VU0152100 is a highly selective positive allosteric modulator (PAM) of the M₄ muscarinic acetylcholine receptor (mAChR). It potentiates the response of M₄ to acetylcholine.

- It shows minimal activity at other mAChR subtypes (M₁, M₂, M₃, M₅) in vitro. [1]
- It has weak affinity for the human dopamine transporter (hDAT), with a Kᵢ of 4.75 μM and an IC₅₀ of 5.98 μM for inhibiting [³H]dopamine uptake in CHO-K1 cells expressing hDAT. [1]
- At the rat dopamine transporter (rDAT), the IC₅₀ for inhibiting [³H]dopamine uptake is >10 μM in HEK293T cells transiently expressing rDAT. [1]

VU0152100 was characterized for its potential off-target effects at the dopamine transporter (DAT).

1. Human DAT Binding and Uptake: In CHO-K1 cells expressing recombinant human DAT, VU0152100 displaced binding of the DAT inhibitor [¹²⁵I]RTI-55 with an estimated Kᵢ of 4.75 μM. It also inhibited [³H]dopamine uptake with an estimated IC₅₀ of 5.98 μM. [1]
2. Rat DAT Uptake: In HEK293T cells transiently transfected with the rat DAT, VU0152100 inhibited [³H]dopamine uptake with an IC₅₀ greater than 10 μM. [1]
3. Pharmacokinetic Verification: The study verified that the batch of VU0152100 used had a brain AUC₀₋ₜ of 36.2 μM·h and a Cmax of 7.6 μM after a 56.6 mg/kg i.p. dose, resulting in a calculated free brain concentration of ~340 nM. This concentration is aligned with M₄ potentiation and well below levels needed to affect DAT. [1]

VU0152100 (10, 30, 56.6 mg/kg; ip; single) counteracts hypermotility brought on by amphetamines [1]. VU0152100 (10, 30, 56.6 mg/kg; ip; single) counteracts hypermotility brought on by amphetamines [1]. In the nucleus accumbens and caudate-putamen, VU0152100 reverses amphetamine-induced elevations in extracellular dopamine levels [1].

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VU0152100 demonstrates robust antipsychotic drug-like effects in multiple rodent models, primarily by reversing amphetamine-induced behaviors and neurochemistry.

1. Reversal of Amphetamine-Induced Hyperlocomotion: In rats, VU0152100 (3-56.6 mg/kg, i.p.) produced a dose-dependent reversal of hyperlocomotion induced by amphetamine (1 mg/kg, s.c.), with significant effects at 30 and 56.6 mg/kg. [1]
2. M4-Dependent Mechanism (KO Mice): In wild-type mice, VU0152100 (30 mg/kg, i.p.) significantly blocked amphetamine-induced hyperlocomotion. This effect was completely absent in M4 knockout mice, confirming that the behavioral effects are mediated specifically through the M₄ receptor. [1]
3. Reversal of Amphetamine-Induced PPI Disruption: In rats, VU0152100 (30 mg/kg, i.p.) significantly attenuated the disruption of prepulse inhibition (PPI) of the acoustic startle reflex caused by amphetamine (3 mg/kg, s.c.) at prepulse intensities of 5 and 10 dB. [1]
4. Reversal of Amphetamine-Induced Contextual Fear Conditioning Deficit: In rats, VU0152100 (10-56.6 mg/kg, i.p.) dose-dependently blocked the disruptive effect of amphetamine (4.8 mg/kg, s.c.) on the acquisition of contextual fear conditioning, with significant reversal at the 56.6 mg/kg dose. When administered alone, VU0152100 had no effect on fear conditioning. [1]
5. Lack of Fos Induction: Unlike the antipsychotics haloperidol and clozapine, VU0152100 (30 and 100 mg/kg, i.p.) did not induce Fos-like immunoreactivity in the prefrontal cortex, nucleus accumbens (core or shell), or caudate-putamen. It also did not induce Fos expression in hypothalamic orexin neurons, a correlate of weight gain liability. [1]
6. Modulation of Amphetamine-Induced Brain Activation (phMRI): In anesthetized rats, pre-treatment with VU0152100 (56.6 mg/kg, i.p.) significantly suppressed amphetamine-induced (1 mg/kg, i.p.) increases in cerebral blood volume (CBV) in the medial thalamus, hippocampus, caudate-putamen, nucleus accumbens, and retrosplenial cortex. Functional connectivity analysis showed that VU0152100 significantly altered amphetamine-induced correlations between several brain region pairs, including retrosplenial cortex-hippocampus, nucleus accumbens-motor cortex, and nucleus accumbens-medial thalamus. [1]
7. Reversal of Amphetamine-Induced Dopamine Release (Microdialysis): In freely moving rats, VU0152100 (56.6 mg/kg, i.p.) significantly reduced the increase in extracellular dopamine levels elicited by amphetamine (1 mg/kg, s.c.) in both the nucleus accumbens and caudate-putamen. VU0152100 alone had no effect on basal dopamine levels. [1]
8. Potentiation of Oxotremorine Effects on Dopamine Utilization: When administered alone, VU0152100 (10-100 mg/kg) did not alter dopamine or serotonin utilization. However, when combined with a sub-threshold dose of the non-selective mAChR agonist oxotremorine (0.01 mg/kg), VU0152100 (56.6 mg/kg) significantly increased dopamine utilization (DOPAC/DA and HVA/DA ratios) in the prefrontal cortex and nucleus accumbens, but decreased it in the dorsal striatum. [1]
9. Lack of Catalepsy: VU0152100 (30-100 mg/kg, i.p.) did not induce catalepsy in rats at any dose or time point tested (up to 240 min). However, it significantly exacerbated haloperidol-induced catalepsy when combined with sub-maximal doses of haloperidol (0.3 and 0.5 mg/kg). [1]
10. Lack of Peripheral Cholinergic Side Effects: Using the Modified Irwin Neurological Test Battery, VU0152100 (56.6 mg/kg, i.p.) produced no changes in salivation, lacrimation, piloerection, respiratory rate, or body temperature. In contrast, the non-selective mAChR agonist oxotremorine (1 mg/kg, s.c.) induced significant changes in all these parameters. [1]
11. Lack of Cardiovascular Effects: In awake, freely moving rats, VU0152100 (56.6 mg/kg, i.p.) did not significantly alter mean arterial blood pressure or heart rate compared to vehicle treatment. [1]

No direct enzyme activity assays were performed. The key in vitro assays were transporter binding and uptake assays.

1. Human DAT Binding Assay: CHO-K1 cells expressing recombinant human DAT were used to assess if VU0152100 displaces binding of the DAT inhibitor [¹²⁵I]RTI-55. IC₅₀ values were estimated by non-linear, least squares regression analysis, and Kᵢ values were calculated using the Cheng-Prusoff equation. [1]
2. Human DAT Uptake Assay: The same cell line was used to determine if VU0152100 interferes with [³H]dopamine uptake. IC₅₀ values were estimated by non-linear, least squares regression. [1]
3. Rat DAT Uptake Assay: HEK293T cells were transiently transfected with rat DAT. Cells were pre-incubated with various concentrations of VU0152100 or the DAT inhibitor GBR12909 before the addition of [³H]dopamine for 15 minutes. Uptake was measured by scintillation counting. [1]

The primary cell-based assays used were for dopamine transporter function.

1. Cell Culture and Transfection (for rDAT): HEK293T cells were cultured in DMEM with 10% FBS and penicillin/streptomycin. Cells were plated in 24-well plates and transiently transfected with pcDNA3-rDAT using Trans-IT LT-1 in serum-free media approximately 24 hours after plating. Assays were conducted ~36 hours post-transfection. [1]
2. [³H]Dopamine Uptake Assay (for rDAT): Cells were washed with KRH buffer and incubated in uptake assay buffer (KRH with glucose, pargyline, ascorbic acid, and tropolone). For inhibition, cells were pre-incubated with GBR12909 or VU0152100 for 10 minutes before the addition of [³H]dopamine for 15 minutes. Uptake was stopped, and radioactivity was measured using a TopCount Scintillation Counter. [1]

Animal/Disease Models: Adult male SD (SD (Sprague-Dawley)) rat (250-275 g; amphetamine-induced hyperlocomotion model) [1].
Doses: 10, 30, 56.6 mg/kg
Route of Administration: intraperitoneal (ip) injection; Single (pre-treatment)
Experimental Results: Powerful dose-dependent reversal of amphetamine-induced hyperkinesis.

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Animal/Disease Models: Adult male SD (SD (Sprague-Dawley)) rat (250-275 g; amphetamine induction) [1].
Doses: 10, 30, 56.6 mg/kg
Route of Administration: intraperitoneal (ip) injection; Single (pre-treatment)
Experimental Results: Blocked amphetamine-induced disruption of prepulse inhibition. Dose-dependently reversed the disruptive effects of amphetamine on the acquisition of context-dependent fear.

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Multiple detailed animal protocols were used to evaluate VU0152100.

1. **Drug Formulation:** VU0152100 was dissolved in 10% Tween 80 plus double deionized water and administered i.p. in a volume of 2 mL/kg. [1]
2. **Amphetamine-Induced Hyperlocomotion (Rats):** Male Sprague-Dawley rats were habituated to open field chambers for 30 min, then pre-treated with vehicle or VU0152100 (3-56.6 mg/kg, i.p.). Thirty minutes later, they received amphetamine (1 mg/kg, s.c.) or vehicle and were monitored for another 60 min. [1]
3. **Amphetamine-Induced Hyperlocomotion (Mice):** Wild-type and M4 KO mice were habituated for 90 min, then injected with vehicle or VU0152100 (30 mg/kg, i.p.). Thirty minutes later, amphetamine (1.8 mg/kg, i.p.) was administered, and activity was monitored for 120 min. [1]
4. **Prepulse Inhibition (PPI):** Rats were pre-treated with vehicle or VU0152100 (3-30 mg/kg, i.p.) for 20 min, then injected with amphetamine (3 mg/kg, s.c.). After 10 min, they were placed in startle chambers for a 20-min session with randomized presentations of various trial types (pulse alone, prepulse alone, prepulse+pulse). [1]
5. **Contextual Fear Conditioning:** Rats were handled and injected with saline for 2 days. On conditioning day, they received vehicle or VU0152100 (10-56.6 mg/kg, i.p.), followed 15 min later by vehicle or amphetamine (4.8 mg/kg, s.c.). After another 15 min, they were placed in chambers for a 7-min conditioning session (four footshocks). Twenty-four hours later, freezing behavior was assessed for 7 min in the same context without shocks. [1]
6. **In Vivo Microdialysis:** Guide cannulae were implanted into the nucleus accumbens or caudate-putamen of rats. After recovery, microdialysis probes were inserted. On the experiment day, rats were placed in open field chambers. After baseline collection, they received vehicle or VU0152100 (56.6 mg/kg, i.p.), followed 30 min later by vehicle or amphetamine (1 mg/kg, s.c.). Dialysate samples were collected every 15 min for 120 min and analyzed for dopamine and its metabolites by HPLC-ECD. [1]
7. **Pharmacological MRI (phMRI):** Anesthetized, ventilated rats with pre-implanted jugular and i.p. catheters were placed in a 9.4T MRI scanner. After iron oxide nanoparticle injection for contrast, a 15-min baseline was collected. Rats then received vehicle or VU0152100 (56.6 mg/kg, i.p.), followed 15 min later by vehicle or amphetamine (1 mg/kg, i.p.), with 45 min of continuous acquisition. Data were processed to calculate fractional cerebral blood volume (CBV) changes. [1]
8. **Catalepsy Test:** Rats received vehicle, VU0152100 (30-100 mg/kg, i.p.), or haloperidol (1.5 mg/kg, i.p.). Catalepsy was assessed at 30, 60, 120, and 240 min post-treatment by placing the forepaws on a bar and measuring the time to remove them. [1]
9. **Modified Irwin Test:** Rats received vehicle, VU0152100 (56.6 mg/kg, i.p.), or oxotremorine (1 mg/kg, s.c.). Autonomic and somatosensory functions (salivation, lacrimation, piloerection, respiratory rate) and body temperature were assessed at 5, 15, 60, 120, and 240 min post-injection. [1]

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Multiple detailed animal protocols were used to evaluate VU0152100.

1. Drug Formulation: VU0152100 was dissolved in 10% Tween 80 plus double deionized water and administered i.p. in a volume of 2 mL/kg. [1]
2. Amphetamine-Induced Hyperlocomotion (Rats): Male Sprague-Dawley rats were habituated to open field chambers for 30 min, then pre-treated with vehicle or VU0152100 (3-56.6 mg/kg, i.p.). Thirty minutes later, they received amphetamine (1 mg/kg, s.c.) or vehicle and were monitored for another 60 min. [1]
3. Amphetamine-Induced Hyperlocomotion (Mice): Wild-type and M4 KO mice were habituated for 90 min, then injected with vehicle or VU0152100 (30 mg/kg, i.p.). Thirty minutes later, amphetamine (1.8 mg/kg, i.p.) was administered, and activity was monitored for 120 min. [1]
4. Prepulse Inhibition (PPI): Rats were pre-treated with vehicle or VU0152100 (3-30 mg/kg, i.p.) for 20 min, then injected with amphetamine (3 mg/kg, s.c.). After 10 min, they were placed in startle chambers for a 20-min session with randomized presentations of various trial types (pulse alone, prepulse alone, prepulse+pulse). [1]
5. Contextual Fear Conditioning: Rats were handled and injected with saline for 2 days. On conditioning day, they received vehicle or VU0152100 (10-56.6 mg/kg, i.p.), followed 15 min later by vehicle or amphetamine (4.8 mg/kg, s.c.). After another 15 min, they were placed in chambers for a 7-min conditioning session (four footshocks). Twenty-four hours later, freezing behavior was assessed for 7 min in the same context without shocks. [1]
6. In Vivo Microdialysis: Guide cannulae were implanted into the nucleus accumbens or caudate-putamen of rats. After recovery, microdialysis probes were inserted. On the experiment day, rats were placed in open field chambers. After baseline collection, they received vehicle or VU0152100 (56.6 mg/kg, i.p.), followed 30 min later by vehicle or amphetamine (1 mg/kg, s.c.). Dialysate samples were collected every 15 min for 120 min and analyzed for dopamine and its metabolites by HPLC-ECD. [1]
7. Pharmacological MRI (phMRI): Anesthetized, ventilated rats with pre-implanted jugular and i.p. catheters were placed in a 9.4T MRI scanner. After iron oxide nanoparticle injection for contrast, a 15-min baseline was collected. Rats then received vehicle or VU0152100 (56.6 mg/kg, i.p.), followed 15 min later by vehicle or amphetamine (1 mg/kg, i.p.), with 45 min of continuous acquisition. Data were processed to calculate fractional cerebral blood volume (CBV) changes. [1]
8. Catalepsy Test: Rats received vehicle, VU0152100 (30-100 mg/kg, i.p.), or haloperidol (1.5 mg/kg, i.p.). Catalepsy was assessed at 30, 60, 120, and 240 min post-treatment by placing the forepaws on a bar and measuring the time to remove them. [1]
9. Modified Irwin Test: Rats received vehicle, VU0152100 (56.6 mg/kg, i.p.), or oxotremorine (1 mg/kg, s.c.). Autonomic and somatosensory functions (salivation, lacrimation, piloerection, respiratory rate) and body temperature were assessed at 5, 15, 60, 120, and 240 min post-injection. [1]

The study provided key pharmacokinetic data for a behaviorally relevant dose.

- Brain and Plasma Exposure: Following a 56.6 mg/kg i.p. dose in rats, VU0152100 achieved a brain AUC₀₋ₜ of 36.2 μM·h and a brain Cmax of 7.6 μM. [1]
- Free Brain Concentration: Taking into account the rat brain unbound fraction (fu,brain = 0.045), the calculated free brain Cmax was approximately 340 nM. This concentration is sufficient for M₄ potentiation but well below that needed for significant DAT inhibition. [1]

VU0152100 was evaluated for several potential adverse effects.

- Catalepsy: No catalepsy was observed at doses up to 100 mg/kg, indicating a low potential for extrapyramidal motor side effects. However, it did potentiate catalepsy induced by haloperidol. [1]
- Peripheral Cholinergic Side Effects: In the Modified Irwin Test, VU0152100 (56.6 mg/kg) did not cause salivation, lacrimation, piloerection, respiratory depression, or hypothermia, confirming its lack of activity at peripheral M₂ and M₃ mAChRs in vivo. [1]
- Cardiovascular Effects: VU0152100 (56.6 mg/kg) did not alter mean arterial blood pressure or heart rate in awake rats. [1]
- Weight Gain Liability: Unlike some antipsychotics, VU0152100 did not induce Fos expression in hypothalamic orexin neurons, which is associated with weight gain. [1]

[1]. Antipsychotic drug-like effects of the selective M4 muscarinic acetylcholine receptor positive allosteric modulator VU0152100. Neuropsychopharmacology. 2014 Jun;39(7):1578-93.

[2]. Centrally active allosteric potentiators of the M4 muscarinic acetylcholine receptor reverse amphetamine-induced hyperlocomotor activity in rats. J Pharmacol Exp Ther. 2008 Dec;327(3):941-53.

VU0152100 is a highly selective, centrally penetrant positive allosteric modulator of the M₄ muscarinic acetylcholine receptor. It was developed as a tool compound to investigate the therapeutic potential of M₄ activation for psychiatric disorders. This study provides a comprehensive preclinical characterization, demonstrating that VU0152100 has an antipsychotic drug-like profile. It reverses a range of amphetamine-induced behavioral (hyperlocomotion, PPI disruption, fear conditioning deficits) and neurochemical (dopamine release, brain activation) effects. Crucially, its efficacy is absent in M₄ knockout mice, confirming its on-target mechanism. It achieves these effects at doses that do not produce catalepsy or peripheral cholinergic side effects associated with non-selective muscarinic agonists. Its favorable profile supports the development of M₄ PAMs as a novel therapeutic strategy for treating psychosis and cognitive impairments in disorders like schizophrenia. [1]

C18H19N3O2S

341.429

341.12

409351-28-6

864492

Light yellow to khaki solid powder

4.406

2

5

4

24

442

0

MDNWGCQSCGNTKH-UHFFFAOYSA-N

InChI=1S/C18H19N3O2S/c1-10-8-11(2)21-18-14(10)15(19)16(24-18)17(22)20-9-12-4-6-13(23-3)7-5-12/h4-8H,9,19H2,1-3H3,(H,20,22)

3-amino-N-[(4-methoxyphenyl)methyl]-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide

VU-152100VU-0152100VU 0152100VU0152100VU152100VU 152100

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)

DMSO : ≥ 50 mg/mL (~146.44 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.32 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 (7.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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