NMDA (N-methyl-D-aspartate) receptor; AP-5 acts as a competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, specifically targeting the glutamate binding site on the NMDA receptor. The compound targets the Glutamate receptor ionotropic NMDA 1 (NMDAR1) subunit. As a competitive antagonist, AP-5 blocks the receptor by competing with the endogenous agonist glutamate for binding at the orthosteric site, thereby preventing receptor activation and the subsequent influx of calcium ions through the receptor channel. No specific IC₅₀ or Kᵢ values for AP-5 at the NMDA receptor were provided in the available literature.

The glutamate-induced increase in Arc/Arg3.1 protein levels is partially inhibited by DL-AP5 (100 μM) [5]. Arc/Arg3.1's NMDA-induced upregulation is reduced by DL-AP5 [5].

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In vitro studies demonstrate that AP-5 functions as a specific NMDA receptor antagonist. At a concentration of 100 μM, AP-5 partially prevents glutamate-induced increases in Arc/Arg3.1 protein levels, a marker of neuronal activity. Additionally, AP-5 reduces NMDA-induced upregulation of Arc/Arg3.1, confirming its ability to block NMDA receptor-mediated signaling pathways. The compound is also reported to specifically block channels in rabbit retina, consistent with its NMDA receptor antagonist properties.

DL-AP5 (0-10 μg/rat, Intra-CA1) greatly diminishes the effect of NMDA [3]. DL-AP5 (0-10 nmol, intracerebroventricular injection) promotes a dose-dependent increase in food consumption [4]. DL-AP5 (5 nmol, intracerebroventricular injection) attenuates the reduction in food consumption generated by intracerebroventricular injection of ghrelin [4].

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This study was designed to examine the effects of intracerebroventricular injection of DL-AP5 (N-methyl-D-aspartate (NMDA) receptor antagonist) and glutamate on ghrelin-induced feeding behavior in 3-h food-deprived (FD3) broiler cockerels. At first, guide cannula was surgically implanted in the right lateral ventricle of chickens. In experiment 1, birds were intracerebroventricularly injected with 0, 2.5, 5, and 10 nmol of DL-AP5. In experiment 2, chickens received 5 nmol DL-AP5 prior to the injection of 0.6 nmol ghrelin. In experiment 3, birds were administered with 0.6 nmol ghrelin after 300 nmol glutamate, and the cumulative feed intake was determined at 3-h postinjection. The results of this study showed that the intracerebroventricular injection of DL-AP5 increased food consumption in FD3 broiler cockerels (P ≤ 0.05), and this increase occurs in a dose-dependent manner. Moreover, the decreased food intake induced with the intracerebroventricular injection of ghrelin was additively enhanced by pretreatment with glutamate, and this effect was attenuated by DL-AP5 administration(P ≤ 0.05).These results suggest that there is an interaction between ghrelin and glutamatergic system (through NMDA receptor) on food intake in broiler cockerels.[4]

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AP-5 exhibits significant in vivo activity across multiple behavioral and physiological paradigms. In pain studies, AP-5 shows significant antinociceptive (pain-relieving) activity. In a rat model of neuropathic pain (sciatic nerve branch selective injury), intraperitoneal administration of AP-5 at 0.7 mg/kg/day for 7 days modulated spinal cord cAMP-PKA-CREB pathway activity, which is involved in pain processing. In learning and memory studies, intracerebroventricular administration of AP-5 (6 μg/2 μL) prior to training impaired passive avoidance retention in rats, demonstrating the critical role of NMDA receptors in memory formation. This memory-impairing effect was dose-dependently prevented by pretreatment with oxiracetam and D-pyroglutamic acid at doses ranging from 50 to 500 mg/kg (subcutaneous). In feeding behavior studies, intracerebroventricular injection of AP-5 (0-10 nmol) caused a dose-dependent increase in food consumption, and AP-5 (5 nmol, intracerebroventricular) attenuated the reduction in food intake induced by ghrelin injection. In memory state-dependent learning studies, intra-CA1 injection of DL-AP5 (0.25 and 0.5 μg/mouse) reversed the memory impairment induced by post-training tramadol (5 mg/kg, intraperitoneal) and also induced tramadol state-dependent memory when co-administered with a sub-effective dose of tramadol (1.25 mg/kg).

The NMDA receptor antagonistic properties of AP-5 have been characterized through functional assays rather than direct binding assays in the available literature. AP-5 is described as a competitive NMDA receptor antagonist, meaning it competes with the endogenous agonist glutamate for binding to the NMDA receptor’s orthosteric (glutamate recognition) site. The compound’s specificity for NMDA receptors over other glutamate receptor subtypes (such as AMPA and kainate receptors) has been well established through electrophysiological and pharmacological studies, making it a standard tool for distinguishing NMDA receptor-mediated responses from other glutamatergic signaling.

Detailed cell assay protocols for AP-5 are limited in the available literature. In cultured neuronal preparations, AP-5 at a concentration of 100 μM partially prevented glutamate-induced increases in Arc/Arg3.1 protein levels, a marker of activity-dependent gene expression. The compound also reduced NMDA-induced Arc/Arg3.1 upregulation, confirming its ability to block NMDA receptor-mediated signaling in cellular models. These assays typically involve treating cultured neurons with AP-5 prior to or concurrent with NMDA or glutamate stimulation, followed by protein extraction and Western blot analysis to measure Arc/Arg3.1 levels.

Animal/Disease Models: Male Wistar rat (180-230 g) [3]
Doses: 1, 3.2 and 10 μg/rat
Route of Administration: Inject into the dorsal hippocampus (within CA1) immediately after electric shock, one time
Experimental Results:Significant reduction There was a significant interaction effect of NMDA (10-2 μg/rat, within CA1).

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Animal/Disease Models: Broiler chicken (FD3) (3 hrs (hrs (hours)) fast, n=8 per group) [4]
Doses: 0, 2.5, 5 and 10 nmol; volume of 10 µL
Route of Administration: intracerebroventricular injection
Experimental Results: dose causing food consumption The dependence increased, being significant for the 5 and 10 nmol doses.

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Animal/Disease Models: broiler rooster (fasted for 3 hrs (hrs (hours)) (FD3), n=8 per group) [4]
Doses: 5 nmol
Route of Administration: intracerebroventricular injection, followed by ghrelin (0.6 nmol)
Experimental Results: Attenuated by Intracerebroventricular injection of ghrelin.

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To determine the involvement of glutamate NMDA receptor in the brain in ghrelin-induced eating response, effects of centrally administered DL-AP5 and glutamate on ghrelin-induced eating response were determined in chickens. Injections were made with a 29-gauge, thin-walled stainless-steel injection cannula which extends 1.0 mm beyond the guide cannula. This injection cannula was connected to a 10-μl Hamilton syringe connected to a 60 cm length of PE-20 tubing. Solutions were injected over a period of 60 s. Another 60-s period was allowed to permit the solution to diffuse from the tip of the cannula into the ventricle. All experimental procedures were performed between 9 a.m. and 2 p.m. Before the injections, the birds were removed from their individual cages, restricted by hand, and after injections were put back into their cages. Birds were handled and mock-injected daily during the 5 days recovery period to habituate them to the injection procedure. Three hours before the beginning of the experiments, animals were deprived of food but with water ad lib. Immediately after injections, the birds were returned to their cages. Fresh food was supplied at the time of injection, and cumulative feed intake (grams) was recorded at 15, 30, 60, 120, and 180 min after injection. Placement of the guide cannula into the ventricle was verified by the presence of cerebrospinal fluid and intracerebroventricular injection of methylene blue and anatomically slicing the frozen brain tissue at the end of the experiments.[4]
Experiment 1 was designed to examine the effect of intracerebroventricular injections of different DL-AP5 doses on cumulative feed intake in 3-h food-deprived (FD3, n = 8 for each group) chickens. For this purpose, the birds received 0, 2.5, 5, and 10 nmol of DL-AP5 in a volume of 10 μl. Control group was injected with 10 μl of 0.9% NaCl solution.[4]
In experiment 2, birds of each group received two injections. The first injection consisted of either 0 or 5 nmol DL-AP5 in a volume of 5 μl. The second injection consisted of either 0 or 0.6 nmol ghrelin in a volume of 5 μl, 15 min after the first injection as described in Table 1 (n = 7–9 for each group).[4]

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Neuropathic pain model: Male Sprague-Dawley rats were subjected to selective nerve injury of the sciatic nerve branches (SNI model). AP-5 was administered intraperitoneally at a dose of 0.7 mg/kg/day for 7 consecutive days. Mechanical pain thresholds were measured before model establishment and at days 10 and 16 post-SNI. Spinal cord tissue was collected for analysis of cAMP content (by radioimmunoassay) and PKA, p-PKA, and CREB protein expression (by Western blot). Passive avoidance learning model: Rats received intracerebroventricular administration of AP-5 (6 μg/2 μL) prior to training. Pretreatment with oxiracetam or D-pyroglutamic acid (50-500 mg/kg, subcutaneous) was administered before AP-5 treatment. Retention was tested after training, and the disruptive effect of AP-5 on passive avoidance was measured. Memory state-dependent learning model: Adult male NMRI mice received post-training intraperitoneal injections of tramadol (2.5 or 5 mg/kg). For state-dependent memory studies, DL-AP5 was injected into the CA1 region of the hippocampus (0.25 or 0.5 μg/mouse) before the memory retention test. NMDA was also injected into the CA1 region (10⁻⁵ or 10⁻⁴ μg/mouse) 5 minutes before tramadol administration. Feeding behavior model: AP-5 was administered via intracerebroventricular injection (0-10 nmol) to rats, and food consumption was measured. In separate experiments, AP-5 (5 nmol, intracerebroventricular) was administered to assess its effect on ghrelin-induced changes in food intake.

No ADME/pharmacokinetic properties (absorption, distribution, metabolism, excretion, half-life, oral bioavailability, plasma protein binding) for AP-5 were described in the available literature. AP-5 is primarily used as a research tool for intracerebroventricular, intrahippocampal, or intraperitoneal administration in animal studies. The compound is noted to be water-soluble, facilitating its use in injection formulations.

No direct toxicity data (such as LD₅₀, hepatotoxicity, nephrotoxicity, or specific organ toxicity) for AP-5 were reported in the available literature. The compound is classified for research use only and is not intended for human therapeutic use. In animal studies, AP-5 has been administered at various doses without acute toxicity being reported: intraperitoneal doses up to 0.7 mg/kg/day for 7 days in rats, intracerebroventricular doses up to 10 nmol, and intra-CA1 doses up to 0.5 μg/mouse. The compound should be handled with standard laboratory safety precautions, including the use of appropriate personal protective equipment.

[1]. Neurokinin and NMDA antagonists (but not a kainic acid antagonist) are antinociceptive in the mouse formalin model. Pain. 1991;44(2):179-185.

[2]. N-methyl-D-aspartate receptors of ganglion cells in rabbit retina. J Neurophysiol. 1990;63(1):16-30.

[3]. Jafari-Sabet M. NMDA receptor blockers prevents the facilitatory effects of post-training intra-dorsal hippocampal NMDA and physostigmine on memory retention of passive avoidance learning in rats. Behav Brain Res. 2006 Apr 25;169(1):120-7.

[4]. The effects of DL-AP5 and glutamate on ghrelin-induced feeding behavior in 3-h food-deprived broiler cockerels. J Physiol Biochem. 2011 Jun;67(2):217-23.

[5]. Glutamate-induced rapid induction of Arc/Arg3.1 requires NMDA receptor-mediated phosphorylation of ERK and CREB. Neurosci Lett. 2017 Nov 20;661:23-28.

2-Amino-5-phosphonovalerate is a 5-phosphonate derivative of 2-aminovalerate and can act as an N-methyl-D-aspartate receptor antagonist. It also has NMDA receptor antagonist activity. Its function is related to that of phosphonates and 2-aminovalerate. 2-Nitro-5-phosphonovalerate has been reported in Euglena gracilis, and relevant data are available.

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NMDA receptor antagonist class: AP-5 belongs to the class of competitive NMDA receptor antagonists, which bind to the same site as the endogenous agonist glutamate. This contrasts with non-competitive antagonists (e.g., MK-801) that bind to other sites on the receptor complex (such as the ion channel pore). Role in synaptic plasticity research: AP-5 has been instrumental in establishing the role of NMDA receptors in long-term potentiation (LTP), a cellular correlate of learning and memory. Studies using AP-5 have shown that NMDA receptor activation is required for the induction of LTP in the hippocampus. Experimental design for memory studies: In educational and research contexts, AP-5 is used as an NMDAR antagonist to block LTP and impair memory formation. A typical experimental design involves two groups of animals: one receiving AP-5 injection and the other receiving saline control, followed by behavioral testing (e.g., hidden platform water maze task) to assess memory performance. Not to be confused with other “AP5” compounds: The CAS number 76326-31-3 specifically refers to the NMDA receptor antagonist AP-5. There are other compounds with similar abbreviated names (e.g., AP5A, diadenosine pentaphosphate) that target different receptors (such as dinucleotide receptors or purinergic receptors) and should not be confused with this agent.

C5H12NO5P

197.1262

197.045

C, 30.47; H, 6.14; N, 7.11; O, 40.58; P, 15.71

76326-31-3

D-AP5;79055-68-8;L-AP5;79055-67-7

1216

White to off-white solid powder

1.5±0.1 g/cm3

482.1±55.0 °C at 760 mmHg

245.4±31.5 °C

0.0±2.6 mmHg at 25°C

1.536

-2.32

4

6

5

12

200

0

C(CC(C(=O)O)N)CP(=O)(O)O

VOROEQBFPPIACJ-UHFFFAOYSA-N

InChI=1S/C5H12NO5P/c6-4(5(7)8)2-1-3-12(9,10)11/h4H,1-3,6H2,(H,7,8)(H2,9,10,11)

2-amino-5-phosphonopentanoic acid

76326-31-3; DL-AP5; 2-Amino-5-phosphonopentanoic acid; DL-2-Amino-5-phosphonopentanoic acid; 5-Phosphononorvaline; 2-AMINO-5-PHOSPHONOVALERATE; 2-Amino-5-phosphovaleric acid; 2-Amino-5-phosphonovaleric Acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~33.33 mg/mL (~169.08 mM)

Solubility in Formulation 1: 50 mg/mL (253.64 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)