mAChR3/4; impurity/metabolite of clozapine

Designer Receptors Exclusively Activated by Designer Drugs (DREADD) is a method for chemical correlation of neural activity in remotely collective freely moving animals. DREADD is an apparently altered category of G-coupled receptors (GPCRs) that are themselves unresponsive to endogenous neurotransmitters but are receptive to other “non” exogenous chemicals [2].

Rhesus monkeys (5 to 6 years old, weight 5.5-7.9 kg) exhibit impaired working memory performance when given Deschloroclozapine (0.3 mg/kg; surgical injection) [3]. In vivo, desclozapine (0.1 mg/kg; im) reversibly senses behavioral effects in monkeys and effectively activates DREADD receptors [3].

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The most common chemogenetic neuromodulatory system, designer receptors exclusively activated by designer drugs (DREADDs), uses a non-endogenous actuator ligand to activate a modified muscarinic acetylcholine receptor that is insensitive to acetylcholine. It is crucial in studies using these systems to test the potential effects of DREADD actuators prior to any DREADD transduction, so that effects of DREADDs can be attributed to the chemogenetic system rather than the actuator drug, particularly in experiments using nonhuman primates. We investigated working memory performance after injections of three DREADD actuators, clozapine, olanzapine, and Deschloroclozapine, in four male rhesus monkeys tested in a spatial delayed response task before any DREADD transduction took place. Performance at 0.1 mg/kg clozapine and 0.1 mg/kg Deschloroclozapine did not differ from vehicle in any of the four subjects. 0.2 mg/kg clozapine impaired working memory function in three of the four monkeys. Two monkeys were impaired after 0.1 mg/kg olanzapine and two were impaired after 0.3 mg/kg Deschloroclozapine. We speculate that the unique neuropharmacology of prefrontal cortex function makes the primate prefrontal cortex especially vulnerable to off-target effects of DREADD actuator drugs with affinity for endogenous monoaminergic receptor systems. These findings underscore the importance of within-subject controls for DREADD actuator drugs in the specific tasks under study to confirm that effects following DREADD receptor transduction are not owing to the actuator drug itself. They also suggest that off-target effects of DREADD actuators may limit translational applications of chemogenetic neuromodulation.

Radioligand binding assays [2]
Human embryonic kidney (HEK-293, ATCC) cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 2 mM L-glutamine, antibiotic/antimycotic (all supplements from Gibco) and 10% heat-inactivated fetal bovine serum and kept in an incubator at 37 °C and 5% CO2. Cells were routinely tested for myclopasma contamination. Cells were seeded on 60 cm2 dishes at 4 × 106 cells/dish 24 h before transfection. The cells were transfected with 5 μg/dish of AAV packaging plasmids encoding for hM3Dq, hM4Di or a control vector and harvested 48 h after transfection. The cells were suspended in Tris-HCl 50 mM pH 7.4 supplemented with protease inhibitor cocktail. HEK-293 cells were disrupted with a Polytron homogenizer. Homogenates were centrifuged at 48,000 g (50 min, 4 °C) and washed twice in the same conditions to isolate the membrane fraction. Protein was quantified by the bicinchoninic acid method. For competition experiments, membrane suspensions (50 μg of protein/mL) were incubated in 50 mM Tris-HCl (pH 7.4) containing 10 mM MgCl2, 2.5 nM of [3H]clozapine (3070 GBq/mmol (83 Ci/mmol)) and increasing concentrations of the competing drugs during 2 h at RT. Nonspecific binding was determined in the presence of 10 μM clozapine. In all cases, free and membrane-bound radioligand were separated by rapid filtration of 500 μL aliquots in a 96-well plate harvester and washed with 2 mL of ice-cold Tris-HCl buffer. Microscint-20 scintillation liquid (65 μL/well) was added to the filter plates. The plates were incubated overnight at RT and radioactivity counts were determined in a MicroBeta2 plate counter with an efficiency of 41%. One-site competition curves were fitted using Prism 7. Ki values were calculated using the Cheng–Prusoff equation.

Drugs were prepared fresh daily, at concentrations so that monkeys received 0.1 ml/kg for injection (e.g., for a 0.2 mg/kg dose of drug, drug solution was prepared at a concentration of 2.0 mg/ml). Solutions were filtered through a 0.22 µm syringe filter and pH was determined before injection. Acetic acid, sodium acetate, and sodium hydroxide (NaOH) were all obtained from Fisher Scientific. Concentrations used for glacial acetic acid, sodium acetate, and NaOH were 99.7% (v/v), 1 m, and 0.2 m, respectively. Clozapine was stored at room temperature. Clozapine was given at 0.1 or 0.2 mg/kg, intramuscularly. For 0.1 mg/kg, clozapine powder was first dissolved in acetic acid and sodium acetate then diluted with NaOH to a final concentration in 0.25/50/49.75 acetic acid/sodium acetate/NaOH (v/v/v). For 0.2 mg/kg, the same reagents were used but the final concentration was 0.5/50/49.5 acetic acid/sodium acetate/NaOH (v/v/v). Olanzapine was stored at room temperature and given at 0.05 or 0.1 mg/kg, intramuscularly. Olanzapine solutions were made using the same method as above for the 0.1 mg/kg clozapine dose. Deschloroclozapine was stored at 4 °C and was given at 0.1 mg/kg and 0.3 mg/kg, intramuscularly. Low dose of Deschloroclozapine was made using the same method as low-dose clozapine and high-dose Deschloroclozapine was made using the same method as the higher dose of clozapine. Vehicle injections consisted of 0.25/50/49.75 acetic acid, sodium acetate, and NaOH and were given at 0.1 ml/kg. Actuator injections were never administered more than twice in 1 week and never on adjacent test days to allow for a washout period, accounting for the half-life of clozapine (14.2 h on average) and olanzapine (33 h on average). There were vehicle or no-injection test days on other days of the test week. Actuators were not counterbalanced; we tested clozapine, olanzapine, and Deschloroclozapine in that order in each monkey. Within drug conditions, however, doses were shuffled in order across drug test days. We also interpolated additional clozapine test days during testing with the other two actuators. Throughout the study, we did not observe any order effects or obvious effects on behavior on the days following actuator injections. Clozapine and olanzapine were given in the home cage 10 min before the start of testing and Deschloroclozapine was given in the home cage 30 min before the start of testing. Clozapine and olanzapine both get into the brain fairly quickly and previous DREADD studies using these actuators have started behavior 10 min post injection. Deschloroclozapine shows a slightly slower timescale with higher plasma concentration ~15–30 min after intramuscular injection and CSF concentration continues to rise between 30 min to 90 min post injection. Accordingly, beginning the delayed response task 30 min post injection for Deschloroclozapine would allow sufficient time for the actuator to get into the brain.[3]

The oral LD50 in mice was 145 mg/kg. (Arzneimittel-Forschung, Drug Research, 15(841), 1965)

[1]. The metabolic formation of reactive intermediates from clozapine, a drug associated with agranulocytosis in man. J Pharmacol Exp Ther. 1995;275(3):1463-1475.

[2]. 18F-labeled radiotracers for in vivo imaging of DREADD with positron emission tomography. Eur J Med Chem. 2021;213:113047.

[3]. Effect of chemogenetic actuator drugs on prefrontal cortex-dependent working memory in nonhuman primates. Neuropsychopharmacology. 2020;45(11):1793-1798.

Clozapine is a dibenzodiazepine antipsychotic drug associated with a 0.8% incidence of agranulocytosis. This clinically limiting toxicity is thought to be related to its chemically active metabolites. This study assessed the generation of these metabolites using human, rat, and mouse liver microsomes, as well as human neutrophils and bone marrow cells, via covalent binding and the formation of thioether adducts. In all cases, the addition of glutathione resulted in the formation of a major clozapine-glutathione adduct—C-6-glutathioneylclozapine. Neutrophil and myeloid cell adduct formation depended on cellular activation by phorbol myristate (PMA). Small amounts of the drug covalently bound to microsomes (1–6.8%) and to proteins co-incubated with neutrophils (0.47%) and myeloid cells (0.21%). Clozapine did not deplete glutathione within activated neutrophils. Clozapine is also metabolized in vivo (in rats and mice) to glutathione conjugates, which are excreted in bile within 3 hours, accounting for 38% and 33% of the administered dose, respectively. In addition to the major clozapine adducts found in vitro, both animals excrete C-8 glutathione derivatives of clozapine. It is concluded that clozapine is bioactivated in a variety of tissues and is significantly bioactivated in vivo. Active metabolites produced by neutrophils and myeloid cells may play an important role in the metabolism of clozapine-induced agranulocytosis. [1] Drug-specific receptor design (DREADD) is a preclinical chemogenetic approach with potential clinical applications in a variety of diseases. In vivo visualization of DREADD has been achieved by positron emission tomography (PET) using 11C radiotracers. This study aims to develop 18F-labeled DREADD radiotracers to extend their isotopic half-life. We synthesized a series of non-radioactive clozapine fluorinated analogs with broad in vitro binding affinity for hM3Dq and hM4Di DREADD receptors for PET imaging. Compound [18F]7b was radiolabeled using a modified 18F deoxyfluorination scheme with commercially available ruthenium reagent. [18F]7b exhibited encouraging PET imaging properties in a DREADD hM3Dq transgenic mouse model, despite lower uptake in the brains of wild-type mice. [18F]7b is a promising long-acting alternative to the DREADD radiotracers [11C]clozapine ([11C]CLZ) and [11C]desclozapine ([11C]DCZ).

C18H20N4

292.3782

292.169

C, 73.94; H, 6.89; N, 19.16

1977-07-7

Deschloroclozapine dihydrochloride

16103

Light yellow to yellow solid powder

1.48E-08mmHg at 25°C

2.518

1

3

1

22

413

0

CN1CCN(CC1)C2=NC3=CC=CC=C3NC4=CC=CC=C42

VQHITFFJBFOMBG-UHFFFAOYSA-N

InChI=1S/C18H20N4/c1-21-10-12-22(13-11-21)18-14-6-2-3-7-15(14)19-16-8-4-5-9-17(16)20-18/h2-9,19H,10-13H2,1H3

6-(4-methylpiperazin-1-yl)-11H-benzo[b][1,4]benzodiazepine

1977-07-7; 11-(4-Methyl-1-piperazinyl)-5H-dibenzo(b,e)(1,4)diazepine; BRN 0761696; DTXSID80173463; 5H-DIBENZO(b,e)(1,4)DIAZEPINE, 11-(4-METHYL-1-PIPERAZINYL)-; 5-25-11-00363 (Beilstein Handbook Reference); 11-(4-Methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine; 5H-Dibenzo[b,e][1,4]diazepine, 11-(4-methyl-1-piperazinyl)-;

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 : ~100 mg/mL (~342.02 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.55 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 (8.55 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.55 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 4: ≥ 2.5 mg/mL (8.55 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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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Solubility in Formulation 5: ≥ 2.08 mg/mL (7.11 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 20.8 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.)