δ Opioid Receptor/DOR; mAChR1

N-desmethylclozapine, a brain penetrant metabolite, was found to bind to M1 muscarinic receptors preferentially, with an IC50 of 55 nM. Compared to clozapine, it was a more potent partial agonist at this receptor, with an EC50 of 115 nM and 50% of acetylcholine response[1].
N-desmethylclozapine has agonistic characteristics at the 5-HT1A receptor in the cerebral cortex and hippocampus, as well as mild agonistic effects on the M1 mAChR. Additionally, this substance exhibits agonistic behavior at the δ-opioid receptor located in the striatum and cerebral cortex[2].
N-desmethylclozapine (3 μM) significantly reduces excitatory neurons' outward current, but not that of inhibitory neurons. When it comes to excitatory neurons, N-desmethylclozapine works better on its own than it does when combined with clozapine or taken alone. One microgram of atropine and 0.1 microgram of pirenzepine both considerably reduce the effects of N-desmethylclozapine on excitatory neurons. In excitatory cells, K¹²⁵ channels were inhibited by N-desmethylclozapine but not by clozapine via M1 receptors[3].
N-desmethylclozapine causes TxB2 levels to drop both in response to TSST-1 stimulation and in unstimulated conditions. The production of TxA2 or TxB2 may be modulated by clozapine, N-desmethylclozapine, and CPZ, potentially affecting neurotransmitter systems[5].
N-desmethylclozapine, fluoxetine hydrochloride, and salmeterol xinafoate have IC50 values of 1 μM, 0.38 μM, and 0.67 μM in Huh-7 cells infected with DENV-2, respectively. When cells treated with all three inhibitors are compared to those treated with DMSO, the levels of NS3 are lower, indicating that the inhibitors function at a stage before the translation of viral proteins. Negative-strand RNA levels are reduced by >75% in cells treated with N-desmethylclozapine[6].

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The atypical antipsychotic clozapine is widely used for treatment-resistant schizophrenic patients. Clozapine and its major active metabolite, N-desmethylclozapine (NDMC), have complex pharmacological properties, and interact with various neurotransmitter receptors. There are several biochemical studies reporting that NDMC exhibits a partial agonist profile at the human recombinant M1 muscarinic receptors. However, direct electrophysiological evidence showing the ability of NDMC to activate native M1 receptors in intact neurons is poor. Using rat hippocampal neurons, we previously demonstrated that activation of muscarinic receptors by a muscarinic agonist, oxotremorine M (oxo-M), induces a decrease in outward K(+)current at -40mV. In the present study, using this muscarinic current response we assessed agonist and antagonist activities of clozapine and NDMC at native muscarinic receptors in intact hippocampal excitatory and inhibitory neurons. Suppression of the oxo-M-induced current response by the M1 antagonist pirenzepine was evident only in excitatory neurons, while the M3 antagonist darifenacin was effective in both types of neurons. Muscarinic agonist activity of NDMC was higher than that of clozapine, higher in excitatory neurons than in inhibitory neurons, sensitive to pirenzepine, and partially masked when co-applied with clozapine. Muscarinic antagonist activity of clozapine as well as NDMC was not different between excitatory and inhibitory neurons, but clozapine was more effective than NDMC. These results demonstrate that NDMC has the ability to activate native M1 receptors expressed in hippocampal excitatory neurons, but its agonist activity might be limited in clozapine-treated patients because of the presence of excessive clozapine with muscarinic antagonist activity. [3]

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Thromboxane A2 (TxA2) and the activation of its receptor have been shown to modulate vasoconstriction and platelet aggregation as well as dopaminergic and serotonergic signalling. Dopaminergic and serotonergic systems play a crucial role in the pathophysiology of schizophrenia and these systems are the main targets of antipsychotics (APs). As the first antipsychotic (AP) chlorpromazine (CPZ) has already been shown to reduce TxA2, we hypothesized that the AP clozapine and its metabolite N-desmethylclozapine (NDMC) might also influence TxA2 production. We measured levels of thromboxane B2 (TxB2), the metabolite of the very unstable molecule TxA2, in unstimulated and stimulated blood samples of 10 healthy female subjects in a whole blood assay using toxic shock syndrome toxin-1 (TSST-1) and monoclonal antibody against surface antigen CD3 combined with protein CD40 (OKT3/CD40) as stimulants. Blood was supplemented with the APs CPZ, clozapine or NDMC in one of four different concentrations. Additionally, thromboxane levels were measured in blood without the addition of APs under different stimulation conditions. Under TSST-1 as well as OKT3/CD40 stimulation, mean TxB2 concentrations were significantly (p < 0.05) decreased by clozapine over all applied concentrations. NDMC led to a decrease in TxB2 levels under unstimulated conditions as well as under TSST-1 stimulation. CPZ reduced TxB2 production at low concentrations under unstimulated and TSST-1- stimulated conditions. Clozapine, NDMC and CPZ possibly act on neurotransmitter systems via modulation of TxA2 or TxB2 production. Additionally, known side effects of APs such as orthostatic hypotension may be a result of changes in the concentrations of TxA2 or TxB2.[5]

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Around 10,000 people die each year due to severe dengue disease, and two-thirds of the world population lives in a region where dengue disease is endemic. There has been remarkable progress in dengue virus vaccine development; however, there are no licensed antivirals for dengue disease, and none appear to be in clinical trials. We took the approach of repositioning approved drugs for anti-dengue virus activity by screening a library of pharmacologically active compounds. We identified N-desmethylclozapine, fluoxetine hydrochloride, and salmeterol xinafoate as dengue virus inhibitors based on reductions in the numbers of infected cells and viral titers. Dengue virus RNA levels were diminished in inhibitor-treated cells, and this effect was specific to dengue virus, as other flaviviruses, such as Japanese encephalitis virus and West Nile virus, or other RNA viruses, such as respiratory syncytial virus and rotavirus, were not affected by these inhibitors. All three inhibitors specifically inhibited dengue virus replication with 50% inhibitory concentrations (IC50s) in the high-nanomolar range. Estimation of negative-strand RNA intermediates and time-of-addition experiments indicated that inhibition was occurring at a postentry stage, most probably at the initiation of viral RNA replication. Finally, we show that inhibition is most likely due to the modulation of the endolysosomal pathway and induction of autophagy [6].

N-desmethylclozapine underlies presynaptic modulation of glutamate release and GABA in humans and rats at M2 and M4 mAChRs, respectively. N-desmethylclozapine, for example, may be an M2 mAChR antagonist in rats, but it is not active at this receptor in the human neocortex. On the other hand, N-desmethylclozapine does not have an agonistic effect at M4 mAChR in the rat neocortex[4].

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The active moiety of clozapine, the prototypical antipsychotic drug, consists of clozapine and its major metabolite, N-desmethylclozapine (NDMC). Previous studies have suggested that NDMC may be more important than the patent compound itself for the improvement in cognition in patients with schizophrenia treated with clozapine. While the pharmacology of clozapine and NDMC are similar in most respects, NDMC has been shown to be an M1 muscarinic receptor partial agonist whereas clozapine is an M1 antagonist in vitro and in vivo. We hypothesized that NDMC may improve cognition by increasing dopamine (DA) and acetylcholine (ACh) release in medial prefrontal cortex (mPFC) via direct stimulation of M1 receptors, whereas both NDMC and clozapine itself would do so by other mechanisms as well, and that clozapine would inhibit the M1 agonist effect of NDMC. In the present study, using microdialysis in awake, freely moving rats, we found that NDMC at doses of 10 and 20, but not 5 mg/kg, significantly increased DA and ACh release in the mPFC and HIP, but not in the nucleus accumbens (NAC). The M1-preferring antagonist, telenzepine (3 mg/kg), completely blocked NDMC (10 mg/kg)-induced increases in cortical DA and ACh release. Clozapine (1.25 mg/kg), which by itself had no effect on DA or ACh release in the cortex, blocked NDMC (10 mg/kg)-induced ACh, but not DA, release in the mPFC. The 5-HT1A receptor antagonist, WAY100635 (0.2 mg/kg) blocked NDMC (20 mg/kg)-induced cortical DA but not ACh release. These findings suggest that: (1) NDMC is an M1 agonist while clozapine is an M1 antagonist in vivo; (2) M1 agonism of NDMC can contribute to the release of cortical ACh and DA release; (3) NDMC, because of its M1 agonism, may more effectively treat the cognitive impairments observed in schizophrenia than clozapine itself; and (4) M1 receptor agonism may be a valuable target for the development of drugs that can improve cognitive deficit in schizophrenia, and perhaps other neuropsychiatric disorders as well. [1]

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Xanomeline had agonistic activity at the M1 muscarinic acetylcholine receptor (mAChR) in all brain regions, as well as at the 5-HT1A receptor in the cerebral cortex and hippocampus. On the other hand, N-desmethylclozapine exhibited slight agonistic effects on the M1 mAChR, and agonistic properties at the 5-HT1A receptor in the cerebral cortex and hippocampus. This compound also behaved as an agonist at the δ-opioid receptor in the cerebral cortex and striatum. In addition, the stimulatory effects of N-desmethylclozapine on [(35)S]GTPγS binding to Gαi/o were partially mediated through mAChRs (most likely M4 mAChR subtype), at least in striatum. Conclusions: The agonistic effects on the mAChRs (particularly M1 subtype, and also probably M4 subtype), the 5-HT1A receptor and the δ-opioid receptor expressed in native brain tissues, some of which are common to both compounds and others specific to either, likely shape the unique beneficial effectiveness of both compounds in the treatment for schizophrenic patients. These characteristics provide us with a clue to develop newer antipsychotics, beyond the framework of dopamine D2 receptor antagonism, that are effective not only on positive symptoms but also on negative symptoms and/or cognitive/affective impairment.[2]

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Cholinergic transmission plays a pivotal role in learning, memory and cognition, and disturbances of cholinergic transmission have been implicated in neurological disorders including Alzheimer's disease, epilepsy and schizophrenia. Pharmacological alleviation of these diseases by drugs including N-desmethylclozapine (NDMC), promising in animal models, often fails in patients. We therefore compared the effects of NDMC on glutamatergic and GABAergic transmission in slices from rat and human neocortex. We used carbachol (CCh; an established agonist at metabotropic muscarinic acetylcholine (ACh) receptors (mAChRs)) as a reference. Standard electrophysiological methods including intracellular and field potential recordings were used. In the rat neocortex, NDMC prevented the CCh-induced decrease of GABAA and GABAB receptor-mediated responses but not the CCh-induced increase of the paired-pulse depression. NDMC reduced neither the amplitude of the excitatory postsynaptic potentials (EPSP) nor antagonized the CCh-induced depression of EPSP. In the human neocortex, however, NDMC failed to prevent CCh-induced decrease of the GABAB responses and directly reduced the amplitude of EPSP. These data suggest distinct effects of NDMC in rat and human at M2 and M4 mAChRs underlying presynaptic modulation of GABA and glutamate release, respectively. In particular, NDMC might be a M2 mAChR antagonist in the rat but has no activity at this receptor in human neocortex. However, NDMC has an agonistic effect at M4 mAChR in the human but no such effect in the rat neocortex. The present study confirms that pharmacology at mAChRs can differ between species and emphasizes the need of studies in human tissue [4].

Inhibitor treatments. [6]
Dengue virus infection and inhibitor treatment experiments were performed with Huh-7 and A549 cells, and viral titers in the culture supernatants were determined by plaque assays on BHK-21 cells. IC50 (50% inhibitory concentration) experiments were performed by using 2-fold dilutions of the inhibitor starting from an 8 μM concentration. A total of 30,000 cells were plated in a 48-well plate, infected with DENV-2 at a multiplicity of infection (MOI) of 3, and incubated with 2% Dulbecco's modified Eagle's medium (DMEM) containing inhibitors. Supernatants were collected at 24 h postinfection (p.i.), and viral titers were estimated by plaque assays on BHK-21 cells. For Western blotting, cell lysates were prepared as described previously (10), under the inhibitor treatment conditions described below, and the membranes were probed with antibodies recognizing DENV nonstructural protein 3 (NS3) (kind gift from Raj Bhatnagar), tubulin monoclonal, calnexin, and 78-kDa glucose-regulated protein (GRP78) antibodies. For LC3 detection, an antibody that has a high specificity for the LC3-II form was used (LC3b [D11]; Cell Signaling Technology). Signals were detected by chemiluminescence. (See the supplemental material for a description of the flow cytometry experiment.)

Drugs [1]
NDMC/N-desmethylclozapine and clozapine was dissolved in a small amount of 0.1 M tartaric acid and the pH was adjusted to 6–7 with 0.1 N NaOH. WAY100635 (Wyeth Laboratories, Philadelphia, PA) and telenzepine (Research Chemical Inc.) were dissolved in deionized water. Vehicle or drugs in a volume of 1.0 ml/kg were administered subcutaneously to randomly assigned rats.
At 3–5 days after cannulation, a dialysis probe was implanted into the mPFC and NAC under slight anesthesia with isoflurane. Rats were then housed individually overnight in a dialysis cage. After the overnight perfusion at 0.4 μl/min of the probe, the flow was increased to 1.5 μl/min. After 1 h, the dialysate samples were collected every 30 min. The perfusion medium was Dulbecco's phosphate-buffered saline solution, including Ca2+ (138 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 0.5 mM MgCl, 1.2 mM CaCl2, pH 7.4). No AChesterase inhibitor in the dialysate is required with this procedure (Ichikawa et al, 2002b). After stable baseline values in the dialysates were obtained, each rat received two injections, vehicle/NDMC (N-desmethylclozapine), WAY100635/NDMC, telenzepine/NDMC, or clozapine/NDMC. The locations of the dialysis probes were verified at the end of each experiment by brain dissection.

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Background: 3(3-Hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) and N-desmethylclozapine are of special interest as promising antipsychotics with better efficacy, especially for negative symptoms and/or cognitive/affective impairment. Methods: The guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding experiments were performed using (1) conventional filtration technique, (2) antibody-capture scintillation proximity assay, and (3) immunoprecipitation method, in brain membranes prepared from rat cerebral cortex, hippocampus, and striatum.[2]

Metabolism / Metabolites
N-Desmethylclozapine has known human metabolites that include Desmethylclozapine N-glucuronide.
N-Desmethylclozapine is a known human metabolite of clozapine.

[1]. N-desmethylclozapine, a major metabolite of clozapine, increases cortical acetylcholine and dopamine release in vivo via stimulation of M1 muscarinic receptors. Neuropsychopharmacology. 2005 Nov;30(11):1986-95.

[2]. Comparative analysis of pharmacological properties of xanomeline and N-desmethylclozapine in rat brain membranes. J Psychopharmacol. 2016 Sep;30(9):896-912.

[3]. Electrophysiological evidence showing muscarinic agonist-antagonist activities of N-desmethylclozapine using hippocampal excitatory and inhibitory neurons. Brain Res. 2016 Jul 1;1642:255-62.

[4]. Different pharmacology of N-desmethylclozapine at human and rat M2 and M 4 mAChRs in neocortex. Naunyn Schmiedebergs Arch Pharmacol. 2015 May;388(5):487-96.

[5]. Impact of clozapine, N-desmethylclozapine and chlorpromazine on thromboxane production in vitro. Med Chem. 2012 Nov;8(6):1032-8.

[6]. N-Desmethylclozapine, Fluoxetine and Salmeterol inhibit post-entry stages of dengue virus life-cycle. Antimicrob Agents Chemother. 2016 Oct 21;60(11):6709-6718.

N-desmethylclozapine is a dibenzodoazepine substituted with chloro and piperazino groups which is a major metabolite of clozapine; a potent and selective 5-HT2C serotonin receptor antagonist. It has a role as a metabolite, a delta-opioid receptor agonist and a serotonergic antagonist. It is a dibenzodiazepine, a member of piperazines and an organochlorine compound.
ACP-104, or N-desmethylclozapine, is the major metabolite of clozapine, and is being developed by ACADIA as a novel, stand-alone therapy for schizophrenia. It combines an atypical antipsychotic efficacy profile with the added potential benefit of enhanced cognition, thereby addressing one of the major challenges in treating schizophrenia today.

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Drug Indication
Investigated for use/treatment in schizophrenia and schizoaffective disorders.

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Mechanism of Action
ACP-104 combines M1 muscarinic agonism, 5-HT2A inverse agonism, and D2 and D3 dopamine partial agonism in a single compound. ACP-104 uniquely stimulates brain cells known as M1 muscarinic receptors that play an important role in cognition. ACP-104 is a partial-agonist that causes weak activation of dopamine D2 and D3 receptors. These partial agonist properties of ACP-104 may lead to less motoric side effects than seen with most other antipsychotic drugs.

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Pharmacodynamics
ACP-104 is a small molecule drug candidate we are developing as a novel therapy for schizophrenia. It is known that large amounts of ACP-104, or N-desmethylclozapine, are formed in the body after administration of clozapine. That is, clozapine is metabolized to ACP-104. We discovered that ACP-104 has a unique ability to stimulate m1 muscarinic receptors, a key muscarinic receptor. The m1 muscarinic receptors are widely known to play an important role in cognition. Since clozapine itself blocks the m1 muscarinic receptor, patients need to extensively metabolize clozapine into ACP-104 to stimulate this receptor and thereby overcome the blocking action of clozapine. Administration of ACP-104 will avoid the variability of this metabolic process and the competing action of clozapine. Like clozapine, ACP-104 is a dopamine antagonist and a 5-HT2A inverse agonist. We believe that ACP-104 represents a new approach to schizophrenia therapy that combines an atypical antipsychotic efficacy profile with the added advantage of beneficial cognitive effects.

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The main findings of the present study are that (1) NDMC, the major active metabolite of clozapine, significantly increased DA and ACh release in the mPFC and HIP, but not the NAC; (2) the M1-preferring antagonist telenzepine completely blocked DA and ACh release in the mPFC produced by NDMC; (3) NDMC (10 mg/kg)-induced ACh release was completely blocked by clozapine (1.25 mg/kg), consistent with previous reports that NDMC is a potent M1 agonist, while clozapine has M1 antagonist properties in vivo; (4) clozapine pretreatment did not block NDMC-induced cortical DA release, indicating M1 agonism did not contribute to this effect of NDMC; and (5) the increases in DA, but not ACh, release in the mPFC produced by NDMC was partially blocked by the 5-HT1A antagonist WAY100635, indicating that cortical DA release is partially dependent upon 5-HT1A-receptor stimulation.
The effect of clozapine on DA or ACh release is most likely the result of the combined effect of clozapine and NDMC, the agonist/antagonist mixing. Thus, high NDMC levels, and particularly high NDMC/clozapine ratios, would increase M1 muscarinic receptor stimulation, as predicted by mass action and by agonist/antagonist mixing studies (Brauner-Osborne et al, 1996). Brain clozapine concentrations in the rat during chronic treatment have been reported to exceed those of NDMC during chronic treatment by three-fold (Weigmann et al, 1999). There is no information on what the relative levels are in man. High concentrations of NDMC are found in plasma samples in some patients treated with clozapine (Hasegawa et al, 1993). High NDMC levels, and a high NDMC/clozapine ratio even more so, would increase M1 muscarinic receptor stimulation. The present data on the blockade of NDMC-induced ACh release by clozapine are consistent with clinical data from our laboratory, which suggest that the NDMC/clozapine ratio is a better predictor of clinical response to clozapine than clozapine levels alone (Frazier et al, 2003; Mauri et al, 2003; Weiner et al, 2004).
In conclusion, NDMC preferentially increased DA and ACh release in the mPFC and HIP but not the NAC, similar to the effect of clozapine and other atypical APDs. The blockade of NDMC-induced ACh release by telezenpine and clozapine indicates that the stimulation of M1 receptors contributes to the ability of NDMC to increase cortical DA and ACh release, confirming that NDMC has significant M1 agonistic actions, whereas the parent compound, clozapine, is an antagonist.[1]

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In the present study, we assessed agonist and antagonist activities of clozapine and its major active metabolite, NDMC, at native muscarinic receptors in rat intact hippocampal excitatory and inhibitory neurons, by monitoring muscarinic current responses. Our data clearly demonstrated that clozapine acts primarily as an antagonist, whereas NDMC acts as a mixed agonist–antagonist. The agonist activity of NDMC, which was explained by its action on the M1, rather than M3, subtype, was largely masked when the same concentration of clozapine was co-applied. These results suggest that the clozapine/NDMC concentration ratio in clozapine-treated patients might determine the degree of M1 receptor activation by NDMC.[3]

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In conclusion, the present data cast some doubts on extrapolating from rat neurons to human neurons, when going beyond the standard compounds, because the pharmacology of NDMC differs markedly in the two species (Thomas et al. 2010). Thus, the present study emphasizes the need of studies in human tissue, despite the inherent limitations of these tissues.[4]

C17H17CLN4

312.797

312.114

C, 65.28; H, 5.48; Cl, 11.33; N, 17.91

6104-71-8

N-Desmethylclozapine-d8; 1189888-77-4; N-Desmethylclozapine-d8 hydrochloride; 2705402-91-9

135409468

Light yellow to green yellow solid powder

1.38g/cm3

490.1ºC at 760 mmHg

120-125°C

250.2ºC

1.709

3.22

2

3

1

22

421

0

ClC1=CC=C2C(N=C(N3CCNCC3)C4=CC=CC=C4N2)=C1

JNNOSTQEZICQQP-UHFFFAOYSA-N

InChI=1S/C17H17ClN4/c18-12-5-6-15-16(11-12)21-17(22-9-7-19-8-10-22)13-3-1-2-4-14(13)20-15/h1-6,11,19-20H,7-10H2

3-chloro-6-piperazin-1-yl-11H-benzo[b][1,4]benzodiazepine

AZD-5991; AZD-5991 S-enantiomer; N-Desmethylclozapine; Norclozapine; 6104-71-8; Desmethylclozapine; N-desmethyl clozapine; N-desmethyl-clozapine; N-DEMETHYLCLOZAPINE; 8-Chloro-11-(1-piperazinyl)-5H-dibenzo(b,e)(1,4)diazepine; AZD 5991 S-enantiomer.

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 (~159.9 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.99 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 (7.99 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.)