Glutamate Carboxypeptidase II Inhibitors Research Articles

Brain N -acetyl-aspartyl-glutamate is associated with cognitive function in older virally suppressed people with HIV.

Cognitive impairment persists in virally suppressed people with HIV (VS-PWH) especially in higher order domains. One cortical circuit, linked to these domains, is regulated by N -acetyl-aspartyl glutamate (NAAG), the endogenous agonist of the metabotropic glutamate receptor 3. The enzyme glutamate carboxypeptidase II (GCPII) catabolizes NAAG and is upregulated in aging and disease. Inhibition of GCPII increases brain NAAG and improves learning and memory in rodent and primate models. As higher order cognitive impairment is present in VS-PWH, and NAAG has not been investigated in earlier magnetic resonance spectroscopy studies (MRS), we investigated if brain NAAG levels measured by MRS were associated with cognitive function. We conducted a retrospective analysis of 7-Tesla MRS data from a previously published study on cognition in older VS-PWH. The original study did not separately quantify NAAG, therefore, work for this report focused on relationships between regional NAAG levels in frontal white matter (FWM), left hippocampus, left basal ganglia and domain-specific cognitive performance in 40 VS-PWH after adjusting for confounds. Participants were older than 50 years, negative for affective and neurologic disorders, and had no prior 3-month psychoactive-substance use. Higher NAAG levels in FWM were associated with better attention/working memory. Higher left basal ganglia NAAG related to better verbal fluency. There was a positive relationship between hippocampal NAAG and executive function which lost significance after correction for confounds. These data suggest brain NAAG serves as a biomarker of cognition in VS-PWH. Pharmacological modulation of brain NAAG warrants investigation as a therapeutic approach for cognitive deficits in VS-PWH.

Chronic GCPII (glutamate-carboxypeptidase-II) inhibition reduces pT217Tau levels in the entorhinal and dorsolateral prefrontal cortices of aged macaques.

Current approaches for treating sporadic Alzheimer's disease (sAD) focus on removal of amyloid beta 1-42 (Aβ1-42) or phosphorylated tau, but additional strategies are needed to reduce neuropathology at earlier stages prior to neuronal damage. Longstanding data show that calcium dysregulation is a key etiological factor in sAD, and the cortical neurons most vulnerable to tau pathology show magnified calcium signaling, for example in dorsolateral prefrontal cortex (dlPFC) and entorhinal cortex (ERC). In primate dlPFC and ERC, type 3 metabotropic glutamate receptors (mGluR3s) are predominately post-synaptic, on spines, where they regulate cAMP-calcium signaling, a process eroded by inflammatory glutamate carboxypeptidase II (GCPII) actions. The current study tested whether enhancing mGluR3 regulation of calcium via chronic inhibition of GCPII would reduce tau hyperphosphorylation in aged macaques with naturally-occurring tau pathology. Aged rhesus macaques were treated daily with the GCPII inhibitor, 2-MPPA (2-3-mercaptopropyl-penanedioic acid (2-MPPA)),Aged rhesus macaques were treated daily with the GCPII inhibitor, 2-MPPA (2-3-mercaptopropyl-penanedioic acid (2-MPPA)). Aged macaques that received 2-MPPA had significantly lower pT217Tau levels in dlPFC and ERC, and had lowered plasma pT217Tau levels from baseline. pT217Tau levels correlated significantly with GCPII activity in dlPFC. Both 2-MPPA- and vehicle-treated monkeys showed cognitive improvement; 2-MPPA had no apparent side effects. Exploratory CSF analyses indicated reduced pS202Tau with 2-MPPA administration, confirmed in dlPFC samples. These data provide proof-of-concept support that GCPII inhibition can reduce tau hyperphosphorylation in the primate cortices most vulnerable in sAD. GCPII inhibition may be particularly helpful in reducing the risk of sAD caused by inflammation. These data in nonhuman primates should encourage future research on this promising mechanism. Inflammation is a key driver of sporadic Alzheimer's disease.GCPII inflammatory signaling in brain decreases mGluR3 regulation of calcium.Chronic inhibition of GCPII inflammatory signaling reduced pT217Tau in aged monkeys.GCPII inhibition is a novel strategy to help prevent tau pathology at early stages.

A gut-restricted glutamate carboxypeptidase II inhibitor reduces monocytic inflammation and improves preclinical colitis.

There is an urgent need to develop therapeutics for inflammatory bowel disease (IBD) because up to 40% of patients with moderate-to-severe IBD are not adequately controlled with existing drugs. Glutamate carboxypeptidase II (GCPII) has emerged as a promising therapeutic target. This enzyme is minimally expressed in normal ileum and colon, but it is markedly up-regulated in biopsies from patients with IBD and preclinical colitis models. Here, we generated a class of GCPII inhibitors designed to be gut-restricted for oral administration, and we interrogated efficacy and mechanism using in vitro and in vivo models. The lead inhibitor, (S)-IBD3540, was potent (half maximal inhibitory concentration = 4 nanomolar), selective, gut-restricted (AUCcolon/plasma > 50 in mice with colitis), and efficacious in acute and chronic rodent colitis models. In dextran sulfate sodium-induced colitis, oral (S)-IBD3540 inhibited >75% of colon GCPII activity, dose-dependently improved gross and histologic disease, and markedly attenuated monocytic inflammation. In spontaneous colitis in interleukin-10 (IL-10) knockout mice, once-daily oral (S)-IBD3540 initiated after disease onset improved disease, normalized colon histology, and attenuated inflammation as evidenced by reduced fecal lipocalin 2 and colon pro-inflammatory cytokines/chemokines, including tumor necrosis factor-α and IL-17. Using primary human colon epithelial air-liquid interface monolayers to interrogate the mechanism, we further found that (S)-IBD3540 protected against submersion-induced oxidative stress injury by decreasing barrier permeability, normalizing tight junction protein expression, and reducing procaspase-3 activation. Together, this work demonstrated that local inhibition of dysregulated gastrointestinal GCPII using the gut-restricted, orally active, small-molecule (S)-IBD3540 is a promising approach for IBD treatment.

Deletion of glutamate carboxypeptidase II (GCPII), but not GCPIII, provided long-term benefits in mice with traumatic brain injury.

N-acetylaspartylglutamate (NAAG) has neuroprotective effects in traumatic brain injury (TBI) by activating metabotropic glutamate receptor 3 (mGluR3) and reducing glutamate release. Glutamate carboxypeptidase II (GCPII) is the primary enzyme responsible for the hydrolysis of NAAG. It remains unclear whether glutamate carboxypeptidase III (GCPIII), a homolog of GCPII, can partially compensate for GCPII's function. GCPII-/- , GCPIII-/- , and GCPII/III-/- mice were generated using CRISPR/Cas9 technology. Mice brain injury model was established through moderate controlled cortical impact (CCI). The relationship between GCPII and GCPIII was explored by analyzing injury response signals in the hippocampus and cortex of mice with different genotypes at the acute (1 day) and subacute (7 day) phase after TBI. In this study, we found that deletion of GCPII reduced glutamate production, excitotoxicity, and neuronal damage and improved cognitive function, but GCPIII deletion had no significant neuroprotective effect. Additionally, there was no significant difference in the neuroprotective effect between the combination of GCPII and GCPIII deletion and GCPII deletion alone. These results suggest that GCPII inhibition may be a therapeutic option for TBI, and that GCPIII may not act as a complementary enzyme to GCPII in this context.

Dendrimer-enabled targeted delivery attenuates glutamate excitotoxicity and improves motor function in a rabbit model of cerebral palsy.

Glutamate carboxypeptidase II (GCPII), localized on the surface of astrocytes and activated microglia, regulates extracellular glutamate concentration in the central nervous system (CNS). We have previously shown that GCPII is upregulated in activated microglia in the presence of inflammation. Inhibition of GCPII activity could reduce glutamate excitotoxicity, which may decrease inflammation and promote a 'normal' microglial phenotype. 2-(3-Mercaptopropyl) pentanedioic acid (2-MPPA) is the first GCPII inhibitor that underwent clinical trials. Unfortunately, immunological toxicities have hindered 2-MPPA clinical translation. Targeted delivery of 2-MPPA specifically to activated microglia and astrocytes that over-express GCPII has the potential to mitigate glutamate excitotoxicity and attenuate neuroinflammation. In this study, we demonstrate that 2-MPPA when conjugated to generation-4, hydroxyl-terminated polyamidoamine (PAMAM) dendrimers (D-2MPPA) localize specifically in activated microglia and astrocytes only in newborn rabbits with cerebral palsy (CP), not in controls. D-2MPPA treatment led to higher 2-MPPA levels in the injured brain regions compared to 2-MPPA treatment, and the extent of D-2MPPA uptake correlated with the injury severity. D-2MPPA was more efficacious than 2-MPPA in decreasing extracellular glutamate level in ex vivo brain slices of CP kits, and in increasing transforming growth factor beta 1 (TGF-β1) level in primary mixed glial cell cultures. A single systemic intravenous dose of D-2MPPA on postnatal day 1 (PND1) decreased microglial activation and resulted in a change in microglial morphology to a more ramified form along with amelioration of motor deficits by PND5. These results indicate that targeted dendrimer-based delivery specifically to activated microglia and astrocytes can improve the efficacy of 2-MPPA by attenuating glutamate excitotoxicity and microglial activation.

GCPII Inhibition Delays Age-related Loss of Muscle Mass and Function in Mice

Sarcopenia, the age-related loss of skeletal muscle mass and function, is a significant problem that reduces the quality of life in the elderly. Despite the growing prevalence of sarcopenia, there is still a lack of effective therapies for this disease. Recent research has focused on the role of the enzyme glutamate carboxypeptidase II (GCPII) in regulating muscle function and neuromuscular junction (NMJ) integrity. We have previously shown that inhibition of GCPII with the potent inhibitor 2-(Phosphonomethyl)-pentanedioic acid (2-PMPA) can significantly delay muscle function loss and NMJ denervation in an animal model of ALS. Given that sarcopenia shares similarities with ALS in terms of NMJ deterioration, we aimed to investigate whether 2-PMPA treatment could also preserve muscle function during aging. We measured the activity of GCPII in the gastrocnemius muscles of 4-month-old and 20-month-old mice, and found that GCPII activity was significantly increased in the 20-month-old mice (3188±182 fmol/mg/h) compared to 4-month-old mice (2009±317 fmol/mg/h). Then we treated 15-month-old C57BL6/J mice with 100mg/kg of 2-PMPA IP daily for 5 months and monitored muscle function. After 5 months of 2-PMPA treatment, we observed preservation of gastrocnemius muscle mass (164.8±5.6 vs. 143.6±6.4 mg, 2-PMPA vs. vehicle treated group) and a significant delay in age-related loss in grip strength (-4.75±3.22% vs. -20.98±3.73%, 2-PMPA vs. vehicle). We also found that 2-PMPA treatment increased the mean single-fiber cross-sectional area (759.4±5.2 vs. 722.4±5.1 μm 2 , 2-PMPA vs. vehicle), delayed the decline of nerve signals to the muscles as observed by compound muscle action potential (CMAP) amplitude decline (-7.37±6.16% vs. -26.05±3.71%, 2-PMPA vs. vehicle), and improved ambulation in old mice (3941±153 vs. 3216±188 beam brakes, 2-PMPA vs. vehicle) as observed by open-field test. Immunofluorescent staining showed that GCPII expression in aged muscle is associated with infiltrating macrophages, indicating inflammation as one potential target for the role of 2-PMPA in preserving muscle function during aging. Overall, our current results support that excessive GCPII activity is associated with muscle aging and GCPII inhibition is a potential therapeutic strategy for sarcopenia. This study highlights the potential of GCPII inhibition as a new therapeutic approach to preserving muscle function in the elderly. This work was supported by NIH R01 AG078181-01 (to BSS), R01 AG068130 (to BSS), R01NS093416 (to BSS), and R25GM109441 (Hopkins PREP support for TRJ). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

THE ORAL GLUTMATE CARBOXYPEPTIDASE II INHIBITOR (S)-IBD3540 ROBUSTLY ATTENUATES INFLAMMATION IN SPONTANEOUS IL10 COLITIS

Glutamate carboxypeptidase II (GCPII) is elevated in active inflamed biopsies of both Crohn’s disease and ulcerative colitis patients and is a promising therapeutic target for the treatment of IBD. We have previously presented the development and characterization of a first-in-class, gut-restricted, and orally active GCPII inhibitor, (S)-IBD3540, and have shown that oral (S)-IBD3540 attenuates disease severity in multiple murine colitis models, including acute dextran sulfate sodium (DSS)-induced colitis. Here, we extend our knowledge regarding (S)-IBD3540’s anti-inflammatory effects in spontaneously occurring colitis of interleukin 10 (IL10-/-) mice by performing longitudinal measurement of inflammatory biomarker fecal lipocalin 2 (LCN2) and terminal colon cytokine and chemokine measurements.

D-DOPA Is a Potent, Orally Bioavailable, Allosteric Inhibitor of Glutamate Carboxypeptidase II.

Glutamate carboxypeptidase-II (GCPII) is a zinc-dependent metalloenzyme implicated in numerous neurological disorders. The pharmacophoric requirements of active-site GCPII inhibitors makes them highly charged, manifesting poor pharmacokinetic (PK) properties. Herein, we describe the discovery and characterization of catechol-based inhibitors including L-DOPA, D-DOPA, and caffeic acid, with sub-micromolar potencies. Of these, D-DOPA emerged as the most promising compound, with good metabolic stability, and excellent PK properties. Orally administered D-DOPA yielded high plasma exposures (AUCplasma = 72.7 nmol·h/mL) and an absolute oral bioavailability of 47.7%. Unfortunately, D-DOPA brain exposures were low with AUCbrain = 2.42 nmol/g and AUCbrain/plasma ratio of 0.03. Given reports of isomeric inversion of D-DOPA to L-DOPA via D-amino acid oxidase (DAAO), we evaluated D-DOPA PK in combination with the DAAO inhibitor sodium benzoate and observed a >200% enhancement in both plasma and brain exposures (AUCplasma = 185 nmol·h/mL; AUCbrain = 5.48 nmol·h/g). Further, we demonstrated GCPII target engagement; orally administered D-DOPA with or without sodium benzoate caused significant inhibition of GCPII activity. Lastly, mode of inhibition studies revealed D-DOPA to be a noncompetitive, allosteric inhibitor of GCPII. To our knowledge, this is the first report of D-DOPA as a distinct scaffold for GCPII inhibition, laying the groundwork for future optimization to obtain clinically viable candidates.

Inhibition of glutamate-carboxypeptidase-II in dorsolateral prefrontal cortex: potential therapeutic target for neuroinflammatory cognitive disorders

Glutamate carboxypeptidase-II (GCPII) expression in brain is increased by inflammation, e.g. by COVID19 infection, where it reduces NAAG stimulation of metabotropic glutamate receptor type 3 (mGluR3). GCPII-mGluR3 signaling is increasingly linked to higher cognition, as genetic alterations that weaken mGluR3 or increase GCPII signaling are associated with impaired cognition in humans. Recent evidence from macaque dorsolateral prefrontal cortex (dlPFC) shows that mGluR3 are expressed on dendritic spines, where they regulate cAMP-PKA opening of potassium (K+) channels to enhance neuronal firing during working memory. However, little is known about GCPII expression and function in the primate dlPFC, despite its relevance to inflammatory disorders. The present study used multiple label immunofluorescence and immunoelectron microscopy to localize GCPII in aging macaque dlPFC, and examined the effects of GCPII inhibition on dlPFC neuronal physiology and working memory function. GCPII was observed in astrocytes as expected, but also on neurons, including extensive expression in dendritic spines. Recordings in dlPFC from aged monkeys performing a working memory task found that iontophoresis of the GCPII inhibitors 2-MPPA or 2-PMPA markedly increased working memory-related neuronal firing and spatial tuning, enhancing neural representations. These beneficial effects were reversed by an mGluR2/3 antagonist, or by a cAMP-PKA activator, consistent with mGluR3 inhibition of cAMP-PKA-K+ channel signaling. Systemic administration of the brain penetrant inhibitor, 2-MPPA, significantly improved working memory performance without apparent side effects, with largest effects in the oldest monkeys. Taken together, these data endorse GCPII inhibition as a potential strategy for treating cognitive disorders associated with aging and/or neuroinflammation.

DISCOVERY OF IBD3540: A NOVEL GUT-RESTRICTED GLUTAMATE CARBOXYPEPTIDASE II INHIBITOR WITH ORAL ACTIVITY IN MOUSE COLITIS MODELS

Glutamate carboxypeptidase II (GCPII) is robustly overexpressed in both Crohn’s disease and ulcerative colitis and is a promising therapeutic target for IBD. We have previously published that systemically or enema administered small molecule GCPII inhibitors have efficacy in multiple mouse colitis models. While this is a highly encouraging activity profile for a candidate IBD drug, an oral dose route would be strongly preferred. Thus, here we develop and characterize a new class of GCPII inhibitors designed to be gut-restricted for oral administration, with the intention of selecting a lead inhibitor for further clinical development.

Dendrimer-2PMPA selectively blocks upregulated microglial GCPII activity and improves cognition in a mouse model of multiple sclerosis.

Cognitive impairment is a common aspect of multiple sclerosis (MS) for which there are no treatments. Reduced brain N-acetylaspartylglutamate (NAAG) levels are linked to impaired cognition in various neurological diseases, including MS. NAAG levels are regulated by glutamate carboxypeptidase II (GCPII), which hydrolyzes the neuropeptide to N-acetyl-aspartate and glutamate. GCPII activity is upregulated multifold in microglia following neuroinflammation. Although several GCPII inhibitors, such as 2-PMPA, elevate brain NAAG levels and restore cognitive function in preclinical studies when given at high systemic doses or via direct brain injection, none are clinically available due to poor bioavailability and limited brain penetration. Hydroxyl-dendrimers have been successfully used to selectively deliver drugs to activated glia.Methods: We attached 2-PMPA to hydroxyl polyamidoamine (PAMAM) dendrimers (D-2PMPA) using a click chemistry approach. Cy5-labelled-D-2PMPA was used to visualize selective glial uptake in vitro and in vivo. D-2PMPA was evaluated for anti-inflammatory effects in LPS-treated glial cultures. In experimental autoimmune encephalomyelitis (EAE)-immunized mice, D-2PMPA was dosed biweekly starting at disease onset and cognition was assessed using the Barnes maze, and GCPII activity was measured in CD11b+ hippocampal cells.Results: D-2PMPA showed preferential uptake into microglia and robust anti-inflammatory activity, including elevations in NAAG, TGFβ, and mGluR3 in glial cultures. D-2PMPA significantly improved cognition in EAE mice, even though physical severity was unaffected. GCPII activity increased >20-fold in CD11b+ cells from EAE mice, which was significantly mitigated by D-2PMPA treatment.Conclusions: Hydroxyl dendrimers facilitate targeted drug delivery to activated microglia. These data support further development of D-2PMPA to attenuate elevated microglial GCPII activity and treat cognitive impairment in MS.

Dendrimer-2PMPA Delays Muscle Function Loss and Denervation in a Murine Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease where muscle weakness and neuromuscular junction (NMJ) denervation precede motor neuron cell death. Although acetylcholine is the canonical neurotransmitter at the mammalian NMJ synapse, glutamate has recently been identified as a critical neurotransmitter for NMJ development and maintenance. One source of glutamate is through the catabolism of N-acetyl-aspartyl-glutamate (NAAG), which is found in mM concentrations in mammalian motoneurons, where it is released upon stimulation and hydrolyzed to glutamate by the glial enzyme glutamate carboxypeptidase II (GCPII). Using the SOD1G93A model of ALS, we found an almost fourfold elevation of GCPII enzymatic activity in SOD1G93A versus WT muscle and a robust increase in GCPII expression which was specifically associated with activated macrophages infiltrating the muscle. 2-(Phosphonomethyl)pentanedioic acid (2PMPA) is a potent GCPII inhibitor which robustly blocks glutamate release from NAAG but is highly polar with limited tissue penetration. To improve this, we covalently attached 2PMPA to a hydroxyl polyamidoamine (PAMAM-G4-OH) dendrimer delivery system (D-2PMPA) which is known to target activated macrophages in affected tissues. Systemic D-2PMPA therapy (20 mg/kg 2PMPA equivalent; IP 2 × /week) was found to localize in muscle macrophages in SOD1G93A mice and completely normalize the enhanced GCPII activity. Although no changes in body weight or survival were observed, D-2PMPA significantly improved grip strength and inhibited the loss of NMJ innervation in the gastrocnemius muscles. Our finding that inhibiting elevated GCPII activity in SOD1G93A muscle can prolong muscle function and delay NMJ denervation may have early therapeutic implications for ALS patients.

Glutamate Carboxypeptidase II in Aging Rat Prefrontal Cortex Impairs Working Memory Performance.

Glutamate carboxypeptidase II (GCPII) expression in brain is increased by inflammation, and reduces NAAG (N-acetyl aspartyl glutamate) stimulation of mGluR3 signaling. Genetic insults in this signaling cascade are increasingly linked to cognitive disorders in humans, where increased GCPII and or decreased NAAG-mGluR3 are associated with impaired prefrontal cortical (PFC) activation and cognitive impairment. As aging is associated with increased inflammation and PFC cognitive deficits, the current study examined GCPII and mGluR3 expression in the aging rat medial PFC, and tested whether GCPII inhibition with 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA) would improve working memory performance. We found that GCPII protein was expressed on astrocytes and some microglia as expected from previous studies, but was also prominently expressed on neurons, and showed increased levels with advancing age. Systemic administration of the GCPII inhibitor, 2-MPPA, improved working memory performance in young and aged rats, and also improved performance after local infusion into the medial PFC. As GCPII inhibitors are well-tolerated, they may provide an important new direction for treatment of cognitive disorders associated with aging and/or inflammation.

P152 EVALUATING THE EFFICACY OF GLUTAMATE CARBOXYPEPTIDASE II (GCPII) INHIBITORS IN “WILD-TYPE” MICE: LESSONS LEARNED FROM THE DEDICATOR OF CYTOKINESIS 2 MUTANT MOUSE

Glutamate carboxypeptidase II (GCPII) is highly upregulated in human IBD and is a therapeutic target under active investigation by our laboratory. We recently published that a spontaneously occurring loss-of-function mutation in dedicator of cytokinesis 2 (Dock2Hsd) that was present in commercially-purchased “wild-type” C57Bl6/NHsd mice increased their sensitivity to DSS-colitis and caused them to closely resemble human IBD with respect to GCPII. The DSS-exposed Dock2Hsd mice had significantly elevated colon GCPII activities and were sensitive to treatment with the GCPII inhibitor, 2-PMPA. We hypothesized that if colitis of the same severity were to be induced in Dock2WT mice, that they would also exhibit heightened colon GCPII activity and would be equally sensitive to 2-PMPA treatment.

Looking for Drugs in All the Wrong Places: Use of GCPII Inhibitors Outside the Brain.

In tribute to our friend and colleague Michael Robinson, we review his involvement in the identification, characterization and localization of the metallopeptidase glutamate carboxypeptidase II (GCPII), originally called NAALADase. While Mike was characterizing NAALADase in the brain, the protein was independently identified by other laboratories in human prostate where it was termed prostate specific membrane antigen (PSMA) and in the intestines where it was named Folate Hydrolase 1 (FOLH1). It was almost a decade to establish that NAALADase, PSMA, and FOLH1 are encoded by the same gene. The enzyme has emerged as a therapeutic target outside of the brain, with the most notable progress made in the treatment of prostate cancer and inflammatory bowel disease (IBD). PSMA-PET imaging with high affinity ligands is proving useful for the clinical diagnosis and staging of prostate cancer. A molecular radiotherapy based on similar ligands is in trials for metastatic castration-resistant prostate cancer. New PSMA inhibitor prodrugs that preferentially block kidney and salivary gland versus prostate tumor enzyme may improve the clinical safety of this radiotherapy. The wide clinical use of PSMA-PET imaging in prostate cancer has coincidentally led to clinical documentation of GCPII upregulation in a wide variety of tumors and inflammatory diseases, likely associated with angiogenesis. In IBD, expression of the FOLH1 gene that codes for GCPII is strongly upregulated, as is the enzymatic activity in diseased patient biopsies. In animal models of IBD, GCPII inhibitors show substantial efficacy, suggesting potential theranostic use of GCPII ligands for IBD.

Spontaneous Loss-of-Function Dock2 Mutation Alters Murine Colitis Sensitivity and Is a Confounding Variable in Inflammatory Bowel Disease Research

AbstractBackgroundDextran sodium sulfate (DSS)-induced colitis is the most commonly used mouse model of inflammatory bowel disease (IBD) due to its acute nature, reproducibility, and phenotypic overlap with human disease. Following an unexpected and sharp decline in DSS-induced colitis susceptibility in our commercially acquired C57Bl/6 wild-type mice, we discovered that a spontaneous loss-of-function mutation in dedicator of cytokinesis 2 (Dock2Hsd) was responsible. Presence of this mutation in research colonies has the capacity to broadly impact preclinical IBD studies.MethodsDSS-colitis was induced in weight-, age-, and gender-matched C57Bl/6NHsd mice. Daily treatment with vehicle or the glutamate carboxypeptidase II (GCPII) inhibitor, 2-PMPA (100 mg/kg IP), was performed and disease activity index was monitored. At termination, colon GCPII activity was measured.ResultsDSS-treated Dock2Hsd mice developed more severe colitis, had significantly increased colon GCPII activity and were more sensitive to pharmacologic inhibition of GCPII.ConclusionsThe Dock2Hsd mutation is a confounding variable of high relevance to the IBD research community. Dock2Hsd mice were distributed as wild-type C57Bl/6 for multiple years and thus it is unknown how prevalent this mutation is in investigator-maintained colonies of C57Bl/6-derived mice. In our research, presence of the Dock2Hsd mutation caused enhanced GCPII colon activity more closely resembling human disease, providing a useful platform for screening GCPII inhibitors for preclinical efficacy. However, unanticipated presence of Dock2Hsd in genetically modified mice used to study IBD pathobiology can confound conclusions. Thus, care must be taken when interpreting studies performed in mice of C57Bl/6 lineage where Dock2 status is unknown.

The NAAG'ing Concerns of Modeling Human Alzheimer's Disease in Mice.

Studies over the past two decades report significant reductions in brain N-acetylaspartyl glutamate (NAAG) levels in neurodegenerative diseases with associated cognitive impairment, including Alzheimer's disease (AD). Because NAAG is cleaved by glutamate carboxypeptidase II (GCPII), restoration of brain NAAG levels via GCPII inhibition is a potential therapeutic strategy for AD. Herein, studies were conducted to identify an appropriate murine model of AD that recapitulates human brain NAAG changes in order to preclinically evaluate the therapeutic benefit of GCPII inhibition. Our opposing findings of brain NAAG changes in human and mouse AD highlights the limited predictive value of AD mouse models.

Uncovering the Role of N-Acetyl-Aspartyl-Glutamate as a Glutamate Reservoir in Cancer

SUMMARYN-acetyl-aspartyl-glutamate (NAAG) is a peptide-based neurotransmitter that has been extensively studied in many neurological diseases. In this study, we show a specific role of NAAG in cancer. We found that NAAG is more abundant in higher grade cancers and is a source of glutamate in cancers expressing glutamate carboxypeptidase II (GCPII), the enzyme that hydrolyzes NAAG to glutamate and N-acetyl-aspartate (NAA). Knocking down GCPII expression through genetic alteration or pharmacological inhibition of GCPII results in a reduction of both glutamate concentrations and cancer growth. Moreover, targeting GCPII in combination with glutaminase inhibition accentuates these effects. These findings suggest that NAAG serves as an important reservoir to provide glutamate to cancer cells through GCPII when glutamate production from other sources is limited. Thus, GCPII is a viable target for cancer therapy, either alone or in combination with glutaminase inhibition.

P135 ORALLY ADMINISTERED FOLH1/GCPII INHIBITORS AS NOVEL THERAPEUTICS IN INFLAMMATORY BOWEL DISEASE (IBD)

The folate hydrolase gene (FOLH1), which encodes for the enzyme glutamate carboxypeptidase II (GCPII), has been implicated in the pathogenesis of inflammatory bowel disease (IBD) where it exhibits a robust 300-1000% increase in enzymatic activity in intestinal biopsies collected from Crohn’s Disease and Ulcerative Colitis patients. We have previously shown that systemic administration of the GCPII inhibitor 2-PMPA (IC50=300 pM) led to significant improvement in disease activity index in three commonly used murine models of IBD: DSS-colitis, TNBS-colitis and spontaneous colitis of IL10-/- mice. While systemic administration of 2-PMPA was effective in preclinical models, in the clinic an oral dosing regimen would be preferred. 2-PMPA is a highly polar compound with minimal oral bioavailability and tissue penetration. Despite displaying systemic restriction in normal animals, we hypothesized that oral administration might provide direct local therapy to inflamed colonic epithelia and be equally effective to systemic dosing in murine colitis models. Age-, gender- and weight-matched wild-type C57Bl/6NHsd mice were exposed to 2.5% DSS in drinking water for 5 days to induce colitis followed by a 2-day exposure to fresh water. Body weight loss, stool consistency and presence of rectal bleeding were scored daily and summated to generate a disease activity index. Daily oral 2-PMPA (0.01 – 100 mg/kg) or vehicle (HEPES buffered saline) was administered. Mice were euthanized at multiple time points (study days 0, 3,5 and 7) and tissues were harvested for pharmacokinetic analysis. Oral 2-PMPA significantly reduced disease activity index in DSS-colitis at doses as low as 1 mg/kg, which is 100-fold lower than the dose utilized for systemic treatment (p<0.01, n=20/group). However, DSS treatment resulted in loss of epithelial barrier integrity and caused increased systemic 2-PMPA plasma exposures, thus we cannot fully determine whether the mechanism of action of oral 2-PMPA was locally- versus systemically-mediated in this model. This is relevant as the barrier disruption after acute DSS treatment in mice is likely to be more severe than what is observed clinically in IBD patients. Therefore, we embarked upon further studies to explore whether 2-PMPA has a direct activity on colonic epithelium. Pilot studies in Caco2 monolayers show that 2-PMPA protects against LPS-induced alterations in epithelial barrier permeability as measured by TEER (p<0.05) and attenuates RNA expression of TNFα (p<0.05), providing support that 2-PMPA has direct effects on colon epithelial cells. The discovery that orally administered 2-PMPA displays robust and potent efficacy in the murine DSS-model, with preliminary evidence of direct epithelial actions, provides strong support that FOLH1/GCPII is a clinically relevant, druggable, target in IBD.

P135 ORALLY ADMINISTERED FOLH1/GCPII INHIBITORS AS NOVEL THERAPEUTICS IN INFLAMMATORY BOWEL DISEASE (IBD)

The folate hydrolase gene (FOLH1), which encodes for the enzyme glutamate carboxypeptidase II (GCPII), has been implicated in the pathogenesis of inflammatory bowel disease (IBD) where it exhibits a robust 300-1000% increase in enzymatic activity in intestinal biopsies collected from Crohn’s Disease and Ulcerative Colitis patients. We have previously shown that systemic administration of the GCPII inhibitor 2-PMPA (IC50=300 pM) led to significant improvement in disease activity index in three commonly used murine models of IBD: DSS-colitis, TNBS-colitis and spontaneous colitis of IL10-/- mice. While systemic administration of 2-PMPA was effective in preclinical models, in the clinic an oral dosing regimen would be preferred. 2-PMPA is a highly polar compound with minimal oral bioavailability and tissue penetration. Despite displaying systemic restriction in normal animals, we hypothesized that oral administration might provide direct local therapy to inflamed colonic epithelia and be equally effective to systemic dosing in murine colitis models. Age-, gender- and weight-matched wild-type C57Bl/6NHsd mice were exposed to 2.5% DSS in drinking water for 5 days to induce colitis followed by a 2-day exposure to fresh water. Body weight loss, stool consistency and presence of rectal bleeding were scored daily and summated to generate a disease activity index. Daily oral 2-PMPA (0.01 – 100 mg/kg) or vehicle (HEPES buffered saline) was administered. Mice were euthanized at multiple time points (study days 0, 3,5 and 7) and tissues were harvested for pharmacokinetic analysis. Oral 2-PMPA significantly reduced disease activity index in DSS-colitis at doses as low as 1 mg/kg, which is 100-fold lower than the dose utilized for systemic treatment (p<0.01, n=20/group). However, DSS treatment resulted in loss of epithelial barrier integrity and caused increased systemic 2-PMPA plasma exposures, thus we cannot fully determine whether the mechanism of action of oral 2-PMPA was locally- versus systemically-mediated in this model. This is relevant as the barrier disruption after acute DSS treatment in mice is likely to be more severe than what is observed clinically in IBD patients. Therefore, we embarked upon further studies to explore whether 2-PMPA has a direct activity on colonic epithelium. Pilot studies in Caco2 monolayers show that 2-PMPA protects against LPS-induced alterations in epithelial barrier permeability as measured by TEER (p<0.05) and attenuates RNA expression of TNFα (p<0.05), providing support that 2-PMPA has direct effects on colon epithelial cells. The discovery that orally administered 2-PMPA displays robust and potent efficacy in the murine DSS-model, with preliminary evidence of direct epithelial actions, provides strong support that FOLH1/GCPII is a clinically relevant, druggable, target in IBD.