Classics in Neuroimaging: Development of PET Tracers for Imaging

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Classics in Neuroimaging: Development of PET Tracers for Imaging Monoamine Oxidases Vidya Narayanaswami,†,∥ Lindsey R. Drake,‡,§,∥ Allen F. Brooks,‡ Jeffrey H. Meyer,† Sylvain Houle,† Michael R. Kilbourn,‡ Peter J. H. Scott,*,‡,§ and Neil Vasdev*,†

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Azrieli Centre for Neuro-Radiochemistry, Research Imaging Centre, Centre for Addiction and Mental Health & Department of Psychiatry, University of Toronto, Toronto, Ontario M5T-1R8, Canada ‡ Division of Nuclear Medicine, Department of Radiology, University of Michigan Medical School, 1301 Catherine Street, Ann Arbor, Michigan 48109, United States § The Interdepartmental Program in Medicinal Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, United States ABSTRACT: In this Viewpoint, we highlight the history of positron emission tomography (PET) radiotracer development to quantify changes in monoamine oxidase (MAO)-A and -B enzyme expression or activity. MAO-A and MAO-B are critical for understanding monoaminergic pathways in psychiatric addiction disorders, and more recently in neurodegenerative disorders with MAO-B expression in astrogliosis. Unique radiochemical innovations have been shown for neuroimaging of MAOs including the clinical translation of irreversible propargylamine-based suicide inhibitors, application of deuterium-substitution to slow down metabolism, development of trapped metabolite imaging agents, and unique 11C-carbonylation chemistry toward novel high-affinity reversibly binding inhibitors. KEYWORDS: Monoamine oxidase, PET, carbon-11, fluorine-18, MAO-A, MAO-B

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The clinical relevance of MAO to numerous neuropsychiatric and substance abuse disorders, such as cigarette smoking, has fostered significant efforts in developing both carbon-11 and fluorine-18 labeled radiotracers for in vivo noninvasive positron emission tomography (PET) neuroimaging studies to investigate alterations of MAO in the human brain.1 Similar to all PET radiotracers for neuroreceptor mapping, the challenge for imaging MAO in the CNS has been in the design of a chemical compound whose kinetics and distribution reflect specificity for a single biochemical process and whose distribution parallels the known regional concentration of the specific MAO isozymes in different brain regions and in peripheral organs. The two isozymes, MAO-A and -B, have long been the target of drug development, with numerous compounds developed and marketed for an array of neurological and psychiatric diseases. This body of work has provided many chemical structures that have served as starting points for the development of PET imaging agents, and include irreversible inhibitors (e.g., propargylamines), reversible inhibitors (e.g., harmine (3) and SL2511.88 (4)), and substrates that form trapped metabolites for imaging (e.g., 1-

onoamine neurotransmitters including catecholamines (dopamine, epinephrine, and norepinephrine), indolamines (serotonin), and imidazoleamines (histamine) as well as trace amines (e.g., phenethylamine and tyramine) play critical roles in the central nervous system (CNS) that include oxidative stress, monoamine metabolism, and intrinsic apoptosis. There are also putative applications for imaging monoamine oxidase (MAO) in sympathetic neurons of the peripheral nervous system. The metabolism of monoamines is key to their importance in various disease states. MAO is an outer-mitochondrial membrane bound, flavin-containing enzyme which oxidizes monoamines. There are two isozymes, termed MAO-A and MAO-B, which are encoded by independent genes, have structural variants, different regional and cellular distributions, and exhibit distinct selectivities toward the various monoamine neurotransmitters. Impaired monoaminergic signaling underlies a large number of behavioral, neurodegenerative, and psychiatric disorders in humans. Specifically, inhibition of MAO-A is utilized in the management of depression and inhibition of MAO-B is one of the therapeutic approaches to treat Parkinson’s disease.1,2 Recent studies demonstrate upregulation of MAO-B in reactive astrocytes that are activated during neuroinflammatory processes, thereby suggesting the potential for measuring alterations in MAO-B as a marker of astrogliosis in Alzheimer’s disease (AD) and related dementias.3,4 © XXXX American Chemical Society

Received: February 4, 2019 Accepted: February 7, 2019

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DOI: 10.1021/acschemneuro.9b00081 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

selectively binds to MAO-A in the human brain, highlighting species differences in the susceptibility of MAO-A to inhibition by clorgyline. Stereoselectivity of MAO-B for L-deprenyl was confirmed by comparison of the brain uptake and retention of the 11Clabeled inactive (D-) and active (L-) enantiomers of deprenyl. Results showed rapid clearance of the inactive enantiomer and retention of the active enantiomer within MAO-B-rich brain regions. Pharmacokinetics of brain uptake and retention in human studies showed that the agents were likely flow limited due to irreversibile kinetics reflected in the time-activity curves. To improve the pharmacokinetics, deuterated analogues were prepared in an attempt to slow the rate of trapping using the kinetic isotope effect (KIE). The KIE can be utilized to slow the rate of reaction observed provided the abstraction of deuterium vs proton is the rate limiting step of the process. In the case of the propargylamines, this is the case and the slower to cleave C−D bonds vs C−H reduced the rate of irreversible inhibition. To that end, deuterium labeled isotopologues ([11C]clorgyline-D2 and [11C]L-deprenyl-D2 ([11C]DED) were successfully translated for human PET studies.1 While this approach had a significant impact for the imaging of MAO-B by [11C]DED, DED is still modeled as an irreversible PET radiotracer. As for [11C]clorgyline-D2, the reduction in the trapping rate resulted in a lower ratio of specific-to-nonspecific signal in the regions of interest, and did not improve its utility for MAO-A imaging. Comparative studies of [11C]clorgyline and [11C]clorgyline-D2 in the human brain revealed significant non-MAO-A binding of [11C]clorgyline-D2 in white matter and cerebellum confirming that the relative strength of the signal due to MAO-A binding is diminished by deuterium substitution as the slower rate of irreversible binding allows more of the PET tracer to wash out of the specific binding sites. As a result, [11C]clorgyline is superior to [11C]clorgylineD2 for human brain studies as the higher rate of covalent attachment is required to attain signal above the background observed due to nonspecific binding in the white matter. On the other hand, studies comparing [11C]L-deprenyl and [11C]DED in the human brain showed a robust deuterium isotope effect and provided a means of selectively controlling the rate of trapping of tracer in brain and enhancing sensitivity of binding to changes in MAO-B. Therefore, [11C]DED is the preferred MAO-B propargylamine agent for human brain imaging studies, and has been successfully employed to study the role of MAO-B in numerous neuro-PET imaging studies including nicotine dependence and aging populations. [11C]DED has also been used to investigate the role of MAO-B in various neurological disorders, with a particular interest in neurodegeneration. The colocalization of MAO-B expressing astrocytes and amyloid plaques has been shown in human brain sections, indicating the possibility of MAO-B involvement in AD. Additionally, [11C]DED binding on human tissue was determined to be highest in the earlier Braak stages. PET imaging using [11C]Pittsburgh compound B ([11C]PiB) has identified amyloid plaque load in AD and mild cognitive impaired patients (MCI). Carter and co-workers performed a multitracer trial using PET imaging with [ 11 C]DED in combination with [ 18 F]fludeoxyglucose ([18F]FDG, which demonstrates glucose metabolism) and [11C]PiB in a small cohort of age-matched normal, MCI and AD subjects.3 Based on the amyloid imaging results, the MCI subjects were then divided into PiB-positive ([11C]PiB+) and PiB-negative ([11C]PiB−) subgroups, and the [11C]DED

methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and a coumarin derivative, Cou, 5) (Figure 1). Each approach has

Figure 1. Selected radioligands for PET imaging of MAO-A and MAO-B.

presented its own challenges and limitations to the radiochemistry community, who have endeavored to develop MAO PET imaging agents useful for understanding MAO pathophysiology for improved patient management, and to support drug development. This Viewpoint aims to highlight the key advancements in the field of radiochemistry and PET imaging that have been instrumental in understanding the role of MAOs in the CNS. Herein, we will follow the history of development of MAO PET radiotracers, beginning with irreversible inhibitors and progressing through the use of reversible inhibitors and substrates. Our Viewpoint aims to serve to contribute toward the series of Viewpoints and Minireviews: Classics in Neuroimaging.

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IRREVERSIBLE INHIBITORS Early human translation of isozyme-selective MAO PET agents concentrated on irreversible (suicide) inhibitors, wherein, the labeled compound results in covalent binding to a catalytically active enzyme residue. Carbon-11 labeled N-methyl propargylanines, clorgyline (N-[3-(2,4-dichloro-phenoxy)propyl]N-methyl-2-propynylamlne; 1), and L-deprenyl (l-N,α-dimethyl-N-2-propynyl phenethylamine; selegiline; 2) (Figure 1) have shown to be selective irreversible inhibitors of MAO-A and MAO-B, respectively. Fowler and co-workers demonstrated the rapid brain uptake of 1 and 2 in humans and confirmed the irreversibility as demonstrated by reaching a plateau in the brain tissue time−activity curves.1 Moreover, the distribution of radioactivity paralleled MAO immunoreactivity in human brain tissue sections and was highest in the striatum, thalamus, and brainstem. These radiotracers were confirmed as being MAO inhibitors through a blocking study with phenelzine, a nonselective MAO inhibitor. It is important to note that interspecies structural differences exist for both MAO isoforms evidenced by immunological data that demonstrate that a monoclonal antibody raised toward human platelet MAO-B binds to human brain MAO-B but does not bind to the rat or mouse enzyme; In addition, in the rat heart, postjunctional extraneuronal MAO is found unlike in primates and humans. These differences should be taken into account during preclinical evaluation of a potential MAO imaging agent and when translating PET radiotracers to clinical studies. For example, [11C]clorgyline was not retained in the brain of baboon or rhesus monkey; however, it B

DOI: 10.1021/acschemneuro.9b00081 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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radiotracers (e.g., [11C]befloxatone, [18F]fluoroethyl-harmol) an important area of radiopharmaceutical chemistry. Elevated MAO-A total distribution volume (VT), measured with [11C]harmine PET, particularly in the prefrontal and anterior cingulate cortex, has also been demonstrated in a number of illnesses associated with depressed mood and in high risk states for major depressive episodes (MDE). Some of these illnesses include early withdrawal from alcohol dependence or heavy cigarette smoking, and borderline personality disorders. While all of these conditions are highly comorbid with MDE, and predispose to MDE, there are other conditions which frequently precede MDE; for example, early postpartum and perimenopause which are associated with elevated MAO-A levels across gray matter regions. Overall, [11C]harmine is a useful PET radiotracer for quantitative evaluation of MAO-A densities in several psychiatric and neurologic illnesses. In addition to the reversible MAO-A PET agents that have been evaluated in humans, a reversible carbamate-based MAOB reversible inhibitor [11C]SL25.1188 (4) was prepared and evaluated (Figure 2). Initial reports by Saba and co-workers

imaging studies were then used to evaluate if MAO-B imaging might be indicative of astrocytosis as an early phenomenon in AD development. Increased [11C]DED binding in the frontal and parietal cortices were found in both PiB+ and PiB− MCI groups, as well as the AD cohort, but with higher and more widespread [11C]DED binding in the PiB+ MCI group. Despite the small cohort sizes, these results suggest that MAO-B PET imaging has potential to measure astrocytosis as an early step in AD progression. Follow up studies conducted by the same research group employed the same radioligands and reported initially high and then declining astrocytosis in autosomal dominant AD carriers, suggesting astrocyte activation in the early stages of AD pathology. Suicide inhibitor-based PET radiotracers such as [11C]clorgyline and [11C]DED have been employed in seminal PET imaging studies to investigate the role of MAO-A and MAO-B in the living human brain. Nevertheless, these irreversibly binding suicide inhibitors are accompanied by significant technical challenges, which include (i) a fast rate of irreversible binding making quantification of binding difficult and (ii) formation of troublesome brain-penetrating radiometabolites, such as labeled (R)-methamphetamine and (R)-amphetamine, that have high affinities toward monoamine transporters. Since such limitations can confound image analysis, other classes of PET imaging agents, such as reversibly binding high-affinity inhibitors were developed as an alternate approach.

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REVERSIBLE INHIBITORS Inhibitors of MAO-A have been recognized for utility in the treatment of psychiatric disorders. As an example, it was theorized that endogenous levels of monoamine neurotransmitters were lower in subjects with major depressive disorder, perhaps explaining why inhibitors of the neuronal membrane monoamine transporters for serotonin and norepinephrine (SSRIs) or inhibitors of MAO-A (MAOIs) are successful drug treatments. Moreover, the provocative changes in MAO-A levels during early postpartum states has led to novel prevention strategies of monoamine precursors and antioxidant therapeutics to improve resiliency against depressed mood. To evaluate the potential differences in MAO-A levels in depression, a new MAO-A radiotracer was needed, and to address this radiochemists turned to the reversible inhibitors of MAO-A. Harmine, a potent and selective MAO-A inhibitor (kD = 2.0 nM) served as a template for development of the reversibly binding MAO-A selective radiotracer [11C]harmine (3) which was synthesized by a simple [11C]methylation of an O-desmethyl precursor. Comparison of [11C]harmine in healthy controls and drugfree depressed subjects showed an average 34% increase in specific binding across multiple brain regions, with significant increases across gray matter regions including prefrontal cortex, anterior cingulate cortex, temporal cortex, posterior cingulate, and thalamus. The authors propose that such elevated levels of MAO-A, which metabolizes all three of the major monoamine neurotransmitters (serotonin, norepinephrine and dopamine), might be a contributing factor in depression along with possible changes in monoaminergic receptors and the neuronal membrane monoamine transporters (these can also be studied using PET imaging and appropriate radiotracers). The ability to image and quantify changes in MAO-A offers the potential to improve our understanding of the role of the enzyme in other psychiatric disorders, making the continued development of MAO-A

Figure 2. Radiotracer uptake of MAO-A and MAO-B PET tracers in human brain.

described the synthesis of [11C]SL25.1188 via [11C]phosgene ([11C]COCl2), a carbon-11 reactant prepared from [11C]CO2, to give access to the labeled carbamate. [11C]SL25.1188 demonstrated favorable properties in preclinical studies (reversible binding, high brain uptake, and slow metabolism) for imaging MAO-B in the brains of nonhuman primates (NHPs). However, the preparation of this radiotracer via [11C]COCl2 was a primary drawback as it involves highly specialized apparatus requiring extensive upkeep, technical expertise, and replacement of key components between production runs. These technical challenges limit the use of [11C]COCl2 to only a few laboratories worldwide. This limitation was overcome by optimizing the radiosynthesis of [11C]SL25.1188 such that it could be accomplished via an intramolecular cyclization reaction in an automated one-pot procedure directly from [11C]CO2, thereby precluding the use of [11C]COCl2. Our Centre in Toronto has successfully translated [11C]SL25.1188 for first-in-human clinical research studies, validating it as the first reversibly binding MAO-B PET imaging agent for human use. [11C]SL25.1188 is presently being explored in several patient populations in clinical PET research studies including most recently in major depressive disorder.2 In this study, we found that 50% of subjects with MDE exhibited MAO-B VT values in the prefrontal cortex that exceeded those of healthy subjects and greater MAO-B VT is an index of MAO-B overexpression. MAO-B expression is C

DOI: 10.1021/acschemneuro.9b00081 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience increased under chronic stress and that greater MAO-B expression in MDE has specific treatment implications given that treatment resistance to serotonin reuptake inhibitor medications is common and MAO-B metabolizes nonserotonergic monoamines, reflecting a mismatch. Overall, these results may contribute toward understanding the pathologies of mitochondrial dysfunction, and impact how we target SSRI treatments. Carbon-11-labeled radiotracers enable PET studies at 2-h intervals on the same day in the same individual. On the other hand, 18F-labeled tracers have a relatively longer half-life and can be distributed to facilities that lack their own production unit thereby facilitating multicenter trials. N-[3-(2′,4′-Dichlorophenoxy)-2- 18 F-fluoropropyl]-N-methylpropargylamine ([18F]fluoroclorgyline) was developed as a potential 18Flabeled radiotracer for MAO-A. In vitro measures showed high affinity for MAO-A and selectivity for MAO-A versus MAO-B. Fluorine-18 labeled aryl-fluorinated derivatives of clorgyline and deprenyl were developed1 via fluorodestannylation using [18F]AcOF. However, this approach poses a methodological drawback of using low molar activity, as [18F]F2 is used as a precursor for the synthesis and is also not commonly used by PET radiochemistry facilities. More recently, a novel bis-deuterium substituted L-deprenyl analog was radiofluorinated on the side-chain by Halldin and co-workers at the Karolinska Institutet, namely, (S)-N-(1-18Ffluoro-3-phenylpropan-2-yl)-N-methyl-[(1,1-2H2)prop-2-yn-1amine; [18F]fluorodeprenyl-D2). This radiotracer was synthesized by [18F]fluoride in high molar activity, and demonstrated improved MAO-B quantitation through the KIE. Favorable pharmacokinetic properties with relatively fast washout from NHP brain and improved sensitivity for MAO-B were noted. These effects were recapitulated in a comparison of [18F]fluororasagiline-D2 vs [18F]fluororasagiline. Nonetheless, the brain-penetrating radioactive metabolites of propargyl compounds can be problematic for quantitation as they were for the carbon-11 agents. To date, no human studies with the 18Flabeled MAO PET tracers have been reported. We and our collaborators, including those at the Karolinska Institutet, have been working toward optimization and preclinical evaluation of fluorine-18 labeled reversible MAO-B tracers, and are focusing our efforts on SL25.1188. Further innovation in this area should make MAO-B PET imaging with reversibly binding radiotracers more accessible for clinical research.

Figure 3. Trapped metabolite imaging agent, [11C]Cou for MAO-B.

to two benign metabolites immediately after MAO-mediated oxidation. A series of 4-aryloxy-MPTP derivatives have been radiolabeled with carbon-11 and evaluated in NHP, including [ 1 1 C]1-methyl-4-phenoxy-1,2,3,6-tetrahydropyridine ([11C]PHXY) and [11C]4-methyl-7-(pyridin-4-yloxy)-2Hchromen-2-one-1,2,3,6-tetrahydropyridine ([11C]Cou). In the NHP brain, in vivo trapping of [11C]PHXY was more sensitive to MAO-A inhibition, and [11C]Cou was more sensitive to MAO-B inhibition. Given the encouraging isozyme selectivity of [11C]PHXY and [11C]Cou in the monkey brain, attempts were then made to improve the in vivo pharmacokinetics through application of the KIE, as previous studies with deuterated MPTP had demonstrated a KIE comparable to that observed for the propargylamines (e.g., deprenyl). However, incorporation of deuterium into the tetrahydropyridine ring of [11C]Cou failed to influence either the in vitro rates of reaction with MAO or the in vivo rate of brain trapping, suggesting that the abstraction of the tetrahydropyridine protons is not the rate limiting step for MAO oxidation of [11C]Cou and that any KIE was masked by a slower step. Further development and evaluation of these radiotracers is underway, with [11C]Cou of interest for the measure of astrogliosis and [11C]PHXY for use in psychiatric illnesses and the measurement of sympathetic neurons in the peripheral nervous system. A challenge to be wary about is that radiotracers that are metabolized intracellularly may have common products of metabolism in the periphery. This can make it difficult to determine whether radioactive metabolites present in brain tissue are of intracellular or extracerebral origin. Throughout this Viewpoint, we have highlighted key advances in the field of radiochemistry that have been instrumental in understanding the role of MAO in the human brain. To summarize, irreversibly binding radioligands and radioligands which are metabolized by MAO to produce trapped metabolites tend to have irreversible time activity curves. The latter may be used as an index of MAO activity. However, time−activity curves reflecting continued greater accumulation of radioactivity are often vulnerable to effects of blood flow. On the other hand, reversible radiotracers with time−activity curves showing peaks and subsequent decline during time of scanning are typically insensitive to blood flow.

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METABOLIC TRAPPING MECHANISM As an alternative strategy for radiotracer development toward MAOs, our program at the University of Michigan has investigated the use of metabolic trapping for imaging MAO enzymatic activity.5 In this approach, a substrate that is freely diffusible across the blood-brain barrier is designed such that the product of MAO oxidation is sufficiently polar to be retained within brain tissues at the sites of enzyme action (Figure 3). The development of a radiotracer substrate is an approach that has been used successfully for most widely used PET radiopharmaceutical (2-fluoro-2-deoxy- D -glucose; [18F]FDG), which is a glucose analog that is metabolically trapped after phosphorylation by hexokinase. Our radiotracer design of MAO substrates was based on MPTP, well-known for forming a toxic metabolite upon oxidation by MAO. In the 1990s, the Castagnoli lab developed 4-aryloxy derivatives of MPTP: these substrates, instead of forming MPP+ (or a related toxic metabolite), are hydrolyzed D

DOI: 10.1021/acschemneuro.9b00081 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience Such radiotracers are more readily optimized for measuring indices related to density of MAO. While some of the tracers discussed herein have been translated to clinical studies, there is continued interest in the development of new optimal MAO radiotracers that could be beneficial for imaging pathological changes in a broad spectrum of neurological diseases. We look forward to this new generation of MAO-B PET radiochemistry developments which offer insights to probe this target via new mechanisms, such as the mechanistically trapped [11C]Cou; reversibly binding [11C]SL25.1188 which uses [11C]CO2 fixation chemistry for human radiopharmaceutical production; new 18 F-tracers; and applications of these probes for neuroinflammatory processes and associated pathologies including depression.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Allen F. Brooks: 0000-0003-3773-3024 Michael R. Kilbourn: 0000-0003-4100-4676 Peter J. H. Scott: 0000-0002-6505-0450 Neil Vasdev: 0000-0002-2087-5125 Author Contributions ∥

V.N. and L.R.D.: Equal contribution. All authors contributed to writing this Viewpoint. Funding

N.V. thanks the National Institute on Aging of the NIH (R01AG054473 and R01AG052414) and the Azrieli Foundation for funding. N.V. and J.H.M. thank the Canada Research Chairs Program for support. P.J.H.S. and M.R.K. thank the NIH for financial support (R21-NS075553). Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Fowler, J. S., MacGregor, R. R., Wolf, A. P., Arnett, C. D., Dewey, S. L., Schlyer, D., Christman, D., Logan, J., Smith, M., Sachs, H., Aquilonius, S. M., Bjurling, P., Halldin, C., Hartvig, P., Leenders, K. L., Lundquist, H., Oreland, L., Stalnacke, C. G., and Langstrom, B. (1987) Mapping human brain monoamine oxidase A and B with carbon eleven-labeled suicide inactivators and PET. Science 235, 481. (2) Moriguchi, S., Wilson, A. A., Miler, L., Rusjan, P. M., Vasdev, N., Kish, S. J., Rajkowska, G., Wang, J., Bagby, M., Mizrahi, R., Varughese, B., Houle, S., and Meyer, J. H. (2019) Greater monoamine oxidase B distribution volume in the prefrontal cortex of major depressive disorder: An [11C]SL2511.88 positron emission tomography study. JAMA Psychiatry, in press. (3) Carter, S. F., Schoell, M., Almkvist, O., Wall, A., Engler, H., Laangstroem, B., and Nordberg, A. (2012) Evidence for astrocytosis in prodromal Alzheimer disease provided by 11C-deuterium-Ldeprenyl: a multitracer PET paradigm combining 11C-Pittsburgh compound B and 18F-FDG. J. Nucl. Med. 53 (1), 37−46. (4) Narayanaswami, V., Dahl, K., Bernard-Gauthier, V., Josephson, L., Cumming, P., and Vasdev, N. (2018) Emerging PET radiotracers and targets for imaging of neuroinflammation in neurodegenerative diseases: Outlook beyond TSPO. Mol. Imaging 17, 1536012118792317. (5) Drake, L. R., Brooks, A. F., Mufarreh, A. J., Pham, J. M., Koeppe, R. A., Shao, X., Scott, P. J. H., and Kilbourn, M. R. (2018) Deuterium kinetic isotope effect studies of a potential in vivo metabolic trapping agent for monoamine oxidase B. ACS Chem. Neurosci. 9, 3024. E

DOI: 10.1021/acschemneuro.9b00081 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX