Selective CNS Uptake of the GCP-II Inhibitor 2-PMPA following Intranasal Administration

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.

Fig 1. Chemical structures and IC₅₀ values of DCMC, 2-MPPA, 2-PMPA.

Fig 2. Mean concentrations of 2-PMPA, 2-MPPA and DCMC in different brain regions.

Concentration were measured in olfactory bulb, cortex and cerebellum following 30mg/kg intranasal administration in rats. Tissues were collected 1h post dose and evaluated via LC/MS/MS.

Fig 3. Mean concentration vs. time profiles for 2-PMPA in rat plasma, olfactory bulb, cortex and cerebellum following (A) 30 mg/kg intraperitoneal (i.p.) and (B) 30 mg/kg intranasal (i.n.) administration.

Fig 4. Brain tissue to plasma (B/P) ratio of 2-PMPA in different brain regions.

B/P ratio was calculated based on area under the curve (AUC_0-t) following 30 mg/kg i.n. or i.p. administration.

Fig 5. Ex vivo GCP-II enzymatic activity following 2-PMPA i.n. administration.

Enzyme activity was measured in olfactory bulb, cortex and cerebellum collected 1 h post dose following 30 mg/kg i.n. administration. Percent inhibition was calculated in all tissue samples relative to brain tissues collected from untreated control rats.