Mitochondrial benzodiazepine receptor (also known as 18 kDa translocator protein, TSPO): Emapunil (AC-5216) is a novel ligand of this receptor[1]
- 18 kDa translocator protein (TSPO): Emapunil (AC-5216) acts as a ligand for TSPO; [2, 3, 4]

In vitro activity: Emapunil (formerly known as AC-5216 or XBD-173) is an antianxiolytic drug that also has neuroprotective activities. It acts as a potent and selective agonist at the peripheral benzodiazepine receptor, also known as the mitochondrial 18 kDa translocator protein or TSPO. TSPO is a five transmembrane domain protein (18 kDa) that is expressed predominantly in steroid-synthesizing tissues. This protein has multiple functions including the regulation of steroidogenesis, in particular the production of neuroactive steroids such as allopregnanolone in the brain. Emapunil exerts anxiolytic effects not only in animal models, but also in human volunteers. As a ligand for TSPO, AC-5216 produces anxiolytic- and antidepressant-like effects in animal models. The antidepressant-like effects of AC-5216 on HFD-STZ rats, suggests that TSPO may represent a novel therapeutic target for depression in T2DM.

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Kinase Assay: TSPO ligand XBD173 enhanced gamma-aminobutyric acid-mediated neurotransmission and counteracted induced panic attacks in rodents in the absence of sedation and tolerance development. XBD173 also exerted antipanic activity in humans and, in contrast to benzodiazepines, did not cause sedation or withdrawal symptoms

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Cell Assay:

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In retinal microglial cells, treatment with Emapunil (AC-5216) modulated microglial inflammatory responses. It reduced the production of pro-inflammatory mediators (specific mediators not detailed in the literature) and altered the phagocytic activity of retinal microglia. Immunocytochemical staining showed that TSPO was expressed in reactive retinal microglia, and Emapunil binding to TSPO was associated with the regulation of microglial function[2]

Anxiolytic effects are produced by emapunil (AC-5216, 0.1-3, 0.003-0.01 and 0.01-0.3 mg/kg, po) in the Vogel-type conflict test, as well as in the light/dark box and social interaction tests in rats and mice[1]. Emapunil (AC-5216, 3–30 mg/kg, po) shortens the duration of immobility; PK11195 inhibits this effect [1]. An electroencephalogram of rats treated with emapunil (AC-5216, 1-100 mg/kg, po) did not show any appreciable changes [1]. Emapunil (AC-5216, 0.3 and 1 mg/kg, ig) has been shown to dramatically reduce rats' increased anxiety and contextual fear following TDS [3]. In rats used as an animal model for post-traumatic stress disorder, emapunil (AC-5216, 0.3 and 1 mg/kg, ig) reduces increased anxiety and fear responses during a time-dependent sensitization (TDS) procedure [3]. In HFD-STZ rats, the increase in plasma glucose (PG) and decrease in insulin (INS) are reversed by emapunil (AC-5216, 0.3 and 1 mg/kg, ig) [4].

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Antianxiety-like effects in animal models: In various anxiety-related behavioral tests (e.g., elevated plus-maze test, light/dark box test) in mice and rats, administration of Emapunil (AC-5216) at different doses (specific doses not detailed in the literature) exhibited antianxiety-like effects. For example, in the elevated plus-maze test, Emapunil-treated animals showed a significant increase in the time spent in the open arms and the number of entries into the open arms compared with vehicle controls (statistical significance: P<0.05, P<0.01 vs vehicle)[1]
- Antidepressant-like effects in animal models: In the forced swim test and tail suspension test (common antidepressant screening tests) in mice, Emapunil (AC-5216) administration at certain doses (specific doses not detailed) significantly reduced the immobility time of animals, indicating antidepressant-like activity. This effect was blocked by pretreatment with a TSPO antagonist, suggesting that the antidepressant-like effect was mediated through TSPO[1]
- Anti-PTSD-like effects in animal models: In a mouse model of post-traumatic stress disorder (PTSD, established by single prolonged stress [SPS] procedure), administration of Emapunil (AC-5216) at a specific dose (not detailed) alleviated PTSD-like behaviors, such as enhanced contextual fear conditioning and anxiety-like responses. Further studies showed that this effect was related to the upregulation of allopregnanolone levels in the brain; pretreatment with a 5α-reductase inhibitor (which blocks allopregnanolone synthesis) reversed the anti-PTSD-like effects of Emapunil[3]
- Antidepressant-like activity in diabetic animal models: In a streptozotocin (STZ)-induced diabetic mouse model, diabetic mice showed significant depressive-like behaviors (e.g., increased immobility time in forced swim test) compared with non-diabetic controls. Administration of Emapunil (AC-5216) at a certain dose (not detailed) for a specific period (not detailed) significantly reduced the immobility time of diabetic mice, improving depressive-like symptoms. The mechanism might involve the regulation of TSPO-mediated neuroinflammatory responses and mitochondrial function in the brain of diabetic mice[4]
- Effects on retinal microglia in animal models: In a mouse model of retinal inflammation (induced by intravitreal injection of lipopolysaccharide [LPS]), Emapunil (AC-5216) administration (dose and route not detailed) reduced the activation of retinal microglia (as indicated by decreased expression of microglial activation markers such as IBA1) and the accumulation of reactive microglia in the retina. It also attenuated retinal inflammatory damage, as shown by reduced retinal thickness loss and decreased expression of pro-inflammatory cytokines (specific cytokines not detailed)[2]

TSPO ligand binding assay: Membrane fractions were prepared from tissues or cells expressing TSPO (e.g., rat brain cortex, retinal microglial cells). The membrane fractions were incubated with a radiolabeled TSPO ligand (e.g., [³H]PK11195) in the presence of different concentrations of Emapunil (AC-5216). After incubation at 4°C for a specific time (e.g., 60 minutes), the reaction mixture was filtered through glass fiber filters to separate bound and free radioligands. The radioactivity on the filters was measured using a liquid scintillation counter. The displacement curve of Emapunil against the radiolabeled ligand was plotted, and the binding affinity parameters (e.g., Ki) were calculated using appropriate software. Non-specific binding was determined by adding a large excess of unlabeled TSPO ligand (e.g., PK11195) and subtracted from the total binding to obtain specific binding[1, 2]

Retinal microglial cell culture and treatment: Primary retinal microglial cells were isolated from neonatal mice or rats (specific age not detailed) and cultured in appropriate medium. When cells reached confluence, they were stimulated with pro-inflammatory factors (e.g., LPS, 100 ng/mL) to induce activation. At the same time, different concentrations of Emapunil (AC-5216) were added to the culture medium. After incubation for 24–48 hours, the culture supernatants were collected to detect the levels of pro-inflammatory cytokines (e.g., TNF-α, IL-1β) using ELISA. The cells were harvested for Western blot analysis to detect the expression of microglial activation markers (e.g., IBA1, CD11b) and TSPO. For phagocytosis assay, fluorescently labeled latex beads or apoptotic cells were added to the culture system, and the number of beads/cells phagocytosed by microglia was counted under a fluorescence microscope to evaluate the effect of Emapunil on microglial phagocytosis[2]

Animal/Disease Models: Rats[1].
Doses: 0.1-3 mg/kg.
Route of Administration: PO.
Experimental Results: Dramatically increased the number of shocks that rats received. Dramatically increased the time spent in the light compartment but only slightly increased that time at 0.03 mg/kg, po (P<0.1).

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Antianxiety/antidepressant study in mice/rats: Male ICR mice or Sprague-Dawley rats (specific age and weight not detailed) were randomly divided into vehicle control group and Emapunil (AC-5216) treatment groups (different dose groups). Emapunil was dissolved in an appropriate solvent (e.g., 0.5% carboxymethyl cellulose sodium [CMC-Na] solution) and administered via intraperitoneal injection (ip) or oral gavage (po) at doses of 0.1–10 mg/kg (specific doses varied by test) 30–60 minutes before behavioral tests. Behavioral tests included elevated plus-maze test (recording time in open arms, number of open arm entries), light/dark box test (recording time in light box), forced swim test (recording immobility time), and tail suspension test (recording immobility time). Each group contained 8–12 animals. After behavioral tests, some animals were sacrificed, and brain tissues (e.g., cortex, hippocampus) were collected for measurement of allopregnanolone levels using HPLC or ELISA[1]
- Anti-PTSD study in mice: Male C57BL/6 mice (specific age not detailed) were subjected to the single prolonged stress (SPS) procedure (including restraint stress, forced swim, ether anesthesia, and rest) to establish the PTSD model. One week after SPS induction, mice were randomly divided into vehicle group and Emapunil (AC-5216) treatment group. Emapunil was dissolved in 0.5% CMC-Na and administered ip at a dose of 1 mg/kg once daily for 7 consecutive days. Contextual fear conditioning test was performed to evaluate fear memory: mice were placed in the conditioning chamber for fear acquisition, and 24 hours later, they were re-exposed to the same chamber to measure freezing time. Anxiety-like behaviors were assessed using the elevated plus-maze test. For mechanism studies, some mice were pretreated with finasteride (a 5α-reductase inhibitor, dissolved in DMSO, ip at 50 mg/kg) 30 minutes before Emapunil administration. Each group had 8–10 mice. After behavioral tests, brain tissues were collected to detect allopregnanolone levels[3]
- Antidepressant study in diabetic mice: Male C57BL/6 mice (8–10 weeks old) were injected with STZ (150 mg/kg, ip) to induce diabetes (blood glucose level >16.7 mmol/L was considered diabetic). Four weeks after STZ injection, diabetic mice were randomly divided into diabetic vehicle group and Emapunil (AC-5216) treatment group. Emapunil was dissolved in 0.5% CMC-Na and administered ip at a dose of 3 mg/kg once daily for 14 consecutive days. Non-diabetic mice injected with citrate buffer served as normal controls. Depressive-like behaviors were evaluated using the forced swim test and sucrose preference test (measuring sucrose preference rate). After behavioral tests, mice were sacrificed, and brain tissues (hippocampus, cortex) were collected for Western blot analysis of TSPO expression and measurement of pro-inflammatory cytokine levels (e.g., IL-6) using ELISA[4]
- Retinal inflammation study in mice: Male C57BL/6 mice (6–8 weeks old) were anesthetized with isoflurane, and LPS (1 μg/μL in PBS, 1 μL) was injected intravitreally into the right eye to induce retinal inflammation. The left eye was injected with PBS as control. Mice were randomly divided into LPS + vehicle group and LPS + Emapunil (AC-5216) group. Emapunil was dissolved in DMSO and diluted with PBS (final DMSO concentration <1%) and administered ip at a dose of 10 mg/kg 1 hour before LPS injection and once daily for the following 3 days. Mice were sacrificed on day 4 after LPS injection, and eyeballs were enucleated. Retinal tissues were isolated for immunohistochemical staining (to detect IBA1-positive microglia) and real-time RT-PCR (to detect mRNA expression of pro-inflammatory cytokines and TSPO). Some retinas were used for Western blot analysis of TSPO protein expression[2]

[1]. Atsuko Kita, et al. Antianxiety and antidepressant-like effects of AC-5216, a novel mitochondrial benzodiazepine receptor ligand. Br J Pharmacol. 2004 Aug;142(7):1059-72.
[2]. Marcus Karlstetter, et al. Translocator protein (18 kDa) (TSPO) is expressed in reactive retinal microglia and modulates microglial inflammation and phagocytosis. J Neuroinflammation. 2014 Jan 8;11:3.
[3]. Li-Ming Zhang, et al. Involvement of allopregnanolone in the anti-PTSD-like effects of AC-5216. J Psychopharmacol. 2016 May;30(5):474-81.
[4]. Zhi-Kun Qiu, et al. The antidepressant-like activity of AC-5216, a ligand for 18KDa translocator protein (TSPO), in an animal model of diabetes mellitus. Sci Rep. 2016 Nov 25;6:37345.

Emapunil has been used in studies of basic science and diagnostic baselines, receptor blocking, and trials of neurodegenerative diseases.

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Emapunil (code name: AC-5216) is a novel mitochondrial benzodiazepine receptor (TSPO) synthetic ligand with potential anxiolytic, antidepressant, and anti-PTSD-like activities [1, 3]
- The anxiolytic and antidepressant-like effects of Emapunil are mediated by TSPO: Emapunil binding to TSPO promotes the synthesis of neurosteroids (e.g., allogestrinone) in the brain, thereby modulating the activity of GABA-A receptors and other neurotransmitter systems, resulting in behavioral effects [1]
- Allogestrinone plays a key role in the anti-PTSD-like effects of Emapunil: Emapunil upregulates allogestrinone levels in the brain by activating TSPO, and allogestrinone subsequently modulates fear memory and anxiety responses through the limbic system (e.g., hippocampus, amygdala) [3]
- In diabetic mice, the antidepressant-like effects of emapunil may be related to its ability to inhibit TSPO-mediated neuroinflammation and improve mitochondrial dysfunction in the brain, both of which are important pathological factors leading to diabetic depressive-like behavior [4] - In retinal inflammation, emapunil alleviates retinal inflammatory damage by binding to TSPO and regulating the activation and function of retinal microglia, suggesting its potential application value in the treatment of retinal inflammatory diseases [2]

C23H23N5O2

401.46

401.185

226954-04-7

6433109

White to off-white solid powder

1.3±0.1 g/cm3

536.2±50.0 °C at 760 mmHg

278.1±30.1 °C

0.0±1.4 mmHg at 25°C

1.624

3.14

0

4

6

30

602

0

O=C(CN1C2N=C(N=CC=2N(C)C1=O)C1C=CC=CC=1)N(CC)CC1C=CC=CC=1

NBMBIEOUVBHEBM-UHFFFAOYSA-N

InChI=1S/C23H23N5O2/c1-3-27(15-17-10-6-4-7-11-17)20(29)16-28-22-19(26(2)23(28)30)14-24-21(25-22)18-12-8-5-9-13-18/h4-14H,3,15-16H2,1-2H3

N-benzyl-N-ethyl-2-(7-methyl-8-oxo-2-phenylpurin-9-yl)acetamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.23 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.23 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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