Additional file 1: Figure S1. of Gastrointestinal delivery of propofol from fospropofol: its bioavailability and activity in rodents and human volunteers

Absolute latency of ipsilateral paw in CCI rats before and after fospropofol or vehicle treatment

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

<div><p>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 (AUC_{0-t, i.n}./AUC_{0-t, i.p.} = 1.0) but dramatically enhanced brain exposures in the olfactory bulb (AUC_{0-t, i.n}./AUC_{0-t, i.p.} = 67), cortex (AUC_{0-t, i.n}./AUC_{0-t, i.p.} = 46) and cerebellum (AUC_{0-t, i.n}./AUC_{0-t, i.p.} = 6.3). Following i.n. administration, the brain tissue to plasma ratio based on AUC_0-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 <i>ex-vivo</i> 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.</p></div

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

<p>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.</p

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

<p>Chemical structures and IC₅₀ values of DCMC, 2-MPPA, 2-PMPA.</p

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

<p>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.</p

Discovery of Orally Available Prodrugs of the Glutamate Carboxypeptidase II (GCPII) Inhibitor 2‑Phosphonomethyl­pentanedioic Acid (2-PMPA)

2-Phosphonomethylpentanedioic acid (<b>1</b>, 2-PMPA) is a potent inhibitor of glutamate carboxypeptidase II which has demonstrated robust neuroprotective efficacy in many neurological disease models. However, <b>1</b> is highly polar containing a phosphonate and two carboxylates, severely limiting its oral bioavailability. We strategized to mask the polar groups via a prodrug approach, increasing the likelihood of passive oral absorption. Our initial strategy was to cover the phosphonate with hydrophobic moieties such as <u>p</u>ivaloyl<u>o</u>xy<u>m</u>ethyl (POM) and iso<u>p</u>ropyl­<u>o</u>xy<u>c</u>arbonyl­oxymethyl (POC) while keeping the α- and γ-carboxylates unsubstituted. This attempt was unsuccessful due to the chemical instability of the bis-POC/POM derivatives. Addition of α,γ-diesters and α-monoesters enhanced chemical stability and provided excellent oral exposure in mice, but these mixed esters were too stable in vivo, resulting in minimal release of <b>1</b>. By introducing POC groups on both the phosphonate and α-carboxylate, we synthesized Tris-POC-2-PMPA (<b>21b)</b>, which afforded excellent release of <b>1</b> following oral administration in both mice and dog

Discovery of a <i>para</i>-Acetoxy-benzyl Ester Prodrug of a Hydroxamate-Based Glutamate Carboxypeptidase II Inhibitor as Oral Therapy for Neuropathic Pain

4-Carboxy-α-[3-(hydroxyamino)-3-oxopropyl]-benzenepropanoic acid <b>1</b> is a potent hydroxamate-based inhibitor of glutamate carboxypeptidase II. In an attempt to improve its poor oral pharmacokinetics, we synthesized a series of prodrugs by masking its hydrophilic hydroxamate group. Prodrugs were evaluated for oral availability in mice and showed varying degree of plasma exposure to <b>1</b>. Of these, <i>para</i>-acetoxybenzyl-based, 4-(5-(((4-acetoxybenzyl)­oxy)­amino)-2-carboxy-5-oxopentyl)­benzoic acid, <b>12</b>, provided 5-fold higher plasma levels of <b>1</b> compared to oral administration of <b>1</b> itself. Subsequently, <i>para</i>-acetoxybenzyl-based prodrugs with additional ester promoiety­(ies) on carboxylate(s) were examined for their ability to deliver <b>1</b> to plasma. Isopropyloxycarbonyloxymethyl (POC) ester <b>30</b> was the only prodrug that achieved substantial plasma levels of <b>1</b>. In vitro metabolite identification studies confirmed stability of the ethyl ester of benzoate while the POC group was rapidly hydrolyzed. At oral daily dose-equivalent of 3 mg/kg, <b>12</b> exhibited analgesic efficacy comparable to dose of 10 mg/kg of <b>1</b> in the rat chronic constrictive injury model of neuropathic pain

Enhanced Brain Delivery of 2‑(Phosphonomethyl)pentanedioic Acid Following Intranasal Administration of Its γ‑Substituted Ester Prodrugs

2-(Phosphonomethyl)­pentanedioic acid (2-PMPA) is a potent and selective inhibitor of glutamate carboxypeptidase-II (GCPII) with efficacy in multiple neurological and psychiatric disease models, but its clinical utility is hampered by low brain penetration due to the inclusion of multiple acidic functionalities. We recently reported an improvement in the brain-to-plasma ratio of 2-PMPA after intranasal (IN) dosing in both rodents and primates. Herein, we describe the synthesis of several 2-PMPA prodrugs with further improved brain delivery of 2-PMPA after IN administration by masking of the γ-carboxylate. When compared to IN 2-PMPA in rats at 1 h post dose, γ-(4-acetoxybenzyl)-2-PMPA (compound <b>1</b>) resulted in significantly higher 2-PMPA delivery to both plasma (4.1-fold) and brain (11-fold). Subsequent time-dependent evaluation of <b>1</b> also showed high brain as well as plasma 2-PMPA exposures with brain-to-plasma ratios of 2.2, 0.48, and 0.26 for olfactory bulb, cortex, and cerebellum, respectively, as well as an improved sciatic nerve to plasma ratio of 0.84. In contrast, IV administration of compound <b>1</b> resulted in similar plasma exposure of 2-PMPA versus the IN route (AUC_IV: 76 ± 9 h·nmol/mL versus AUC_IN: 99 ± 24 h·nmol/mL); but significantly lower nerve and brain tissue exposures with tissue-to-plasma ratios of 0.21, 0.03, 0.04, and 0.04 in nerve, olfactory bulb, cortex, and cerebellum, respectively. In primates, IN administration of <b>1</b> more than doubled 2-PMPA concentrations in the cerebrospinal fluid relative to previously reported levels following IN 2-PMPA. The results of these experiments provide a promising strategy for testing GCPII inhibition in neurological and psychiatric disorders