2-(Phosphonomethyl)-pentanedioic acid (2-PMPA) is a potent (IC50 = 300 pM) and selective inhibitor of glutamate carboxypeptidase II (GCPII) with efficacy in multiple neurological and psychiatric disease preclinical models and more recently in models of inflammatory bowel disease (IBD) and cancer. 2-PMPA (1), however, has not been clinically developed due to its poor oral bioavailability (<1%) imparted by its four acidic functionalities (c Log P = -1.14). In an attempt to improve the oral bioavailability of 2-PMPA, we explored a prodrug approach using (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (ODOL), an FDA-approved promoiety, and systematically masked two (2), three (3), or all four (4) of its acidic groups. The prodrugs were evaluated for in vitro stability and in vivo pharmacokinetics in mice and dog. Prodrugs 2, 3, and 4 were found to be moderately stable at pH 7.4 in phosphate-buffered saline (57, 63, and 54% remaining at 1 h, respectively), but rapidly hydrolyzed in plasma and liver microsomes, across species. In vivo, in a single time-point screening study in mice, 10 mg/kg 2-PMPA equivalent doses of 2, 3, and 4 delivered significantly higher 2-PMPA plasma concentrations (3.65 ± 0.37, 3.56 ± 0.46, and 17.3 ± 5.03 nmol/mL, respectively) versus 2-PMPA (0.25 ± 0.02 nmol/mL). Given that prodrug 4 delivered the highest 2-PMPA levels, we next evaluated it in an extended time-course pharmacokinetic study in mice. 4 demonstrated an 80-fold enhancement in exposure versus oral 2-PMPA (AUC0-t: 52.1 ± 5.9 versus 0.65 ± 0.13 h*nmol/mL) with a calculated absolute oral bioavailability of 50%. In mouse brain, 4 showed similar exposures to that achieved with the IV route (1.2 ± 0.2 versus 1.6 ± 0.2 h*nmol/g). Further, in dogs, relative to orally administered 2-PMPA, 4 delivered a 44-fold enhanced 2-PMPA plasma exposure (AUC0-t for 4: 62.6 h*nmol/mL versus AUC0-t for 2-PMPA: 1.44 h*nmol/mL). These results suggest that ODOL promoieties can serve as a promising strategy for enhancing the oral bioavailability of multiply charged compounds, such as 2-PMPA, and enable its clinical translation.

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

Zobrazit více v PubMed

Slusher BS; Rojas C; Coyle JT Glutamate Carboxypeptidase II. In Handbook of Proteolytic Enzymes; Elsevier, 2013; Vol. 2, pp 1620–1627.

Urazaev AK; Buttram J Jr,; Deen J; Gafurov BS; Slusher B; Grossfeld R; Lieberman E Mechanisms for clearance of released N-acetylaspartylglutamate in crayfish nerve fibers: implications for axon–glia signaling. Neuroscience 2001, 107, 697–703. PubMed

Lieberman EM; Achreja M; Urazaev AK Synthesis of N-acetylaspartylglutamate (NAAG) and N-acetylaspartate (NAA) in Axons and Glia of the Crayfish Medial Giant Nerve Fiber. In N-Acetylaspartate; Springer, 2006; pp 303–315. PubMed

Adedoyin MO; Vicini S; Neale JH Endogenous N-acetylaspartylglutamate (NAAG) inhibits synaptic plasticity/transmission in the amygdala in a mouse inflammatory pain model. Mol. Pain 2010, 6, No. 60. PubMed PMC

Kozikowski AP; Zhang J; Nan F; Petukhov PA; Grajkowska E; Wroblewski JT; Yamamoto T; Bzdega T; Wroblewska B; Neale JH Synthesis of urea-based inhibitors as active site probes of glutamate carboxypeptidase II: efficacy as analgesic agents. J. Med. Chem 2004, 47, 1729–1738. PubMed

Nagel J; Belozertseva I; Greco S; Kashkin V; Malyshkin A; Jirgensons A; Shekunova E; Eilbacher B; Bespalov A; Danysz W Effects of NAAG peptidase inhibitor 2-PMPA in model chronic pain–relation to brain concentration. Neuropharmacology 2006, 51, 1163–1171. PubMed

Yamamoto T; Hirasawa S; Wroblewska B; Grajkowska E; Zhou J; Kozikowski A; Wroblewski J; Neale JH Antinociceptive effects of N-acetylaspartylglutamate (NAAG) peptidase inhibitors ZJ-11, ZJ-17 and ZJ-43 in the rat formalin test and in the rat neuropathic pain model. Eur. J. Neurosci 2004, 20, 483–494. PubMed

Yamamoto T; Nozaki-Taguchi N; Sakashita Y; Inagaki T Inhibition of spinal N-acetylated-α-linked acidic dipeptidase produces an antinociceptive effect in the rat formalin test. Neuroscience 2001, 102, 473–479. PubMed

Slusher BS; Vornov JJ; Thomas AG; Hurn PD; Harukuni I; Bhardwaj A; Traystman RJ; Robinson MB; Britton P; Lu X-CM Selective inhibition of NAALADase, which converts NAAG to glutamate, reduces ischemic brain injury. Nat. Med 1999, 5, No. 1396. PubMed

Carpenter KJ; Sen S; Matthews EA; Flatters SL; Wozniak KM; Slusher BS; Dickenson AH Effects of GCP-II inhibition on responses of dorsal horn neurones after inflammation and neuropathy: an electrophysiological study in the rat. Neuropeptides 2003, 37, 298–306. PubMed

Zhang W; Murakawa Y; Wozniak K; Slusher B; Sima A The preventive and therapeutic effects of GCPII (NAALADase) inhibition on painful and sensory diabetic neuropathy. J. Neurol. Sci 2006, 247, 217–223. PubMed

Zhang W; Slusher B; Murakawa Y; Wozniak K; Tsukamoto T; Jackson P; Sima A GCPII (NAALADase) inhibition prevents long-term diabetic neuropathy in type 1 diabetic BB/Wor rats. J. Peripher. Nerv. Syst 2002, 7, 211. PubMed

Olszewski RT; Bzdega T; Neale JH mGluR3 and not mGluR2 receptors mediate the efficacy of NAAG peptidase inhibitor in validated model of schizophrenia. Schizophr. Res 2012, 136, 160–161. PubMed

McKinzie D; Li TK; McBride W; Slusher B NAALADase inhibition reduces alcohol consumption in the alcohol-preferring (P) line of rats. Addict. Biol 2000, 5, 411–416. PubMed

Shippenberg TS; Rea W; Slusher BS Modulation of behavioral sensitization to cocaine by NAALADase inhibition. Synapse 2000, 38, 161–166. PubMed

Xi Z-X; Kiyatkin M; Li X; Peng X-Q; Wiggins A; Spiller K; Li J; Gardner EL N-acetylaspartylglutamate (NAAG) inhibits intravenous cocaine self-administration and cocaine-enhanced brain-stimulation reward in rats. Neuropharmacology 2010, 58, 304–313. PubMed PMC

Ha D; Bing SJ; Ahn G; Kim J; Cho J; Kim A; Herath KH; Yu HS; Jo SA; Cho IH Blocking glutamate carboxypeptidase II inhibits glutamate excitotoxicity and regulates immune responses in experimental autoimmune encephalomyelitis. FEBS J. 2016, 283, 3438–3456. PubMed

Rahn KA; Watkins CC; Alt J; Rais R; Stathis M; Grishkan I; Crainiceau CM; Pomper MG; Rojas C; Pletnikov MV Inhibition of glutamate carboxypeptidase II (GCPII) activity as a treatment for cognitive impairment in multiple sclerosis. Proc. Natl. Acad. Sci. U.S.A 2012, 109, 20101–20106. PubMed PMC

Hollinger KR; Alt J; Riehm AM; Slusher BS; Kaplin AI Dose-dependent inhibition of GCPII to prevent and treat cognitive impairment in the EAE model of multiple sclerosis. Brain Res. 2016, 1635, 105–112. PubMed

Feng J.-f.; Gurkoff GG; Van KC; Song M; Lowe DA; Zhou J; Lyeth BG NAAG peptidase inhibitor reduces cellular damage in a model of TBI with secondary hypoxia. Brain Res. 2012, 1469, 144–152. PubMed PMC

Gao Y; Xu S; Cui Z; Zhang M; Lin Y; Cai L; Wang Z; Luo X; Zheng Y; Wang Y Mice lacking glutamate carboxypeptidase II develop normally, but are less susceptible to traumatic brain injury. J. Neurochem 2015, 134, 340–353. PubMed

Ben-Shachar S; Yanai H; Baram L; Elad H; Meirovithz E; Ofer A; Brazowski E; Tulchinsky H; Pasmanik-Chor M; Dotan I Gene expression profiles of ileal inflammatory bowel disease correlate with disease phenotype and advance understanding of its immunopathogenesis. Inflamm. Bowel Dis 2013, 19, 2509–2521. PubMed

Noble CL; Abbas AR; Lees CW; Cornelius J; Toy K; Modrusan Z; Clark HF; Arnott ID; Penman ID; Satsangi J; Diehl L Characterization of intestinal gene expression profiles in Crohn’s disease by genome-wide microarray analysis. Inflamm. Bowel Dis 2010, 16, 1717–1728. PubMed

Zhang T; Song B; Zhu W; Xu X; Gong QQ; Morando C; Dassopoulos T; Newberry RD; Hunt SR; Li E An ileal Crohn’s disease gene signature based on whole human genome expression profiles of disease unaffected ileal mucosal biopsies. PLoS One 2012, 7, No. e37139. PubMed PMC

Novakova Z; Wozniak K; Jancarik A; Rais R; Wu Y; Pavlicek J; Ferraris D; Havlinova B; Ptacek J; Vavra J Unprecedented binding mode of hydroxamate-based inhibitors of glutamate carboxypeptidase II: structural characterization and biological activity. J. Med. Chem 2016, 59, 4539–4550. PubMed

Nguyen T; Kirsch BJ; Asaka R; Nabi K; Quinones A; Tan J; Antonio MJ; Camelo F; Li T; Nguyen S; Hoang G; Nguyen K; Udupa S; Sazeides C; Shen YA; Elgogary A; Reyes J; Zhao L; Kleensang A; Chaichana KL; Hartung T; Betenbaugh MJ; Marie SK; Jung JG; Wang TL; Gabrielson E; Le A Uncovering the Role of N-Acetyl-Aspartyl-Glutamate as a Glutamate Reservoir in Cancer. Cell Rep. 2019, 27, 491–501. PubMed PMC

Ferraris DV; Shukla K; Tsukamoto T Structure-activity relationships of glutamate carboxypeptidase II (GCPII) inhibitors. Curr. Med. Chem 2012, 19, 1282–1294. PubMed

Wang H; Byun Y; Barinka C; Pullambhatla M; Hyo-eun CB; Fox JJ; Lubkowski J; Mease RC; Pomper MG Bioisosterism of urea-based GCPII inhibitors: synthesis and structure–activity relationship studies. Bioorg. Med. Chem. Lett. dependence are inhibited by the selective glutamate carboxypeptidase II (GCP II, NAALADase) inhibitor, 2-PMPA. Neuropsychopharmacology 2003, 28, 457–467. PubMed

Majer P; Jancari k A; Krecmerova M.; Tichy T; Tenora L; Wozniak K; Wu Y; Pommier E; Ferraris D; Rais R Discovery of orally available prodrugs of the glutamate carboxypeptidase II (GCPII) inhibitor 2-phosphonomethylpentanedioic acid (2-PMPA). J. Med. Chem 2016, 59, 2810–2819. PubMed

Vornov J; Hollinger K; Jackson P; Wozniak K; Farah M; Majer P; Rais R; Slusher B Still NAAG’ing after all These years: the Continuing Pursuit of GCPII Inhibitors. In Advances in Pharmacology; Elsevier, 2016; Vol. 76, pp 215–255. PubMed

Barinka C; Rojas C; Slusher B; Pomper M Glutamate carboxypeptidase II in diagnosis and treatment of neurologic disorders and prostate cancer. Curr. Med. Chem 2012, 19, 856–870. PubMed PMC

Zhou J; Neale JH; Pomper MG; Kozikowski AP NAAG peptidase inhibitors and their potential for diagnosis and therapy. Nat. Rev. Drug Discovery 2005, 4, No. 1015. PubMed

Ferraris DV; Majer P; Ni C; Slusher CE; Rais R; Wu Y; Wozniak KM; Alt J; Rojas C; Slusher BS; Tsukamoto T delta-Thiolactones as prodrugs of thiol-based glutamate carboxypeptidase II (GCPII) inhibitors. J. Med. Chem 2014, 57, 243–247. PubMed PMC

Rais R; Vavra J; Tichy T; Dash RP; Gadiano AJ; Tenora L; Monincova L; Barinka C; Alt J; Zimmermann SC; Slusher CE; Wu Y; Wozniak K; Majer P; Tsukamoto T; Slusher BS Discovery of a para-acetoxy-benzyl ester prodrug of a hydroxamate-based glutamate carboxypeptidase II inhibitor as oral therapy for neuropathic pain. J. Med. Chem 2017, 60, 7799–7809. PubMed PMC

Chen SR; Wozniak KM; Slusher BS; Pan HL Effect of 2-(phosphono-methyl)-pentanedioic acid on allodynia and afferent ectopic discharges in a rat model of neuropathic pain. J. Pharmacol. Exp. Ther 2002, 300, 662–667. PubMed

Luszczki JJ; Mohamed M; Czuczwar SJ 2-phosphonomethyl-pentanedioic acid (glutamate carboxypeptidase II inhibitor) increases threshold for electroconvulsions and enhances the antiseizure action of valproate against maximal electroshock-induced seizures in mice. Eur. J. Pharmacol 2006, 531, 66–73. PubMed

Popik P; Kozela E; Wrobel M; Wozniak KM; Slusher BS Morphine tolerance and reward but not expression of morphine Sebastian, S.; Varma MS; Venkateswarlu G; Bhattacharya A; Reddy PP; Anand RVJSC Efficient synthesis of olmesartan medoxomil, an antihypertensive drug. Synth. Commun 2008, 39,

Tortella FC; Lin Y; Ved H; Slusher BS; Dave JR Neuroprotection produced by the NAALADase inhibitor 2-PMPA in rat cerebellar neurons. Eur. J. Pharmacol 2000, 402, 31–37. PubMed

Witkin JM; Gasior M; Schad C; Zapata A; Shippenberg T; Hartman T; Slusher BS NAALADase (GCP II) inhibition prevents cocaine-kindled seizures. Neuropharmacology 2002, 43, 348–356. PubMed

Wozniak KM; Rojas C; Wu Y; Slusher BS The role of glutamate signaling in pain processes and its regulation by GCP II inhibition. Curr. Med. Chem 2012, 19, 1323–1334. PubMed

Rais R; Jiang W; Zhai H; Wozniak KM; Stathis M; Hollinger KR; Thomas AG; Rojas C; Vornov JJ; Marohn M; Li X; Slusher BS FOLH1/GCPII is elevated in IBD patients, and its inhibition ameliorates murine IBD abnormalities. JCI insight 2016, 1, No. 88634. PubMed PMC

Date AA; Rais R; Babu T; Ortiz J; Kanvinde P; Thomas AG; Zimmermann SC; Gadiano AJ; Halpert G; Slusher BS; Ensign LM Local enema treatment to inhibit FOLH1/GCPII as a novel therapy for inflammatory bowel disease. J. Controlled Release 2017, 263, 132–138. PubMed PMC

Kratochwil C; Giesel FL; Leotta K; Eder M; Hoppe-Tich T; Youssoufian H; Kopka K; Babich JW; Haberkorn U PMPA for nephroprotection in PSMA-targeted radionuclide therapy of prostate cancer. J. Nucl. Med 2015, 56, 293–298. PubMed

Nedelcovych M; Dash RP; Tenora L; Zimmermann SC; Gadiano AJ; Garrett C; Alt J; Hollinger KR; Pommier E; Jancǎrǐ′k A. Enhanced Brain Delivery of 2-(Phosphonomethyl) pentanedioic Acid Following Intranasal Administration of Its γ-Substituted Ester Prodrugs. Mol. Pharm 2017, 14, 3248–3257. PubMed PMC

Babu KS; Reddy MS; Tagore AR; Reddy GS;Sebastian S; Varma MS; Venkateswarlu G; Bhattacharya A;Reddy PP; Anand RVJSC Efficient synthesis of olmesartan medoxomil, an antihypertensive drug. Synth. Commun 2008, 39, 291–298.

Garaga S; Misra NC; Raghava Reddy AV; Prabahar KJ; Takshinamoorthy C; Sanasi PD; Babu KR Commercial synthesis of Azilsartan Kamedoxomil: An angiotensin II receptor blocker. Org. Process Res. Dev 2015, 19, 514–519.

Brousil JA; Burke JMJC Olmesartan medoxomil: an angiotensin II-receptor blocker. Clin. ther 2003, 25, 1041–1055. PubMed

McKenna CE; Higa MT; Cheung NH; McKenna M-C The facile dealkylation of phosphonic acid dialkyl esters by bromotrimethylsilane. Tetrahedron Lett. 1977, 18, 155–158.

Campbell DA The synthesis of phosphonate esters; an extension of the Mitsunobu reaction. J. Org. Chem 1992, 57, 6331–6335.

Harsańyi K.; Domańy G.; Greiner I; Forintos H; Keglevich G Synthesis of 2-phosphinoxidomethyl-and 2-phosphonomethyl glutaric acid derivatives. Heteroatom Chem. 2005, 16, 562–565.

Rais R; Wozniak K; Wu Y; Niwa M; Stathis M; Alt J; Giroux M; Sawa A; Rojas C; Slusher BS Selective CNS uptake of the GCP-II inhibitor 2-PMPA following intranasal administration. PLoS One 2015, 10, No. e0131861. PubMed PMC

Daina A; Michielin O; Zoete V SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep 2017, 7, No. 42717. PubMed PMC

Gabrielson SW SciFinder. J. Med. Libr. Assoc 2018, 106, No. 588.

Aungst BJ; Hussain MA Use of Prodrugs of 3-Hydroxymorphinans to Prevent Bitter Taste upon Buccal, Nasal or Sublingual Administration. U.S Patent US46736791987.

Hussain AA; AL-Bayatti AA; Dakkuri A; Okochi K; Hussain MA Testosterone 17β-N, N-dimethylglycinate hydro-chloride: A prodrug with a potential for nasal delivery of testosterone. J. Pharm. Sci 2002, 91, 785–789. PubMed

Kao HD; Traboulsi A; Itoh S; Dittert L; Hussain A Enhancement of the systemic and CNS specific delivery of L-dopa by the nasal administration of its water soluble prodrugs. Pharm. Res 2000, 17, 978–984. PubMed

Rautio J; Meanwell NA; Di L; Hageman MJ The expanding role of prodrugs in contemporary drug design and development. Nat. Rev. Drug Discovery 2018, No. 46. PubMed

Krise JP; Stella VJ Prodrugs of phosphates, phosphonates, and phosphinates. Adv. Drug Delivery Rev 1996, 19, 287–310.

Hecker SJ; Erion MD Prodrugs of phosphates and phosphonates. J. Med. Chem 2008, 51, 2328–2345. PubMed

Dando TM; Plosker GL Adefovir dipivoxil. Drugs 2003, 63, 2215–2234. PubMed

Noble S; Goa KL Adefovir dipivoxil. Drugs 1999, 58, 479–487. PubMed

Gallant JE; Deresinski S Tenofovir disoproxil fumarate. Clin. Infect. Dis 2003, 37, 944–950. PubMed

Shaw J-P; Sueoka CM; Oliyai R; Lee WA; Arimilli MN; Kim CU; Cundy KC Metabolism and pharmacokinetics of novel oral prodrugs of 9-[(R)-2-(phosphonomethoxy) propyl] adenine (PMPA) in dogs. Pharm. Res 1997, 14, 1824–1829. PubMed

Chapman TM; McGavin JK; Noble S Tenofovir disoproxil fumarate. Drugs 2003, 63, 1597–1608. PubMed

Naesens L; Bischofberger N; Augustijns P; Annaert P; Van den Mooter G; Arimilli MN; Kim CU; De Clercq E Antiretroviral efficacy and pharmacokinetics of oral bis (isopropyloxycarbonyloxymethyl) 9-(2-phosphonylmethoxypropyl) adenine in mice. Antimicrob. Agents Chemother. 1998, 42, 1568–1573. PubMed PMC

Rautio J; Mannhold R; Kubinyl H; Folkers G Prodrugs and Targeted Delivery; Wiley VCH: Hoboken, 2010; p 97.

Assessment report of Edarbi; European Medicines Agency: United Kingdom, 2011.

Rautio J; Kumpulainen H; Heimbach T; Oliyai R; Oh D; vinen T; Savolainen J. Prodrugs: design and clinical applications. Nat. Rev. Drug Discovery 2008, 7, No. 255. 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

Nedelcovych MT; Tenora L; Kim BH; Kelschenbach J; Chao W; Hadas E; Jancarik A; Prchalova E; Zimmermann SC; Dash RP; Gadiano AJ; Garrett C; Furtmuller G; Oh B; Brandacher G; Alt J; Majer P; Volsky DJ; Rais R; Slusher BS N-(pivaloyloxy)alkoxy-carbonyl prodrugs of the glutamine antagonist 6-Diazo-5-oxo-l-norleucine (DON) as a potential treatment for HIV associated neurocognitive disorders. J. Med. Chem 2017, 60, 7186–7198. PubMed PMC

Zimmermann SC; Tichy T; Vavra J; Dash RP; Slusher CE; Gadiano AJ; Wu Y; Jancarik A; Tenora L; Monincova L; Prchalova E; Riggins GJ; Majer P; Slusher BS; Rais R N-substituted prodrugs of mebendazole provide improved aqueous solubility and oral bioavailability in mice and dogs. J. Med. Chem 2018, 61, 3918–3929. PubMed

Combination of In Silico and In Vitro Screening to Identify Novel Glutamate Carboxypeptidase II Inhibitors

Phosphonates and Phosphonate Prodrugs in Medicinal Chemistry: Past Successes and Future Prospects