Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14) attenuates glutamate synthesis in HIV-infected microglia/macrophages, offering therapeutic potential for HAND. We show that 14 prevents manifestation of spatial memory deficits in chimeric EcoHIV-infected mice, a model of HAND. 14 is not clinically available, however, because its development was hampered by peripheral toxicities. We describe the synthesis of several substituted N-(pivaloyloxy)alkoxy-carbonyl prodrugs of 14 designed to circulate inert in plasma and be taken up and biotransformed to 14 in the brain. The lead prodrug, isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d), was stable in swine and human plasma but liberated 14 in swine brain homogenate. When dosed systemically in swine, 13d provided a 15-fold enhanced CSF-to-plasma ratio and a 9-fold enhanced brain-to-plasma ratio relative to 14, opening a possible clinical path for the treatment of HAND.

Department of Medicine Icahn School of Medicine at Mount Sinai New York New York 10029 United States

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic vvi 166 10 Prague Czech Republic

Zobrazit více v PubMed

Zink MC. Translational research models and novel adjunctive therapies for neuroAIDS. J Neuroimmune Pharmacol. 2007;2:14–19. PubMed

Heaton RK, Clifford DB, Franklin DR, Jr, Woods SP, Ake C, Vaida F, Ellis RJ, Letendre SL, Marcotte TD, Atkinson JH, Rivera-Mindt M, Vigil OR, Taylor MJ, Collier AC, Marra CM, Gelman BB, McArthur JC, Morgello S, Simpson DM, McCutchan JA, Abramson I, Gamst A, Fennema-Notestine C, Jernigan TL, Wong J, Grant I. HIV-associated neurocognitive disorders persist in the era of potent antiretroviral therapy: CHARTER Study. Neurology. 2010;75:2087–2096. PubMed PMC

Harezlak J, Buchthal S, Taylor M, Schifitto G, Zhong J, Daar E, Alger J, Singer E, Campbell T, Yiannoutsos C, Cohen R, Navia B. Persistence of HIV-associated cognitive impairment, inflammation, and neuronal injury in era of highly active antiretroviral treatment. AIDS. 2011;25:625–633. PubMed PMC

Heaton RK, Franklin DR, Ellis RJ, McCutchan JA, Letendre SL, Leblanc S, Corkran SH, Duarte NA, Clifford DB, Woods SP, Collier AC, Marra CM, Morgello S, Mindt MR, Taylor MJ, Marcotte TD, Atkinson JH, Wolfson T, Gelman BB, McArthur JC, Simpson DM, Abramson I, Gamst A, Fennema-Notestine C, Jernigan TL, Wong J, Grant I. HIV-associated neurocognitive disorders before and during the era of combination antiretroviral therapy: differences in rates, nature, and predictors. J NeuroVirol. 2011;17:3–16. PubMed PMC

Robertson KR, Smurzynski M, Parsons TD, Wu K, Bosch RJ, Wu J, McArthur JC, Collier AC, Evans SR, Ellis RJ. The prevalence and incidence of neurocognitive impairment in the HAART era. AIDS. 2007;21:1915–1921. PubMed

Potter MC, Figuera-Losada M, Rojas C, Slusher BS. Targeting the glutamatergic system for the treatment of HIV-associated neurocognitive disorders. J Neuroimmune Pharmacol. 2013;8:594–607. PubMed PMC

Vazquez-Santiago FJ, Noel RJ, Jr, Porter JT, Rivera-Amill V. Glutamate metabolism and HIV-associated neurocognitive disorders. J NeuroVirol. 2014;20:315–331. PubMed PMC

Doi A, Iijima K, Kano S, Ishizaka Y. Viral protein R of HIV type-1 induces retrotransposition and upregulates glutamate synthesis by the signal transducer and activator of transcription 1 signaling pathway. Microbiol Immunol. 2015;59:398–409. PubMed

Erdmann N, Tian C, Huang Y, Zhao J, Herek S, Curthoys N, Zheng J. In vitro glutaminase regulation and mechanisms of glutamate generation in HIV-1-infected macrophage. J Neurochem. 2009;109:551–561. PubMed PMC

Erdmann N, Zhao J, Lopez AL, Herek S, Curthoys N, Hexum TD, Tsukamoto T, Ferraris D, Zheng J. Glutamate production by HIV-1 infected human macrophage is blocked by the inhibition of glutaminase. J Neurochem. 2007;102:539–549. PubMed PMC

Erdmann NB, Whitney NP, Zheng J. Potentiation of excitotoxicity in HIV-1 associated dementia and the significance of glutaminase. Clin Neurosci Res. 2006;6:315–328. PubMed PMC

Huang Y, Zhao L, Jia B, Wu L, Li Y, Curthoys N, Zheng JC. Glutaminase dysregulation in HIV-1-infected human microglia mediates neurotoxicity: relevant to HIV-1-associated neurocognitive disorders. J Neurosci. 2011;31:15195–15204. PubMed PMC

Thomas AG, O’Driscoll CM, Bressler J, Kaufmann W, Rojas CJ, Slusher BS. Small molecule glutaminase inhibitors block glutamate release from stimulated microglia. Biochem Biophys Res Commun. 2014;443:32–36. PubMed PMC

Tian C, Erdmann N, Zhao J, Cao Z, Peng H, Zheng J. HIV-infected macrophages mediate neuronal apoptosis through mitochondrial glutaminase. J Neurochem. 2008;105:994–1005. PubMed PMC

Tian C, Sun L, Jia B, Ma K, Curthoys N, Ding J, Zheng J. Mitochondrial glutaminase release contributes to glutamate-mediated neurotoxicity during human immunodeficiency virus-1 infection. J Neuroimmune Pharmacol. 2012;7:619–628. PubMed PMC

Wu B, Huang Y, Braun AL, Tong Z, Zhao R, Li Y, Liu F, Zheng JC. Glutaminase-containing microvesicles from HIV-1-infected macrophages and immune-activated microglia induce neurotoxicity. Mol Neurodegener. 2015;10:61. PubMed PMC

Ye L, Huang Y, Zhao L, Li Y, Sun L, Zhou Y, Qian G, Zheng JC. IL-1beta and TNF-alpha induce neurotoxicity through glutamate production: a potential role for neuronal glutaminase. J Neurochem. 2013;125:897–908. PubMed PMC

Zhao L, Huang Y, Tian C, Taylor L, Curthoys N, Wang Y, Vernon H, Zheng J. Interferon-alpha regulates glutaminase 1 promoter through STAT1 phosphorylation: relevance to HIV-1 associated neurocognitive disorders. PLoS One. 2012;7:e32995. PubMed PMC

Zhao L, Huang Y, Zheng J. STAT1 regulates human glutaminase 1 promoter activity through multiple binding sites in HIV-1 infected macrophages. PLoS One. 2013;8:e76581. PubMed PMC

Musante V, Summa M, Neri E, Puliti A, Godowicz TT, Severi P, Battaglia G, Raiteri M, Pittaluga A. The HIV-1 viral protein Tat increases glutamate and decreases GABA exocytosis from human and mouse neocortical nerve endings. Cereb Cortex. 2010;20:1974–1984. PubMed

Anderson ER, Boyle J, Zink WE, Persidsky Y, Gendelman HE, Xiong H. Hippocampal synaptic dysfunction in a murine model of human immunodeficiency virus type 1 encephalitis. Neuroscience. 2003;118:359–369. PubMed

Anderson ER, Gendelman HE, Xiong H. Memantine protects hippocampal neuronal function in murine human immunodeficiency virus type 1 encephalitis. J Neurosci. 2004;24:7194–7198. PubMed PMC

Behnisch T, Francesconi W, Sanna PP. HIV secreted protein Tat prevents long-term potentiation in the hippocampal CA1 region. Brain Res. 2004;1012:187–189. PubMed

Dong J, Xiong H. Human immunodeficiency virus type 1 gp120 inhibits long-term potentiation via chemokine receptor CXCR4 in rat hippocampal slices. J Neurosci Res. 2006;83:489–496. PubMed

Fitting S, Ignatowska-Jankowska BM, Bull C, Skoff RP, Lichtman AH, Wise LE, Fox MA, Su J, Medina AE, Krahe TE, Knapp PE, Guido W, Hauser KF. Synaptic dysfunction in the hippocampus accompanies learning and memory deficits in human immunodeficiency virus type-1 Tat transgenic mice. Biol Psychiatry. 2013;73:443–453. PubMed PMC

Keblesh JP, Dou H, Gendelman HE, Xiong H. 4-Aminopyridine improves spatial memory in a murine model of HIV-1 encephalitis. J Neuroimmune Pharmacol. 2009;4:317–327. PubMed PMC

Li ST, Matsushita M, Moriwaki A, Saheki Y, Lu YF, Tomizawa K, Wu HY, Terada H, Matsui H. HIV-1 Tat inhibits long-term potentiation and attenuates spatial learning [corrected] Ann Neurol. 2004;55:362–371. PubMed

Tang H, Lu D, Pan R, Qin X, Xiong H, Dong J. Curcumin improves spatial memory impairment induced by human immunodeficiency virus type 1 glycoprotein 120 V3 loop peptide in rats. Life Sci. 2009;85:1–10. PubMed

Cassol E, Misra V, Dutta A, Morgello S, Gabuzda D. Cerebrospinal fluid metabolomics reveals altered waste clearance and accelerated aging in HIV patients with neurocognitive impairment. AIDS. 2014;28:1579–1591. PubMed PMC

Bairwa D, Kumar V, Vyas S, Das BK, Srivastava AK, Pandey RM, Sharma SK, Jagannathan NR, Sinha S. Case control study: magnetic resonance spectroscopy of brain in HIV infected patients. BMC Neurol. 2016;16:99. PubMed PMC

Ernst T, Jiang CS, Nakama H, Buchthal S, Chang L. Lower brain glutamate is associated with cognitive deficits in HIV patients: a new mechanism for HIV-associated neurocognitive disorder. J Magn Reson Imaging. 2010;32:1045–1053. PubMed PMC

Mohamed MA, Barker PB, Skolasky RL, Selnes OA, Moxley RT, Pomper MG, Sacktor NC. Brain metabolism and cognitive impairment in HIV infection: a 3-T magnetic resonance spectroscopy study. Magn Reson Imaging. 2010;28:1251–1257. PubMed PMC

Sailasuta N, Shriner K, Ross B. Evidence of reduced glutamate in the frontal lobe of HIV-seropositive patients. NMR Biomed. 2009;22:326–331. PubMed

Schifitto G, Navia BA, Yiannoutsos CT, Marra CM, Chang L, Ernst T, Jarvik JG, Miller EN, Singer EJ, Ellis RJ, Kolson DL, Simpson D, Nath A, Berger J, Shriver SL, Millar LL, Colquhoun D, Lenkinski R, Gonzalez RG, Lipton SA. Memantine and HIV-associated cognitive impairment: a neuropsychological and proton magnetic resonance spectroscopy study. AIDS. 2007;21:1877–1886. PubMed

Stankoff B, Tourbah A, Suarez S, Turell E, Stievenart JL, Payan C, Coutellier A, Herson S, Baril L, Bricaire F, Calvez V, Cabanis EA, Lacomblez L, Lubetzki C. Clinical and spectroscopic improvement in HIV-associated cognitive impairment. Neurology. 2001;56:112–115. PubMed

Gelman BB, Chen T, Lisinicchia JG, Soukup VM, Carmical JR, Starkey JM, Masliah E, Commins DL, Brandt D, Grant I, Singer EJ, Levine AJ, Miller J, Winkler JM, Fox HS, Luxon BA, Morgello S. The National NeuroAIDS Tissue Consortium brain gene array: two types of HIV-associated neurocognitive impairment. PLoS One. 2012;7:e46178. PubMed PMC

Gelman BB, Lisinicchia JG, Chen T, Johnson KM, Jennings K, Freeman DH, Jr, Soukup VM. Prefrontal dopaminergic and enkephalinergic synaptic accommodation in HIV-associated neurocognitive disorders and encephalitis. J Neuroimmune Pharmacol. 2012;7:686–700. PubMed PMC

Borjabad A, Morgello S, Chao W, Kim SY, Brooks AI, Murray J, Potash MJ, Volsky DJ. Significant effects of antiretroviral therapy on global gene expression in brain tissues of patients with HIV-1-associated neurocognitive disorders. PLoS Pathog. 2011;7:e1002213. PubMed PMC

Conti F, Minelli A. Glutamate immunoreactivity in rat cerebral cortex is reversibly abolished by 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of phosphate-activated glutaminase. J Histochem Cytochem. 1994;42:717–726. PubMed

Chung SH, Johnson MS. Studies on sound-induced epilepsy in mice. Proc R Soc London, Ser B. 1984;221:145–168. PubMed

Zhao J, Lopez AL, Erichsen D, Herek S, Cotter RL, Curthoys NP, Zheng J. Mitochondrial glutaminase enhances extracellular glutamate production in HIV-1-infected macrophages: linkage to HIV-1 associated dementia. J Neurochem. 2004;88:169–180. PubMed

Potash MJ, Chao W, Bentsman G, Paris N, Saini M, Nitkiewicz J, Belem P, Sharer L, Brooks AI, Volsky DJ. A mouse model for study of systemic HIV-1 infection, antiviral immune responses, and neuroinvasiveness. Proc Natl Acad Sci U S A. 2005;102:3760–3765. PubMed PMC

He H, Sharer LR, Chao W, Gu CJ, Borjabad A, Hadas E, Kelschenbach J, Ichiyama K, Do M, Potash MJ, Volsky DJ. Enhanced human immunodeficiency virus Type 1 expression and neuropathogenesis in knockout mice lacking Type I interferon responses. J Neuropathol Exp Neurol. 2014;73:59–71. PubMed PMC

Earhart RH, Amato DJ, Chang AY, Borden EC, Shiraki M, Dowd ME, Comis RL, Davis TE, Smith TJ. Phase II trial of 6-diazo-5-oxo-L-norleucine versus aclacinomycin-A in advanced sarcomas and mesotheliomas. Invest New Drugs. 1990;8:113–119. PubMed

Magill GB, Myers WP, Reilly HC, Putnam RC, Magill JW, Sykes MP, Escher GC, Karnofsky DA, Burchenal JH. Pharmacological and initial therapeutic observations on 6-diazo-5-oxo-1-norleucine (DON) in human neoplastic disease. Cancer. 1957;10:1138–1150. PubMed

Rahman A, Smith FP, Luc PT, Woolley PV. Phase I study and clinical pharmacology of 6-diazo-5-oxo-L-norleucine (DON) Invest New Drugs. 1985;3:369–374. PubMed

Rais R, Jancarik A, Tenora L, Nedelcovych M, Alt J, Englert J, Rojas C, Le A, Elgogary A, Tan J, Monincova L, Pate K, Adams R, Ferraris D, Powell J, Majer P, Slusher BS. Discovery of 6-Diazo-5-oxo-l-norleucine (DON) prodrugs with enhanced CSF delivery in monkeys: a potential treatment for glioblastoma. J Med Chem. 2016;59:8621–8633. PubMed

Rasheed A, Kumar CKA. Novel approaches on prodrug based drug design. Pharm Chem J. 2008;42:677–686.

Li F, Maag H, Alfredson T. Prodrugs of nucleoside analogues for improved oral absorption and tissue targeting. J Pharm Sci. 2008;97:1109–1134. PubMed

Fattash B, Karaman R. Chemical approaches used in prodrugs design. In: Karaman R, editor. Prodrugs Design – A New Era ; Pharmacology—Research, Safety Testing and Regulation. Nova Science Publishers Inc.; New York: 2014. pp. 103–138.

Liederer BM, Borchardt RT. Enzymes involved in the bioconversion of ester-based prodrugs. J Pharm Sci. 2006;95:1177–1195. PubMed

Rautio J, Laine K, Gynther M, Savolainen J. Prodrug approaches for CNS delivery. AAPS J. 2008;10:92–102. PubMed PMC

Vlieghe P, Khrestchatisky M. Medicinal chemistry based approaches and nanotechnology-based systems to improve CNS drug targeting and delivery. Med Res Rev. 2013;33:457–516. PubMed

Fu J, Pacyniak E, Leed MGD, Sadgrove MP, Marson L, Jay M. Interspecies differences in the metabolism of a multiester prodrug by carboxylesterases. J Pharm Sci. 2016;105:989–995. PubMed PMC

Van Gelder J, Shafiee M, De Clercq E, Penninckx F, Van den Mooter G, Kinget R, Augustijns P. Species-dependent and site-specific intestinal metabolism of ester prodrugs. Int J Pharm. 2000;205:93–100. PubMed

Suenderhauf C, Parrott N. A physiologically based pharmacokinetic model of the minipig: data compilation and model implementation. Pharm. Res. 2013;30:1–15. PubMed

Gallop MA, Yao F, Ludwikow MJ, Phan T, Peng G. Acyloxyalkyl Carbamate Prodrugs, Methods of Synthesis and Use. WO Pat Appl. 2005;2005019163:A2.

Gallop MA, Xu F, Phan T, Dilip U, Peng G. Acyloxyalkyl Carbamate Prodrugs, Methods of Synthesis and Use. WO Pat Appl. 2008;2008033572:A1.

Gallop M, Cundy K, Zhou C, Yao F, Xiang JN, Ollman I, Qiu F. Prodrugs of GABA Analogs, Compositions and Uses Thereof. US Pat Appl. 2006;29361:A1. 2006/02.

Woods SP, Moore DJ, Weber E, Grant I. Cognitive neuropsychology of HIV-associated neurocognitive disorders. Neuropsychol Rev. 2009;19:152–168. PubMed PMC

Lee CF, Lo YC, Cheng CH, Furtmuller GJ, Oh B, Andrade-Oliveira V, Thomas AG, Bowman CE, Slusher BS, Wolfgang MJ, Brandacher G, Powell JD. Preventing allograft rejection by targeting immune metabolism. Cell Rep. 2015;13:760–770. PubMed PMC

Baxter VK, Glowinski R, Braxton AM, Potter MC, Slusher BS, Griffin DE. Glutamine antagonist-mediated immune suppression decreases pathology but delays virus clearance in mice during nonfatal alphavirus encephalomyelitis. Virology. 2017;508:134–149. PubMed PMC

Annaert P, Tukker JJ, van GelderJ, Naesens L, de ClercqE, van DenMooter G, Kinget R, Augustijns P. In vitro, ex vivo, and in situ intestinal absorption characteristics of the antiviral ester prodrug adefovir dipivoxil. J Pharm Sci. 2000;89:1054–1062. PubMed

Masuda K, Naiki M. Method for Promoting the Synthesis of Collagen and Proteoglycan in Chondrocytes. US Pat Appl. 2013;64383:A1. 2013/01.

Potash MJ, Chao W, Bentsman G, Paris N, Saini M, Nitkiewicz J, Belem P, Sharer L, Brooks AI, Volsky DJ. A mouse model for study of systemic HIV-1 infection, antiviral immune responses, and neuroinvasiveness. Proc Natl Acad Sci U S A. 2005;102:3760–3765. PubMed PMC

Shijie J, Takeuchi H, Yawata I, Harada Y, Sonobe Y, Doi Y, Liang J, Hua L, Yasuoka S, Zhou Y, Noda M, Kawanokuchi J, Mizuno T, Suzumura A. Blockade of glutamate release from microglia attenuates experimental autoimmune encephalomyelitis in mice. Tohoku J Exp Med. 2009;217:87–92. PubMed

Manivannan S, Baxter VK, Schultz KL, Slusher BS, Griffin DE. Protective Effects of Glutamine Antagonist 6-Diazo-5-Oxo-l-Norleucine in Mice with Alphavirus Encephalomyelitis. J Virol. 2016;90:9251–9262. PubMed PMC

Potter MC, Baxter VK, Mathey RW, Alt J, Rojas C, Griffin DE, Slusher BS. Neurological sequelae induced by alphavirus infection of the CNS are attenuated by treatment with the glutamine antagonist 6-diazo-5-oxo-l-norleucine. J NeuroVirol. 2015;21:159–173. PubMed PMC

Alt J, Potter MC, Rojas C, Slusher BS. Bioanalysis of 6-diazo-5-oxo-l-norleucine in plasma and brain by ultra-performance liquid chromatography mass spectrometry. Biochem. 2015;474:28–34. PubMed PMC

Arendash GW, Gordon MN, Diamond DM, Austin LA, Hatcher JM, Jantzen P, DiCarlo G, Wilcock D, Morgan D. Behavioral assessment of Alzheimer’s transgenic mice following long-term Abeta vaccination: task specificity and correlations between Abeta deposition and spatial memory. DNA Cell Biol. 2001;20:737–744. PubMed

Hadas E, Borjabad A, Chao W, Saini M, Ichiyama K, Potash MJ, Volsky DJ. Testing antiretroviral drug efficacy in conventional mice infected with chimeric HIV-1. AIDS. 2007;21:905–909. PubMed

He H, Sharer LR, Chao W, Gu CJ, Borjabad A, Hadas E, Kelschenbach J, Ichiyama K, Do M, Potash MJ, Volsky DJ. Enhanced human immunodeficiency virus type 1 expression and neuropathogenesis in knockout mice lacking type I interferon responses. J Neuropathol Exp Neurol. 2014;73:59–71. PubMed PMC

Kelschenbach JL, Saini M, Hadas E, Gu C-J, Chao W, Bentsman G, Hong JP, Hanke T, Sharer LR, Potash MJ, Volsky DJ. Mice chronically infected with chimeric HIV resist peripheral and brain superinfection: A model of protective immunity to HIV. J Neuroimmune Pharmacol. 2012;7:380–387. PubMed PMC

Rais R, Hoover R, Wozniak K, Rudek MA, Tsukamoto T, Alt J, Rojas C, Slusher BS. Reversible disulfide formation of the glutamate carboxypeptidase II inhibitor E2072 results in prolonged systemic exposures in vivo. Drug Metab. Dispos. 2012;40:2315–2323. PubMed PMC

Baranczewski P, Stanczak A, Sundberg K, Svensson R, Wallin A, Jansson J, Garberg P, Postlind H. Introduction to in vitro estimation of metabolic stability and drug interactions of new chemical entities in drug discovery and development. Pharmacol Rep. 2006;58:453–472. PubMed

Pro-905, a Novel Purine Antimetabolite, Combines with Glutamine Amidotransferase Inhibition to Suppress Growth of Malignant Peripheral Nerve Sheath Tumor

Discovery of DRP-104, a tumor-targeted metabolic inhibitor prodrug

The Novel Glutamine Antagonist Prodrug JHU395 Has Antitumor Activity in Malignant Peripheral Nerve Sheath Tumor

Enhanced Oral Bioavailability of 2-(Phosphonomethyl)-pentanedioic Acid (2-PMPA) from its (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl (ODOL)-Based Prodrugs

Tumor-Targeted Delivery of 6-Diazo-5-oxo-l-norleucine (DON) Using Substituted Acetylated Lysine Prodrugs

JHU-083 selectively blocks glutaminase activity in brain CD11b+ cells and prevents depression-associated behaviors induced by chronic social defeat stress