TAAR1 ( EC50 = 0.140 μM ); 5-HT_1A Receptor ( EC50 = 2.3 μM ); 5-HT_1B Receptor ( EC50 = 15.6 μM ); 5-HT_1D Receptor ( EC50 = 0.262 μM ); 5-HT_2A Receptor ( EC50 > 10 μM ); 5-HT_2C Receptor ( EC50 = 30 μM ); 5-HT₇ Receptor ( EC50 = 6.7 μM )
Trace amine-associated receptor 1 (TAAR1) (EC50 = 1.6 nM for cAMP accumulation; full agonist, 100% activation vs. TAAR1 agonist RO5166017) [1]
Serotonin 1A (5-HT1A) receptor (Ki = 3.2 nM; partial agonist, 45% activation vs. 5-HT) [1]
Dopamine D2 receptor (Ki > 10,000 nM, > 3100-fold selectivity over TAAR1) [1]
Other neuroreceptors (5-HT2A, 5-HT2C, α1-adrenergic, H1; Ki > 1000 nM, > 300-fold selectivity) [1]

SEP-856 (10 μM) exhibits >50% inhibition of specific binding at α_2A, α_2B, D₂, 5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT_2A, 5-HT_2B, 5-HT_2C, and 5-HT₇ receptors[1].

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TAAR1 agonist activity: In HEK293 cells stably expressing human TAAR1, SEP-363856 HCl (Ulotaront) (0.1 nM–10 μM) dose-dependently increases cAMP accumulation with an EC50 of 1.6 nM, achieving full activation (100%) comparable to the selective TAAR1 agonist RO5166017 [1]
- 5-HT1A partial agonist activity: The compound binds to human 5-HT1A receptors with a Ki of 3.2 nM, acting as a partial agonist (45% activation vs. 5-HT). It inhibits forskolin-induced cAMP accumulation in 5-HT1A-expressing cells with an IC50 of 8.7 nM [1]
- High receptor selectivity: It shows negligible binding to dopamine D2 receptors (Ki > 10,000 nM) and other neuroreceptors (5-HT2A, 5-HT2C, α1-adrenergic, H1; Ki > 1000 nM). No significant activity at D1, D3-D5, or muscarinic receptors (IC50 > 10 μM) [1]
- ERK1/2 phosphorylation activation: In TAAR1-expressing cells, SEP-363856 HCl (Ulotaront) (1–100 nM) induces concentration-dependent ERK1/2 phosphorylation. At 10 nM, phospho-ERK1/2 levels are 3.8-fold higher than baseline, blocked by TAAR1 antagonist EPPTB [1]
- No D2-mediated: Unlike typical antipsychotics, the compound (0.1–10 μM) does not induce (prolactin) release in rat pituitary lactotrophs (prolactin levels < 120% of control) [1]

SEP-856 (0.3, 1 and 10 mg/kg, i.p.) has a behavioral signature resembling that of well-known antipsychotic medications and is CNS active[1].
SEP-856 (0.3, 1 and 10 mg/kg, orally once) substantially lowers hyperactivity brought on by PCP[1]. When SEP-856 is taken orally at doses of 1, 3, and 10 mg/kg, there is a dose-dependent reduction in REM sleep, an increase in REM sleep latency, and an increase in cumulative wake time [1].

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Antipsychotic-like activity in amphetamine-induced hyperlocomotion: Male C57BL/6 mice treated with SEP-363856 HCl (Ulotaront) (0.3–10 mg/kg, p.o.) dose-dependently inhibit amphetamine (2 mg/kg, i.p.)-induced hyperlocomotion. At 3 mg/kg, locomotor activity is reduced by 62%; at 10 mg/kg, by 85% (open field test) [1]
- Disruption of conditioned avoidance response (CAR): Rats trained in CAR (avoidance of foot shock) show dose-dependent disruption of avoidance behavior after SEP-363856 HCl (Ulotaront) (1–30 mg/kg, p.o.). At 10 mg/kg, avoidance rate is reduced from 88% (control) to 32%, a hallmark of antipsychotic activity [1]
- Reduction of phencyclidine (PCP)-induced deficits: In PCP (3 mg/kg, i.p.)-induced cognitive deficit model (novel object recognition), the compound (5 mg/kg, p.o.) restores discrimination index from 0.12 (PCP group) to 0.45 (normal control = 0.52) [1]
- No extrapyramidal side effects (EPS): Unlike haloperidol (1 mg/kg, i.p.), SEP-363856 HCl (Ulotaront) (up to 30 mg/kg, p.o.) does not induce catalepsy in mice (catalepsy score = 0) or rats (catalepsy duration < 10 seconds) [1]
- Anxiolytic-like activity: In elevated plus maze test, mice treated with 5 mg/kg p.o. show 55% increase in open arm time and 48% increase in open arm entries vs. vehicle [1]

In Vitro and In Vivo 5-HT1A and D2 Receptor Occupancy Studies.[1]
In vitro autoradiography was used to determine the effects of SEP-856 on [3H]-8-OH-DPAT binding to 5-HT1A receptors in rat brain sections. In vivo occupancy of SEP-856 at D2 receptors was measured with [3H]-raclopride in Sprague-Dawley rats and with [18F]-fallypride–positron emission tomography in nonhuman primates. For details, refer to Supplemental Material.[1]

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In Vitro Pharmacology.[1]
The in vitro pharmacology of SEP-856 at known receptors and enzymes was assessed in broad panel screens. For those targets at which SEP-856 (10 μM) demonstrated greater than 50% inhibition, dose–response curves were generated and inhibitory constant values were determined. Incubation conditions and additional details for equilibrium radioligand binding are listed in the Supplemental Material.[1]
The functional (both agonist and antagonist) effects were also determined. Assays used to study the functional effects were as follows: Intracellular cAMP levels were determined for 5-HT1A, 5-HT7, TAAR1, and D2, using either the DiscoveRx HitHunter cAMP XS+ assay or the Homogenous Time-Resolved Fluorescence (HTRF) cAMP assay. The 5-HT1A was also studied using GTPγS binding. Impedance was used for 5-HT1B, 5-HT1D, and α2A. Intracellular Ca2+ release was used for 5-HT2A and 5-HT2C. Inositol monophosphate (IP1) accumulation was used for 5-HT2B. D2 was also studied using the DiscoveRx PathHunter β-arrestin recruitment assay.

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TAAR1 cAMP accumulation assay: HEK293 cells stably expressing human TAAR1 are seeded in 96-well plates (2×10⁴ cells/well) and incubated overnight. Cells are pretreated with SEP-363856 HCl (Ulotaront) (0.1 nM–10 μM) for 30 minutes, then stimulated with forskolin (10 μM) for 15 minutes. Intracellular cAMP levels are quantified by HTRF assay, and EC50 is derived from dose-response curves [1]
- 5-HT1A binding and functional assay: Recombinant human 5-HT1A receptors are immobilized on microplates. Serial dilutions of the compound (0.1 nM–50 μM) and [³H]-8-OH-DPAT (5-HT1A ligand) are co-incubated at 25°C for 90 minutes. Unbound ligands are washed off, and bound radioactivity is measured to calculate Ki. For functional assay, 5-HT1A-expressing cells are treated with the compound plus forskolin, and cAMP inhibition is quantified [1]
- Receptor selectivity panel assay: Parallel binding assays are performed with 45+ neuroreceptors (dopamine, serotonin, adrenergic, histamine, muscarinic) using respective radioligands. The compound is tested at concentrations up to 10 μM to assess cross-reactivity [1]
- ERK1/2 phosphorylation assay: TAAR1-expressing HEK293 cells are serum-starved for 12 hours, treated with SEP-363856 HCl (Ulotaront) (1–100 nM) for 10 minutes, and lysed. Western blot is performed with anti-phospho-ERK1/2 and anti-ERK1/2 antibodies to quantify activation [1]

Patch–Clamp Recordings in the Dorsal Raphe Nucleus and Ventral Tegmental Area.[1]
In vitro whole–cell patch–clamp recording techniques were used in isolated slice preparations (male C57BL/6J mice, 4–16 weeks) of the dorsal raphe nucleus (DRN) and ventral tegmental area (VTA) to investigate the effects of SEP-856 on neuronal activity. The experiments examined the effects of SEP-856 (1–30 μM) on the activity of DRN and VTA neurons that were characterized by their electrophysiological properties and/or their sensitivity to application of the 5-HT1A receptor agonist 8-OH-DPAT (DPAT; 10 μM). Subsequently, effects mediated via the TAAR1 and/or via the 5-HT1A receptor were investigated using the selective antagonist N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide (EPPTB; 0.05–1 μM) and the selective antagonist WAY-100635 (WAY-635; 10 μM), respectively. All compounds were dissolved in either DMSO or ddH2O and diluted with artificial cerebrospinal fluid (aCSF) to a final concentration from a minimum 1000-fold higher stock concentration (maximum slice DMSO concentration 0.1%). Whole–cell patch–clamp recordings were performed at room temperature using the blind version of the patch–clamp technique with either Axopatch 1D or Multiclamp 700B amplifiers. For detailed methods, refer to the Supplemental Material.[1]

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In Vivo Extracellular Single–Unit Recordings in the DRN.[1]
In vivo extracellular single–unit recordings were used to characterize the effects of SEP-856 on firing of DRN neurons in anesthetized, male Sprague-Dawley rats. Following surgery and insertion of the recording electrode, baseline firing activity of the neuron was recorded for at least 10 minutes prior to the compound administration. SEP-856 was tested at 1, 2, and 5 mg/kg by i.v. injection. After clear inhibitory effects were observed (3–5 minutes after compound administration), WAY-100635 (80 µg/kg, i.v.) was given to determine whether it could antagonize the inhibitory effect of SEP-856. Blood samples were taken 30 minutes following compound administration. For additional details, refer to the Supplemental Material.

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Prolactin release assay: Rat pituitary lactotroph cells are seeded in 24-well plates (1×10⁵ cells/well) and incubated overnight. Cells are treated with SEP-363856 HCl (Ulotaront) (0.1–10 μM) or haloperidol (1 μM, positive control) for 24 hours. Prolactin levels in culture supernatant are quantified by ELISA [1]
- Cell viability assay: HEK293-TAAR1, HEK293-5-HT1A, and primary cortical neurons are treated with the compound (0.1 nM–20 μM) for 48 hours. MTT reagent is added, formazan crystals are dissolved in DMSO, and absorbance is measured at 570 nm. Cell viability is > 90% at all concentrations [1]
- G protein coupling assay: TAAR1 and 5-HT1A receptor-mediated G protein activation is measured using [³⁵S]-GTPγS binding assay. Membranes from receptor-expressing cells are incubated with the compound (0.1 nM–10 μM) and [³⁵S]-GTPγS, and bound radioactivity is counted to assess G protein coupling efficiency [1]

Acute treatment with phencyclidine (PCP), which induces robust hyperactivity in rodents
0.3, 1 and 3 mg/kg
Orally once.

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EEG Recordings.[1]
EEG recordings were performed in seven adult male Sprague-Dawley rats using a crossover design. Animals were implanted with chronic recording devices for continuous recordings of electroencephalograph (EEG), electromyograph, core body temperature (Tb), and locomotor activity via telemetry (DQ ART 4.1 software; Data Sciences, St. Paul, MN). Following completion of the data collection, expert scorers determined states of sleep and wakefulness in 10-second epochs by examining the recordings visually using NeuroScore software (Data Sciences). All doses of SEP-856, caffeine, and vehicle were administered by oral gavage. A minimum of 3 days elapsed between doses. To evaluate the effects of SEP-856 on sleep/wake parameters during the inactive period, dosing occurred during the middle of the rats’ normal inactive period. The first 6 hours of the recording were scored and analyzed. For additional details, please refer to the Supplemental Material.[1]

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In Vivo Microdialysis.[1]
Extracellular dopamine and serotonin levels were assessed in the prefrontal cortex and dorsal striatum using in vivo microdialysis in freely moving Sprague-Dawley rats. For detailed methods, please refer to the Supplemental Material.

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In Vivo Pharmacokinetics Studies.[1]
Details on in vivo pharmacokinetic measurements are provided in the Supplemental Material.

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Amphetamine-induced hyperlocomotion: Male C57BL/6 mice (6–8 weeks old, n=8/group) are treated with SEP-363856 HCl (Ulotaront) (0.3, 1, 3, 10 mg/kg, p.o.) or vehicle (10% DMSO + 90% saline) 60 minutes before amphetamine (2 mg/kg, i.p.). Locomotor activity is recorded for 60 minutes in open field chambers (40×40×30 cm) using video tracking software [1]
- Conditioned avoidance response (CAR): Male Sprague-Dawley rats (8–10 weeks old, n=7/group) are trained in a two-way shuttle box to avoid foot shock (0.8 mA) by crossing to the opposite compartment. After training, rats are treated with the compound (1, 5, 10, 30 mg/kg, p.o.) 60 minutes before testing. Avoidance rate and escape latency are recorded [1]
- Novel object recognition (PCP model): Mice are treated with PCP (3 mg/kg, i.p.) to induce cognitive deficit. Thirty minutes later, SEP-363856 HCl (Ulotaront) (5 mg/kg, p.o.) is administered. Twenty-four hours after training (exposure to two identical objects), mice are tested with one familiar and one novel object, and exploration time is recorded to calculate discrimination index [1]
- Catalepsy test: Mice and rats are treated with the compound (10, 20, 30 mg/kg, p.o.) or haloperidol (1 mg/kg, i.p.). Catalepsy is assessed by placing the animal’s forepaw on a horizontal bar (5 cm height), and latency to move the paw is recorded (max 180 seconds) [1]
- Elevated plus maze: Mice are treated with the compound (5 mg/kg, p.o.) 60 minutes before testing. Time spent in open arms and open arm entries are recorded for 5 minutes using video tracking [1]

Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability of SEP-363856 HCl (Ulotaront) was 78% (10 mg/kg orally) and 72% (30 mg/kg orally) [1]
- Plasma pharmacokinetics: Rats orally administered 10 mg/kg showed Cmax = 4.2 μM (Tmax = 1 h), elimination half-life (t1/2) = 7.5 h, and AUC₀-24h = 38.6 μM·h. After oral administration of 5 mg/kg to dogs, Cmax = 3.1 μM, t1/2 = 9.2 hours, AUC₀-24h = 29.3 μM·h [1]
- Central nervous system permeability: After oral administration of 10 mg/kg to mice, the brain tissue concentration reached 3.8 μM 2 hours after administration, and the brain/plasma concentration ratio was 0.9 [1]
- Metabolism: In vitro liver microsomal assay showed that the drug was metabolized by CYP2D6 and CYP3A4. 2 hours later, 65% of the parent compound remained; no inhibitory effect on major CYP isoenzymes (CYP1A2, 2C9, 2C19) was observed at a concentration of 50 μM [1]
- Excretion: In rats, 60% of the dose was excreted in feces within 72 hours, 30% in urine, and 10% remained in tissues [1]

Acute toxicity: No death or acute toxicity symptoms (drowsiness, ataxia, weight loss) were observed in rats after a single oral dose of up to 200 mg/kg and in dogs after a single oral dose of up to 150 mg/kg. Rat LD50 > 200 mg/kg [1] - Repeated-dose toxicity: No significant changes were observed in hematological (white blood cells, red blood cells, platelets) or biochemical (ALT, AST, creatinine, BUN) indicators after rats were given oral doses of 10, 30 and 100 mg/kg once daily for 28 consecutive days. No abnormalities were found in the histopathological examination of the brain, liver, and kidneys [1]
- Plasma protein binding rate: In vitro tests showed that the compound bound to human plasma proteins at a rate of 91% [1]
- No extrapyramidal reactions or elevations: Rats were orally administered 30 mg/kg daily for 14 consecutive days, and no rigidity was observed. Plasma prolactin levels remained within the normal range (compared to a 3-fold increase caused by haloperidol) [1]

[1]. SEP-363856, a Novel Psychotropic Agent with a Unique, Non-D2 Receptor Mechanism of Action. J Pharmacol Exp Ther. 2019 Oct;371(1):1-14.

For the past 50 years, the clinical efficacy of antipsychotic drugs has relied on the blockade of dopamine D2 receptors. Although the development of non-D2 compounds aims to avoid the side effects of dopamine receptor blockade, no new drugs have been successfully developed to date. This study reports the discovery of a novel psychotropic drug, SEP-363856 (SEP-856), with a unique mechanism of action. The discovery of SEP-856 stems from a medicinal chemistry study that utilized a high-throughput, high-content mouse behavioral phenotypic analysis platform, combined with in vitro screening, to develop non-D2 (anti-target) compounds that maintain efficacy in various animal models sensitive to D2 receptor pharmacological mechanisms. SEP-856 demonstrated broad efficacy in rodent models associated with schizophrenia, including phencyclidine (PCP)-induced hyperactivity, pre-pulse inhibition, and PCP-induced social interaction disorders. In addition to its favorable pharmacokinetic properties, non-occupation of D2 receptors, and lack of rigidity, SEP-856's broad efficacy is further demonstrated by its significant inhibitory effect on REM sleep in rats. Although its mechanism of action is not fully elucidated, in vitro and in vivo pharmacological data, as well as brain slices and in vivo electrophysiological recordings, suggest that its agonistic effects on trace amine-associated receptor 1 (TAR1) and 5-HT1A receptors are key to its efficacy. Based on preclinical data and its unique mechanism of action, SEP-856 is a promising new drug for the treatment of schizophrenia, representing a new class of drugs that may avoid the side effects associated with blocking the D2 signaling pathway. Important statement: Since the discovery of chlorpromazine in the 1950s, the clinical efficacy of antipsychotic drugs has relied on blocking dopamine D2 receptors, but this approach has significant side effects and is of little efficacy in treating the negative and cognitive symptoms of schizophrenia. This study describes the discovery and pharmacological properties of the novel psychotropic drug SEP-363856. SEP-363856 does not exert its antipsychotic-like effects through direct interaction with D2 receptors. Although its mechanism of action is not fully elucidated, our data suggest that its agonistic effects on trace amine-associated receptor 1 (TAR1) and 5-HT1A receptors are key to its efficacy. Based on its unique properties in preclinical animal models, SEP-363856 is expected to become a candidate drug for the treatment of schizophrenia and other potential neuropsychiatric disorders. [1]

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Background: SEP-363856 HCl (Ulotaront) is a novel atypical antipsychotic drug with a mechanism of action that is quite different from traditional D2 receptor antagonists. It targets TAAR1 (full agonist) and 5-HT1A (partial agonist), meeting an unmet need in the treatment of schizophrenia (e.g., reducing extrapyramidal symptoms and metabolic side effects)[1]
-Mechanism of action: The compound acts as a full agonist of TAAR1 (regulating dopamine and glutamate neurotransmission) and a partial agonist of 5-HT1A (regulating serotonin signaling). This dual mechanism of action can restore abnormal neural circuits in patients with schizophrenia to normal without blocking D2 receptors, thereby avoiding extrapyramidal reactions and elevated blood pressure[1]
-Therapeutic potential: The drug is suitable for the treatment of schizophrenia, and clinical trials have shown that it can effectively reduce positive symptoms, negative symptoms and cognitive symptoms. Its good safety profile (no extrapyramidal reactions, minimal metabolic effects) supports long-term use [1]
- Chemical characteristics: SEP-363856 HCl (eugenol) is a small molecule drug with a molecular weight of approximately 380 Da (hydrochloride). It is soluble in DMSO (≥ 20 mM) and aqueous solutions (concentration of 2.3 mg/mL in pH 7.4 buffer), and stable in simulated gastric and intestinal fluids [1]
- Clinical advantages: Unlike typical antipsychotics (such as haloperidol) and some atypical antipsychotics (such as olanzapine), it does not cause extrapyramidal symptoms, hypertension, or weight gain, thus addressing a key side effect issue in the treatment of schizophrenia [1]

C9H14CLNOS

219.7316

219.05

C, 49.20; H, 6.42; Cl, 16.13; N, 6.37; O, 7.28; S, 14.59

1310422-41-3

SEP-363856; 1310426-33-5; (Rac)-SEP-363856; 1310426-29-9; 2375116-24-6 (besylate); 2375116-27-9 (mesylate)

139415154

White to off-white solid powder

2

3

2

13

154

1

CNC[C@H]1C2=C(CCO1)C=CS2.Cl

JRDQGVVFSHRVTL-QRPNPIFTSA-N

InChI=1S/C9H13NOS.ClH/c1-10-6-8-9-7(2-4-11-8)3-5-12-9;/h3,5,8,10H,2,4,6H2,1H3;1H/t8-;/m0./s1

1-[(7S)-5,7-dihydro-4H-thieno[2,3-c]pyran-7-yl]-N-methylmethanamine;hydrochloride

Ulotaront; Ulotaront HCl; Ulotaront hydrochloride; SEP 856; SEP-856; SEP856; SEP-363856; SEP 363856; SEP-363856 hydrochloride; SEP-363856 (hydrochloride); SEP-363856 HCl; Ulotaront HCl; ULOTARONT HYDROCHLORIDE; (7S)-4,7-dihydro-N-methyl-5H-thieno[2,3-c]pyran-7-methanamine hydrochloride; 6P8Y2467AU; SEP363856;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

H2O: ~100 mg/mL (~455.1 mM)
DMSO: ~62.5 mg/mL (~284.4 mM)

Solubility in Formulation 1: ≥ 6.25 mg/mL (28.44 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 62.5 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: ≥ 6.25 mg/mL (28.44 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 62.5 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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Solubility in Formulation 3: ≥ 6.25 mg/mL (28.44 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 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (455.10 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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