Abstract
Glutamate carboxypeptidase II (GCP-II) is a brain metallopeptidase that hydrolyzes the abundant neuropeptide N-acetyl-aspartyl-glutamate (NAAG) to NAA and glutamate. Small molecule GCP-II inhibitors increase brain NAAG, which activates mGluR3, decreases glutamate, and provide therapeutic utility in a variety of preclinical models of neurodegenerative diseases wherein excess glutamate is presumed pathogenic. Unfortunately no GCP-II inhibitor has advanced clinically, largely due to their highly polar nature resulting in insufficient oral bioavailability and limited brain penetration. Herein we report a non-invasive route for delivery of GCP-II inhibitors to the brain via intranasal (i.n.) administration. Three structurally distinct classes of GCP-II inhibitors were evaluated including DCMC (urea-based), 2-MPPA (thiol-based) and 2-PMPA (phosphonate-based). While all showed some brain penetration following i.n. administration, 2-PMPA exhibited the highest levels and was chosen for further evaluation. Compared to intraperitoneal (i.p.) administration, equivalent doses of i.n. administered 2-PMPA resulted in similar plasma exposures (AUC0-t, i.n./AUC0-t, i.p. = 1.0) but dramatically enhanced brain exposures in the olfactory bulb (AUC0-t, i.n./AUC0-t, i.p. = 67), cortex (AUC0-t, i.n./AUC0-t, i.p. = 46) and cerebellum (AUC0-t, i.n./AUC0-t, i.p. = 6.3). Following i.n. administration, the brain tissue to plasma ratio based on AUC0-t in the olfactory bulb, cortex, and cerebellum were 1.49, 0.71 and 0.10, respectively, compared to an i.p. brain tissue to plasma ratio of less than 0.02 in all areas. Furthermore, i.n. administration of 2-PMPA resulted in complete inhibition of brain GCP-II enzymatic activity ex-vivo confirming target engagement. Lastly, because the rodent nasal system is not similar to humans, we evaluated i.n. 2-PMPA also in a non-human primate. We report that i.n. 2-PMPA provides selective brain delivery with micromolar concentrations. These studies support intranasal delivery of 2-PMPA to deliver therapeutic concentrations in the brain and may facilitate its clinical development.
Highlights
Elevated levels of glutamate, a major neurotransmitter in the central and peripheral nervous system, is often associated with excitotoxicity, which is a hallmark of many neurological and psychiatric disorders [1,2,3]
One strategy to reduce the levels of extracellular glutamate involves the inhibition of the brain enzyme glutamate carboxypeptidase II (GCP-II) (EC 3.4.12.21), a membrane bound zinc metalloprotease involved in the hydrolysis of the abundant neuropeptide N-acetylaspartylglutamate (NAAG) to N-acetylaspartate (NAA) and L-glutamate [1,4,5]
Released NAAG can be catabolized by GCP-II, liberating glutamate, which can serve as an agonist at various glutamate receptors
Summary
A major neurotransmitter in the central and peripheral nervous system, is often associated with excitotoxicity, which is a hallmark of many neurological and psychiatric disorders [1,2,3]. Potent GCP-II inhibitors identified to date have required two functionalities–a glutarate moiety that binds the C-terminal glutamate recognition site of GCP-II, and a zinc chelating group to engage the divalent zinc atoms at the enzyme’s active site [5] Inclusion of these functionalities has led to highly potent inhibitors, the compounds suffer from being exceedingly hydrophilic and show low membrane permeability. We first compared the brain penetration of three potent and widely used GCP-II inhibitors using intranasal delivery in a single dose and single timepoint study. We report that compared to i.p. administration, equivalent i.n. administered dose of 2-PMPA resulted in similar plasma but dramatically enhanced brain exposures. These findings were confirmed in non-human primate studies
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