel activity [35], with contribution by other Ca 2+ transporters [36,37] in disease. Interestingly, the utility of MnDPDP in tracking Ca 2+ channel activity has been confirmed in a meticulous study of retinal function in light-vs. darkadapted rats [83].

Detection of Myocardial Ischemia by Stress Testing.
In animals, MEMRI can detect myocardial ischemia on its own [9,40] by revealing diminished Mn 2+ uptake and ΔR 1 in an ischemic region. Detection is strengthened, however, by infusion of the β-adrenergic agonist dobutamine which enhances inotropy and Mn 2+ uptake in nonischemic remote regions. Efficacy of MEMRI in dobutamine testing requires highly mobile Mn 2+ in plasma and interstitium, as was first demonstrated by Hu and Koretsky with MnCl 2 in rats [12] and later confirmed by Eriksson and Johansson with a low affinity Mn-chelate in pigs [84]. With MnDPDP, however, Mn 2+ release is too slow as documented by Amundsen et al. in human volunteers [71]. Hence, infusion of MnDPDP (5 μmol/kg in 5 min) during dobutamine stress (10 min) did not raise myocardial R 1 above the rest level.
Interestingly, native T 1 mapping in patients with coronary artery disease [52] has shown that increases in myocardial blood volume (MBV) during vasodilation by adenosine, minimal in infarcted vs. maximal in remote regions, were paralleled by transient increases in T 1 (0.2% vs. 6.2%). With infusion of adenosine in due time after MnDPDP infusion, an infarct-to-remote T 1 gradient may be no less. Stress testing with adenosine after myocardial Mn 2+ enhancement with MnDPDP may thus be an interesting option to pursue.

Cardiac Injury and Repair in Patients.
Clinical reports with MnDPDP or other Mn 2+ -releasing agents concern cardiac remodeling following a previous AMI [19,[72][73][74]. In 2003, a congress abstract from Abolmaali et al. [72] reported that MnDPDP (10 μmol/kg) reduced LV myocardial T 1 at 1.5 T, from 550 ms to 450 ms in healthy volunteers (n � 9) and from 815 ms to 630 ms in patients with impending heart      Contrast Media & Molecular Imaging failure (n � 7). Unfortunately, these early data were not presented in a complete paper. Present MRI techniques to describe the complex pathophysiology of cardiac remodeling [85][86][87] are based on signs of edema and fibrosis by delayed contrast enhancement with EC Gd agents or by native T 1 mapping and detection of deficient contractile function by cine-MRI [1,5,[80][81][82]. In 2007 Skjold et al. [19] applied MnDPDP to measure sectorwise myocardial viability by R 1 and systolic wall thickening (SWT) in patients 3-12 weeks after AMI treated with primary Percutaneous Coronary Intervention (pPCI). Ten patients were examined by dual imaging, i.e., before and after i.v. infusion (5 min) of MnDPDP (5 μmol/kg). T 1 WI after MnDPDP (Figure 10) demarcated infarcts in 4 patients only but revealed increase in remote wall thickening in 9. Importantly, in these 9 patients sectorial LV maps of R 1 and SWT showed identical directions of growing infarct-to-remote gradients. Mn 2+ -uptake was biphasic in remote sectors but monophasic and smaller in the infarcted sectors. In one patient no change from normal appeared, and confirmed clinical indices of myocardial salvage.
A limitation to the above technique is the lack of finer details in R 1 distribution since only a single mean R 1 value represented each sector and more detailed R 1 guided colour coding was not applied. Still, the accumulated data from all patients and sectors showed that SWT (range 0-5 mm) correlated significantly with both native R 1 and R 1 after MnDPDP. Moreover, infarct-toremote R 1 gradients (Figure 11(a)) were significant both before, 0.87-0.96 s −1 (ΔR 1 0.09 s −1 ), and after, 1.11-1.35 s −1 (ΔR 1 0.24 s −1 ), MnDPDP.
ese findings, as also presented in a T 1 -SWT diagram (Figure 11(b)), illustrate in a quantitative manner parallel but supplementary aspects of myocardial injury and remodeling. While native T 1 maps present overall tissue conditions rather evenly [1,81,82] with main emphasis on EC events, T 1 maps after Mn 2+ enhancement encompass conditions in the major IC compartment. Accordingly, native MRI reflects edema plus fibrosis whereas MEMRI mainly reveals energy state and Ca 2+ control in cardiomyocytes. R 1 elevation in revascularized infarct sectors with assumedly dead tissue (Figures 10 and 11) seemed a puzzling finding. Partial elevation of R 1 in the infarct, as also observed in rat hearts (Figure 4) [85,87].
Altogether, although small the study Skjold et al. provides a snapshot of how MEMRI might be exploited in the human heart. Both single imaging (MEMRI delayed or online) and dual imaging (native MRI + online MEMRI) may become attractive tools for an in-depth analysis of myocardial pathophysiology, not least when combined with more recently developed mapping techniques.
3.6. Experience with DEMRI plus MEMRI. In 2014, Matsuura et al. [73] reported dual contrast imaging in patients (N = 5) with ischemic cardiomyopathy using delayed enhancement MRI (DEMRI) with gadopentetate dimeglumine to be followed by MEMRI with use of EVP1001. e latter is a rapid Mn 2+ -releasing gluconate salt supplemented with Ca 2+ (SeeMore ™ , Eagle Vision Pharmaceuticals, USA). e DEMRI, infarct plus peri-infarct (PIR), region and the infarcted MEMRI region measured by T 1 mapping at 3.0 T revealed these volumes: DEMRI 34%, MEMRI 14%, and by subtraction PIR 20%. However, being effective in detecting the PIR for potential revascularization, the reported procedure required administering two contrast agents in two separate imaging sessions.

Recent Studies of MEMRI with MnDPDP in Animals.
Two recent reports from in vivo rats deserve comment as they apply current techniques to provide up-to-date information on MnDPDP as a biomarker of widely differing tissue injuries.
In 2018, Spath et al. published an in vivo rat heart study [88] with measurement of myocardial infarct size (IS) 3 and 12 weeks after AMI. In introductory experiments, the T 1 reducing capacity of EVP1001 (22 μmol/kg) and MnCl 2 (22 μmol/kg) in normal myocardium at 7.0 T was twice that of MnDPDP (44 μmol/kg). Still, AMI measurements of IS by use of EVP1001 (n � 6) and MnDPDP (n � 7) were obtained with equally high accuracy when compared to histology. DEMRI with gadobenate dimeglumine (500 μmol/kg) applied in prior separate experiments was reported as less accurate than MEMRI in defining IS by including peri-infarct edema and fibrosis. In 2020, Liu et al. [89] reported on the use of MnDPDP (25 μmol/kg) and MEMRI to predict the therapeutic efficacy of a vascular disrupting anticancer agent (VDA) in rats with primary and secondary malignancies of liver. Tumor-to-liver contrast at 3.0 T was judged by tissue SI, and results were closely compared with postmortem microangiography and histology. VDA-mediated intratumoral necrosis was imaged by use of gadoterate meglumine (200 μmol/kg). Important findings ( Figure 12) were first that tumor-toliver contrast enhancement by MnDPDP was strong in highly (grade I) and weak in lowly (grade III-IV) differentiated hepatocellular carcinoma (HCC) before treatment. Secondly, the necrotic responses to the VDA assessed by Gd-MRI correlated with the grade of differentiation, i.e., major in high and minor in low grade HCC. 24-hour delay in imaging after infusion of MnDPDP avoided transient blood pool effects and improved the contrast between the HCCs and liver. e study confirms that MEMRI with MnDPDP represents a noninvasive surrogate for biopsy taking in primary liver cancer.

Therapy in Humans
ree small scale feasibility studies [23−25] and one case report [22] indicate that MnDPDP may provide clinically relevant cytoprotection in humans. [25]. With the aim of preventing reperfusion injury during pPCI, patients submitted with their first episode of AMI were randomized to receive 2 min i.v. infusion of MnDPDP (2 μmol/kg) or placebo (NaCl) immediately after angiography but prior to the reopening of a culprit coronary artery branch. e infusions were without side effects. As reported by Karlsson JE et al., the MnDPDP group revealed an unfavorable distribution of patients (Table 1), fewer intraventricular thrombi, and a trend towards more rapid reversal of ECG changes, but the remaining results did not reveal differences between groups. us, a tendency to potential benefit in few patients needs confirmation in a larger phase II trial, preferably based on an improved protocol.

Chemotherapy of Cancer and Adverse Events (AEs).
MnDPDP has been applied to patients with colorectal adenocarcinoma undergoing repeated treatment cycles with the platinum derivative oxaliplatin and 5-fluorouracil [22][23][24]. Severe adverse events (AEs) of oxaliplatin like painful acute or chronic peripheral sensory neuropathy (PSN) and bone marrow depression are closely related to oxidative stress [24,62,65,66]. Importantly, chronic PSN may be caused by prooxidant platinum ions (Pt 2+ ) accumulating in pain-conducting dorsal root ganglion cells [24].

Case Report.
e first patient to receive MnDPDP for therapy was a young male who received palliation by 14 cycles of oxaliplatin, each supplemented with MnDPDP HCC grade I HCC grade III T 2 WI T 1 WI MnDPDP T 2 WI T 1 WI MnDPDP Figure 12: Predictive imaging prior to therapy of rat livers with hepatocellular carcinoma (HCC) of high (I) and low (III) grade of differentiation [89]. MnDPDP raised tumor-to-liver contrast in T 1 WIs, see arrow, in grade I HCC to the left, but hardly in grade III HCC as depicted to the right (reproduced with permission from Transl Oncol). Contrast Media & Molecular Imaging 11 e regimen went without PSN or reduction in white blood cell count (WBC), and there was a surprising lowering of pain. After 8 months, the patient developed a mild hand tremor as a potential early sign of Parkinsonism. en, MRI of the brain ( Figure 13) showed widely distributed Mn deposits [44,45] with maximal SI in basal ganglia including dentate nucleus and globus pallidum. As recently discussed by Blomlie et al. [90] these basal ganglia sites are also noted for deposition of Gd 3+ [91] indicating a common, possibly Ca 2+ related, pathway for focal brain storage of these metals.
Mn deposition outside the basal ganglia indicated a most extensive brain overload due to additive predisposing factors: a too high total dose vs. time of MnDPDP; a marked influence by concomitant liver failure; and probably also a BBB weakened by disease and/or by chemotherapy [33,44]. e case illustrates that, with a potential exception for end stage palliation, there is a need for dose reduction and attention to liver function and BBB integrity in multiple administrations of MnDPDP.

Prevention of Acute Toxicity.
In the first feasibility study of cytoprotection of normal tissues, Karlsson et al. [23] examined a small group of patients with locally advanced cancer receiving 3 cycles of oxaliplatin, with each cycle preceded by a low dose of MnDPDP (2 μmol/kg) or saline (placebo). Main significant findings with MnDPDP compared to placebo were a higher WBC after these cycles and almost absence of grade II-IV AEs. In particular, life threatening or severe AEs were only observed in the placebo group (Figure 14(a)).

Prevention and Reversal of Neurotoxicity.
In another feasibility study, Coriat et al. [24] examined patients with PSN already detected in prior oxaliplatin cycles who received 4-8 further cycles, but now with preinfusion of MnDPDP (5 μmol/kg). After introducing MnDPDP, the PSNs became fewer and less severe (Figure 14(b)), indicating both prevention and reversal of nerve toxicity. ese benefits were partly explained by acute MnSOD mimetic actions. Another likely mechanism implies chelation and elimination of oxidizing metals including platinum ions (Pt 2+ ) released from oxaliplatin, an interpretation supported by EPR analysis revealing a Pt 2+ affinity to DPDP close to that of Cu + [92]. With an accumulated MnDPDP dose up to 40 μmol/kg over 4 months in Coriat's study, plasma Mn (Figure 14(c)) rose gradually without exceeding normal levels [33]. ere were no signs of Parkinsonism or bone marrow depression. e two latter studies indicate that MnDPDP in a low imaging dose (2-5 μmol/kg) at timely intervals (2-4 weeks) and with attention to liver function may prevent and reduce severe AEs in repeated (4-8) cycles of chemotherapy without causing any undue Mn accumulation as shown in the case report. e studies were too small, however, to indicate any effect upon tumor growth.

Experience with a Derivative of MnDPDP.
[Ca 4 Mn(DPDP) 5 ] (calmangafodipir, PledOx ™ , Aladote ™ , PledPharma AB, Sweden) was developed with the aim of combining efficacy in therapy with reduced brain Mn 2+ uptake [59]. In a phase II trial, PledOx seemingly prevented oxaliplatin-induced PSN after 3 and 6 months of follow-up, but after Figure 13: Brain MRI in a patient receiving MnDPDP 140 μmol/kg over 8 months [22,90]. MnDPDP (10 μmol/kg) was applied as cytoprotective adjunct to 14 cycles of chemotherapy with oxaliplatin as the primary drug in a patient with cancer of colon. MRI of the brain (1.5 T) was undertaken after the last cycle. Sagittal and parasagittal images (A-B, a-b) were obtained by T 1 W-FLAIR and descending axial images (C-D, c-d) by T 1 W-SE. High SI reflects marked Mn deposition in: A-a, corpus callosum (open arrow), mesencephalon (thick white arrow), and pituitary gland (thin white arrow); B-b, C-c, putamen and globus pallidus (L nucleus lentiformis) and caput nucleus caudatus (N); D-d, cerebellum with nucleus dentatus (curved white arrow) and brain stem (white angled arrow) (Blomlie V, Jynge P., unpublished images). 9 and 12 months, there were no differences between treated and nontreated groups [93]. In ongoing trials, paracetamol-overdose patients are given Aladote as supplement to the standard antidote N-acetyl-cysteine (NAC), and initial phase I data indicate suppression of early biomarkers of liver injury [94].

Back to the Future
In reappraising principle and agent for diagnostic imaging MEMRI and MnDPDP provide unique possibilities to quantify tissue function and viability at a cellular and subcellular level, with T 1 mapping being more effective than T 1 WI. Administration of MnDPDP outside or inside the magnet enables examinations ranging from screening of heart disease and of arrhythmias to in-depth studies of cell Ca 2+ fluxes and possibly measurement of ECV. Detailed information about injury, repair, and remodeling may also be obtained by dual imaging combining native MRI with MEMRI. e above options may benefit from and potentially improve recent achievements in native MRI. With sharper delineation of cardiac anatomy, cine imaging and tagging of regional contractile function are distinct possibilities to exploit [51]. e same applies to myocardial T 1 mapping in general and during adenosine stress to quantify MBV [52] or to measure perfusion by arterial spin labeling [95]. Hence, MEMRI with MnDPDP may give comprehensive information about myocardial viability, function, and perfusion, i.e., key indicators predicting the need for invasive coronary angiography or reducing the need for endomyocardial biopsies.
Against a future breakthrough speak a renewed position of Gd based MRI and the greater T 1 shortening capacity of Gd agents compared to MnDPDP. In addition, recent improvements in native MRI may question the need for contrast agents [1,51,52,82]. Notwithstanding, the IC approach with direct access to cardiomyocytes, multifunctional properties, and a potential to replace isotope scanning support a future role of cardiac MEMRI with MnDPDP. Likewise, quantification of viability is a unique principle which may be adopted for other organs like liver, pancreas, kidney, endocrine, and exocrine glands, subjected to tissue injury and repair.
Of particular advantage is that cytoprotection offered by MnDPDP may both increase the safety and extend the diagnostic applications. A major problem in cardiovascular disease and in diabetes refers to the use of contrast media in patients with impaired kidney function. At present, the intravascular, nanoparticular, and iron oxide-containing compound Ferumoxytol, mainly a T 2 or T 2 * agent, serves as a safe substitute for Gd compounds in MRI of kidney [96]. Interestingly, with transient renal perfusion with MnDPDP including MnPLED and uptake/retention of paramagnetic Mn 2+ in the cortex, MnDPDP might become attractive as a safe alternative. What is essential for safety is conservation of NO, a mediator of intrarenal perfusion and key to kidney preservation [97]. With an apparent cortex-to-medulla T 1 gradient and long imaging window [13,16], MnDPDP might also be effective in imaging of renal diseases. Altogether, combining imaging with potential tissue protection, hitherto not tested in the human kidney, may become an important option to pursue.
Since MnDPDP both images and preserves viable myocardium, theragnostic use seems a distinct possibility, for example, in AMI, the post-cardiac-arrest syndrome, and heart failure with inflammation and oxidative stress. A particularly important scenario may be its use as cytoprotective and diagnostic adjunct to chemotherapy with anthracyclines [58,70,98] which cause both acute and chronic heart failure at least partly due to production of ROS-RNS. In spite of limited or no success with scavenging agents [98], it still seems rational to attack the problem with a potent catalytic antioxidant acting at both initial and subsequent steps in a prooxidant cascade. MnDPDP may  [23]. AEs of grade I (mild), II (moderate), III (severe), and IV (life-threatening) were recorded in 14 patients during 3 therapy cycles with oxaliplatin and with preinfusion of MnDPDP 2 μmol/kg or saline (placebo). ere was a major reduction in AEs grade II-IV with MnDPDP. Also plasma leukocyte content was maintained at a higher level with MnDPDP (reprinted with permission from Translational Oncology). (b) Peripheral sensory neuropathy (PSN) [24]. Patients that experienced PSN during previous oxaliplatin cycles were followed for up to 8 further cycles, each with preinfusion of MnDPDP 5 μmol/kg. In these cycles, MnDPDP gradually reduced the initial severity of PSN (black > dark gray > light gray) indicating a reversal of the underlying nerve injuries (reprinted with permission from J Clin Invest). (c) Plasma [Mn] (nmol/L) during therapy with oxaliplatin and MnDPDP [24]. Patients cited in B showed a gradual rise in plasma [Mn] over 8 cycles in 4 months without exceeding normal levels of 10-20 nmol/L [29,33] (reprinted with permission from J Clin Invest).
here be given as a cytoprotectant at onset of each treatment cycle while serving as a contrast agent for delayed imaging and monitoring of myocardial viability.
A parallel indication concerns the liver in abdominal cancer. In hepatic failure induced by paracetamol [66,94] or by other etiology (hepatitis), low-dose MnDPDP may become both therapeutic drug and biomarker. A further option is in the transplantation field with imaging and protection of donor cells and organs as well as of the recipient. Stem cells in general [99] and pancreatic islets [100] together with cardiac, liver, and kidney transplants might become likely candidates.
"Manganese and MRI" reveals a current annual publication rate of about 100, but with more focus on new and stable macrocyclic chelates or (nano)particulate matter than on Mn 2+ -releasing agents as is required in MEMRI. us Mn 2+ apparently substitutes for Gd 3+ in novel highly stable complexes designed for EC, intravascular, or molecular-targeted deliveries [101,102]. With exception of EVP1001 [73] MEMRI has not materialized in new i.v. formulations for trial in humans. Of considerable interest, though, is the recent indication in animals [103] of efficacy of a miniature dose of a 52 Mn tracer with MEMRI-like properties in PET of the brain, thereby offering promise for functional PET/MRI.

Conclusion
Attempts are now made to reposition MnDPDP for diagnostic use in both the USA [46] and Europe [104]. With current insight into its work mode in MEMRI and in treating conditions of oxidative stress, previous indications are open for immediate use and new possibilities appear ready for offlabel assessment of a future potential. e challenge will be to develop MEMRI and MnDPDP for use in daily routine and not only as exciting tools in clinical research. orough clinical trials are thus required.

Conflicts of Interest
Jynge, Skjold, and Eidsaunet own shares in the Norwegian R&D company IC Targets AS that attempts to reintroduce MnDPDP for diagnostic use. Andersson and Karlsson own shares in the Swedish company PledPharma AB that promotes derivatives of MnDPDP for therapy. Jynge, Skjold, Andersson, and Karlsson are inventors of patents involving MnDPDP for diagnosis and/or therapy. Falkmer, Bruvold, Seland, and Blomlie declare no conflicts of interest.