Demeclocycline exerts its antibacterial effect by binding to the 30S subunit of the bacterial ribosome, specifically the 16S rRNA molecule, which prevents the binding of aminoacyl-tRNA to the ribosome A-site and thereby inhibits bacterial protein synthesis . This mechanism is bacteriostatic rather than bactericidal. In mammalian cells, tetracyclines including demeclocycline target the 28S small subunit of the mitochondrial ribosome (specifically the 12S rRNA), thereby deactivating mitochondrial protein synthesis and exerting cytotoxic effects on metabolically active cells . Additionally, demeclocycline has been identified as an inhibitor of EphB kinases (EphB1, EphB2, EphB3, EphB4), with varying IC₅₀ values in the low micromolar range . For the treatment of SIADH, demeclocycline acts on the renal collecting duct cells to induce nephrogenic diabetes insipidus by inhibiting the action of antidiuretic hormone (ADH) on the V2 receptor, an effect independent of its antibiotic activity.
Aquaporin-2 (AQP2) water channel in renal collecting duct principal cells – demeclocycline decreases AQP2 abundance and gene transcription. [1]
Adenylate cyclase 3 (AC3) and adenylate cyclase 5/6 (AC5/6) – demeclocycline reduces their abundance. [1]

In mpkCCD cells, treatment with demetocycline (0-100 μM) for 24 hours decreases the amount of AQP2 [3]. Monocyte and macrophage activity is enhanced by demeclocycline (10 μM; 24 h) therapy [4]. Treatment with demeclocycline (1-10 μM; 72 h) directly impacts the development of cells that initiate brain tumors [4].

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Demeclocycline decreased AQP2 abundance in mpkCCD cells (mouse cortical collecting duct cells) in a concentration-dependent manner, with significant effects observed at 50 μM. At 100 μM, transepithelial resistance was significantly decreased. The electrical potential difference (dependent on ENaC) was significantly decreased by 50 and 100 μM demeclocycline. [1]
Demeclocycline (50 μM) significantly reduced AQP2 abundance after 8 hours of incubation, as determined by immunoblot analysis. Coomassie brilliant blue staining confirmed equal protein loading, indicating the effect was not due to cell loss. [1]
In the presence of the protein synthesis inhibitor cycloheximide, demeclocycline did not affect AQP2 abundance, whereas it decreased AQP2 abundance in control cells, indicating that demeclocycline does not increase AQP2 degradation but affects AQP2 transcription or RNA stability. [1]
In mpkCCD cells stably transfected with a 3.0 kb AQP2 promoter-luciferase construct, dDAVP induced a twofold increase in luciferase activity. Demeclocycline reduced luciferase activity, indicating it affects AQP2 transcription. [1]
Demeclocycline (50 μM) significantly reduced cAMP levels in both unstimulated cells and dDAVP-stimulated mpkCCD cells (with IBMX present). [1]
Demeclocycline reduced AC3 abundance to <40% of control levels after 24 hours, with reduced abundance initiating at 8 hours of incubation. Demeclocycline reduced AC5/6 levels to <25% of control levels after 24 hours. AC5/6 expression was significantly decreased after 8 hours, in line with the decrease in AQP2. [1]
Demeclocycline affected AQP2 localization; cells treated with dDAVP for 4 days showed predominantly apical AQP2 expression, while cells incubated with demeclocycline during the last 24 hours showed lower AQP2 abundance with AQP2 spread throughout the cell and less apical expression. [1]
The addition of 8-Br-cAMP (100 μM) to demeclocycline-treated cells induced AQP2 translocation to the apical membrane and increased AQP2 abundance to control levels, but AQP2 abundance was still significantly lower than in cells treated with 8-Br-cAMP only, suggesting demeclocycline may also affect AQP2 abundance at a post-AC level. [1]
Demeclocycline (10 μM) enhanced TNF-α production in human monocytes stimulated with IL-1β/IFN-γ (100 ng/ml each) or LPS (100 ng/ml) compared to stimulation alone. Demeclocycline alone did not increase TNF-α secretion by unstimulated monocytes. [4]
Demeclocycline (10 μM) promoted chemotaxis of human monocytes toward a CCL2 gradient (10 ng/ml) in IL-1β/IFN-γ-primed conditions. [4]
Conditioned medium from demeclocycline-treated monocytes (Demec/MonoCM) significantly reduced BTIC growth (BT025 and BT048 lines) in neurosphere assays compared to conditioned medium from untreated monocytes. [4]
Demeclocycline (5 and 10 μM) directly decreased sphere formation and cell number of BTICs. At 1 μM, demeclocycline reduced BTIC growth as measured by alamarBlue assay. [4]
Demeclocycline (10 μM) added to growing BTIC spheres 3 days after their formation from singly dissociated cells still reduced further growth of spheres (BT025 and BT048 lines). [4]
Demeclocycline (10 μM) was not toxic to human MAP-2-positive neurons in culture. [4]
Microarray analysis of three BTIC lines treated with demeclocycline (10 μM for 6 hours) identified 301 genes commonly affected (fold change ≥1.3 or ≤-1.3). Down-regulated genes included TGFB1I1, FZD5, EMR2, ROMO1, and BCL3. Up-regulated genes included CHAC1, DDIT4, and CLEC2A. [4]
Among several tetracycline derivatives tested at 10 μM, only demeclocycline consistently inhibited sphere-forming capacity across all three BTIC lines, whereas tetracycline and oxytetracycline reduced sphere-forming capacity to varying extents across different lines. [4]

Demeclocycline (ip; 40 mg/kg; once daily; 48 hours) treatment resulted in a considerable reduction in hyponatremia and a significant correction of hypoosmolality without nephrotoxicity [3].

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In a rat model of SIADH (induced by dDAVP infusion via subcutaneous osmotic minipumps at 5 ng/hour and liquid diet for 8 days), rats receiving daily intraperitoneal injections of demeclocycline (40 mg/kg body weight) showed: significant reduction of hyponatremia (serum Na⁺: control 98.7 ± 0.8 mmol/l vs demeclocycline 117.3 ± 4.5 mmol/l, p<0.01); nearly significant correction of hyposomolality; significantly increased urine volume (control 44.8 ± 9.3 vs demeclocycline 91.3 ± 15.9 ml•kg⁻¹•24 h⁻¹, p<0.05); significantly decreased urine osmolality (control 1374 ± 117 vs demeclocycline 820 ± 148 mosM/kg, p<0.05); significantly increased fractional excretion of water (control 0.53 ± 0.14 vs demeclocycline 2.07 ± 0.44, p<0.01); and significantly increased fractional Na⁺ excretion (control 0.17 ± 0.06 vs demeclocycline 0.59 ± 0.13, p<0.05). Blood K⁺ levels were significantly increased (control 3.67 ± 0.29 vs demeclocycline 4.56 ± 0.23 mmol/l, p<0.05). [1]
Immunoblot analysis revealed a significant 75% reduction in AQP2 abundance in the inner medulla of demeclocycline-treated rats compared to controls. Demeclocycline treatment led to significant 2.5-fold and 2.0-fold increases in AQP2 abundance in the outer medulla and cortex, respectively. [1]
Immunohistochemistry showed reduced AQP2 labeling intensity in kidney sections from demeclocycline-treated rats, with AQP2 less prominent at the apical and basolateral plasma membrane and greater numbers of AQP2-positive intracellular vesicles observed. pS256 AQP2 labeling intensity was largely decreased in demeclocycline-treated rats compared to controls. [1]
AC5/6 abundance was 50% reduced in the inner medulla of demeclocycline-treated rats. In the outer medulla, AC5/6 expression was increased. [1]
No clear morphological differences between kidneys from demeclocycline-treated and control rats were observed. Picrosirius red staining (collagen, increased with toxicity/fibrosis) and inducible nitric oxide synthase staining (inflammation) did not differ between groups. AQP4 and β-catenin abundances and distributions were similar in both groups. [1]

Methodology for EphB Kinase Inhibition Assay: The inhibitory activity of demeclocycline against EphB kinases can be determined using a radiometric in vitro protein kinase assay (33PanQinase Activity Assay). Four different EphB kinase domains (EphB1, EphB2, EphB3, EphB4) are incubated with varying concentrations of demeclocycline ranging from 3 × 10⁻⁹ M to 1 × 10⁻⁴ M in the presence of a peptide substrate and [γ-³³P]ATP. The reaction is carried out in a buffer containing 50 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM DTT, and 0.01% Triton X-100. After incubation at 30°C for 30 minutes, the reaction is stopped by the addition of phosphoric acid. The phosphorylated peptide is captured onto P81 filter papers, washed extensively, and the incorporated radioactivity is measured by liquid scintillation counting. IC₅₀ values are calculated from concentration-response curves by fitting the residual activity percentage using non-linear regression analysis .

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cAMP measurement: mpkCCD cells were grown with or without dDAVP (1 nM for 4 days), with or without demeclocycline (50 μM for the last day), with the last 30 minutes in combination with the phosphodiesterase inhibitor IBMX (0.5 mM). After lysis, intracellular cAMP levels were measured using a cAMP-Glo assay or cAMP enzyme immunoassay according to manufacturer's instructions. Results were related to a standard curve based on measurement of defined cAMP solutions. [1]
Luciferase assay: mpkCCD cells containing a 3.0 kb AQP2 promoter-luciferase construct were grown and treated without or with dDAVP (1 nM for 4 days) without or with demeclocycline (50 μM during the last 24 hours). Cells were lysed, and luciferase activity was measured using a Luciferase Assay System. Luminescence was measured for 10 seconds. Protein concentrations were determined to verify equal amounts of protein per sample. [1]

Western Blot Analysis [3]
Cell Types: MpkCCD Cell
Tested Concentrations: 0-100 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: AQP2 abundance diminished in mpkCCD cells, with a significant effect at 50 μM.

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Cell viability assay [4]
Cell Types: Mouse bone marrow-derived macrophages and monocytes
Tested Concentrations: 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Enhanced TNF-α production and modulated monocyte function.

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Cell viability assay[4]
Cell Types: Brain Tumor Starting Cell
Tested Concentrations: 1, 5 and 10 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibition of cell growth in two ways: mediated by monocytes, and directly by affecting proliferation and Spheroid-forming ability of brain tumor-initiating cells.

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MpkCCD cell culture: cells were seeded at 1.5 × 10⁵ cells/cm² on semipermeable filters (pore size 0.4 μm) and cultured for 8 days. Unless stated otherwise, cells were exposed to 1 nM dDAVP at the basolateral side for the last 4 days to induce AQP2 expression. Demeclocycline was added to both sides of the filters for the last 2-24 hours with or without cycloheximide (50 μM), forskolin (10 μM), or 8-Br-cAMP (100 μM). Transcellular electrical resistance and electrical potential difference were measured using a meter at the end of the experiment. [1]
Immunoblot analysis: mpkCCD cells from a 1.13 cm² filter or 5-10 μg of kidney material were lysed in Laemmli buffer, sonicated, and denatured for 30 minutes at 37°C. For PNGase-F digestion, cell lysate was incubated with PNGase-F for 1 hour at 37°C. Samples were subjected to PAGE, blotting, and membranes were blocked. Membranes were incubated for 16 hours at 4°C with primary antibodies (anti-AQP2, anti-AC3, anti-AC5/6) in TBST with 1% nonfat dried milk. Blots were incubated for 1 hour with secondary antibodies coupled to horseradish peroxidase. Proteins were visualized using enhanced chemiluminescence. Densitometric analyses were performed. Equal loading was confirmed by Coomassie brilliant blue staining. [1]
Immunocytochemistry: mpkCCD cells were fixed in 3% paraformaldehyde in PBS. For AQP2 detection, fixed cells were incubated with affinity-purified rabbit AQP2 antibodies (1:100) and goat anti-rabbit antibodies coupled to Alexa 488 (1:100). For V2R-GFP localization, GFP expression was analyzed by confocal microscopy. [1]
Human monocyte isolation and culture: human monocytes were isolated from venous blood of healthy adults. Monocytes (100,000 cells/well/100 μl) were plated in RPMI medium supplemented with 20% human serum in 96-well plates. After 24 hours, cells were switched to BTIC medium. Treatment involved administering demeclocycline (10 or 1 μM) with or without LPS (100 ng/ml), IL-1β (100 ng/ml), or IFN-γ (100 ng/ml) for 48 hours, and conditioned media were collected. TNF-α levels were measured by ELISA. [4]
Chemotaxis assay: human monocytes were treated with demeclocycline (10 μM) for 1 hour, then IFN-γ/IL-1β (100 ng/ml each) was added. After 24 hours, 200,000 cells were plated onto filters (5 μm pore size) of ChemoTx plates. Recombinant human CCL2 (10 ng/ml) was added below the filter as a chemotactic stimulus. Cells were incubated for 16 hours. alamarBlue was added (1:10) for 4 hours, and signal was read at 570 nm. [4]
BTIC culture: BTICs were isolated from resected glioblastoma specimens. Cells were dissociated and plated in serum-free medium supplemented with EGF and FGF-2. For neurosphere assays, BTIC cells (10,000 cells/well/100 μl) were plated into 96-well plates. The number of spheres above 60 μm diameter cutoff was monitored after 72 hours. For total cell counts, BTIC spheres were collected, centrifuged, resuspended in Accumax, mixed with Trypan Blue (1:1), and counted using an automated cell counter. alamarBlue assay was also used: dye (1:10) was added for 4-6 hours, and readings were taken at excitation 544 nm/emission 590 nm. [4]
Human neuron toxicity assay: human fetal brain neurons (15-20 weeks) were isolated and cultured. Demeclocycline (10 μM) was added to neurons for 24 hours. Cells were fixed with 4% paraformaldehyde and stained for MAP-2 and Hoechst. Cells were imaged and quantified. [4]
Microarray: BTICs (BT012, BT025, BT048) were treated with demeclocycline (10 μM) for 6 hours. RNA was extracted, purified, and labeled. Samples were hybridized to GeneChip Human Gene 2.0 ST Arrays at 45°C for 16 hours. Arrays were stained, washed, and scanned. Data were analyzed for fold change between treatment and control (p<0.05 from t-test). [4]

Animal/Disease Models: Male Wistar rat hyponatremia [3]
Doses: 40 mg/kg
Route of Administration: intraperitoneal (ip) injection; 40 mg/kg; one time/day; 48 hrs (hrs (hours))
Experimental Results: increased urine output, diminished urine osmotic pressure, water The fractional excretion increased Dramatically.

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Animal/Disease Models: Male Wistar rat hyponatremia [3]
Doses: 40 mg/kg
Route of Administration: intraperitoneal (ip) injection; 40 mg/kg; one time/day; 48 hrs (hrs (hours))
Experimental Results: Specifically demonstrated the intrarenal medulla on AQP2 and AC5/6 and no secondary toxic effects.

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To induce hyponatremia in male Wistar rats (150-200 g), rats were infused with dDAVP in isotonic saline at a rate of 5 ng/hour via subcutaneously implanted osmotic minipumps (model 1002) and were fed a nutritionally balanced rodent liquid formula (1.0 kcal/ml) for a total of 8 days. For demeclocycline therapy, rats received daily intraperitoneal injections with 40 mg/kg body weight demeclocycline hydrochloride dissolved in PBS. For the last 48 hours, rats were housed in metabolic cages to measure water intake and urine output during the last 24 hours. Rats were anesthetized with isoflurane, blood was removed by heart puncture, and rats were killed by cervical dislocation. Kidneys were rapidly removed; one kidney was prepared for immunohistochemistry by overnight immersion in 4% paraformaldehyde in PBS, and the inner medulla, outer medulla, and cortex of the other kidney were dissected. [1]

The pharmacokinetic profile of demeclocycline is distinct from other tetracyclines. Following oral administration, absorption is slower than tetracycline, with a time to reach peak concentration (Tmax) of approximately 4 hours. After a single 150 mg oral dose, mean plasma concentrations are 0.46 mcg/mL at 1 hour and 1.22 mcg/mL at 3 hours . The serum elimination half-life (t₁/₂) ranges between 10 and 16 hours . Plasma protein binding varies by method: approximately 40% by dialysis equilibrium method and 90% by ultra-filtration method . The renal clearance rate is 35 mL/min/1.73 m², less than half that of tetracycline . Following a single 150 mg dose, 44% of the dose is excreted in urine within 96 hours, with 13-46% excreted in feces as active drug . Food, especially dairy products, and antacids containing aluminum, calcium, or magnesium reduce absorption by more than 50% . The drug concentrates in the liver and is excreted into bile at concentrations higher than in blood .

Effects During Pregnancy and Lactation
◉ Overview of Medication Use During Lactation
Some reviews suggest that tetracycline antibiotics are contraindicated during lactation due to the potential for staining of infant tooth enamel or bone deposition. However, a careful review of existing literature indicates that short-term use of demeclocycline during lactation is unlikely to cause harm because the drug concentration in breast milk is low, and the infant's absorption of the drug is inhibited by calcium in breast milk. Short-term use of demeclocycline in lactating women is acceptable. As a theoretical precaution, prolonged or repeated use during lactation should be avoided. Closely monitor the infant for rashes and potential effects on the gut microbiota, such as diarrhea or candidiasis (thrush, diaper rash).
◉ Effects on Breastfed Infants
No relevant published information was found as of the revision date.
◉ Effects on Lactation and Breast Milk
No relevant published information was found as of the revision date.

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At 100 μM demeclocycline in mpkCCD cells, transcellular resistance was significantly decreased, indicating the cell monolayer was seriously affected. [1]
Demeclocycline at 10 μM was not toxic to human MAP-2-positive neurons in culture. [4]
In the rat SIADH model, no clear morphological differences between kidneys from demeclocycline-treated and control rats were observed. Picrosirius red staining (collagen, increased with toxicity/fibrosis) and inducible nitric oxide synthase staining (inflammation) did not differ between groups. AQP4 and β-catenin abundances and distributions were similar in both groups, indicating specific effects on AQP2 and AC5/6 without general nephrotoxicity in this model. [1]
Despite clinical reports of nephrotoxicity in humans (Miller et al. 1980; Roth et al. 1967), no nephrotoxic effects were observed in this rat model. [1]

[1]. Tetracyclines, molecular and clinical aspects. J Antimicrob Chemother. 1992 Mar;29(3):245-77.
[2]. Tetracyclines: antibiotic action, uptake, and resistance mechanisms. Arch Microbiol. 1996 Jun;165(6):359-69.
[3]. Demeclocycline attenuates hyponatremia by reducing aquaporin-2 expression in the renal inner medulla. Am J Physiol Renal Physiol. 2013 Dec 15;305(12):F1705-18.
[4]. Demeclocycline Reduces the Growth of Human Brain Tumor-Initiating Cells: Direct Activity and Through Monocytes. Front Immunol. 2020 Feb 21;11:272.

Demeclocycline is a tetracycline antibiotic, lacking a methyl substituent at position 7 and having a chlorine-substituted hydrogen atom at the para-position of the phenolic hydroxyl group. Like tetracycline, it is an antibiotic, but due to its slower excretion rate, it maintains effective blood concentrations for a longer period. It (primarily in hydrochloride form) is used to treat Lyme disease, acne, and bronchitis, as well as hyponatremia (low blood sodium concentration) caused by syndrome of dysregulation of antidiuretic hormone secretion (SIADH), particularly when simple fluid restriction is ineffective. It has antibacterial, anti-aging, and diuretic effects. Demeclocycline is a tetracycline antibacterial drug. A tetracycline analog with a chlorine atom at position 7 and a methyl group at position 6. Due to its slower excretion rate than tetracycline, it maintains effective blood concentrations for a longer period.

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Demeclocycline is a bacteriostatic antibiotic of the tetracycline group. The structures of demeclocycline, minocycline, and tetracycline are shown in Figure 1 of reference [1]. [1]
The aquatic effect of tetracycline antibiotics such as demeclocycline and minocycline is mediated via downregulation of the AVP-regulated water channel AQP2. Demeclocycline decreases AQP2 gene transcription by decreasing AC5/6 expression and cAMP generation and possibly by affecting a post-AC target, resulting in less AQP2 protein expression in the inner medulla. This results in increased water loss and attenuates hyponatremia in SIADH. [1]
Demeclocycline is a derivative of meclocycline (which has significant toxicity and is limited to topical use). Demeclocycline can be administered systemically and is used off-label to manage chronic SIADH. [4]
Demeclocycline is a candidate to reactivate compromised immune cells (monocytes/macrophages) to improve the prognosis of patients with gliomas. It reduces BTIC growth through direct effects and through indirect stimulation of monocytes. Elevated DDIT4 expression (upregulated by demeclocycline in BTICs) was associated with improved survival in glioma patients based on TCGA database analysis. [4]

C21H21N2O8CL

464.85304

464.098

C, 54.26; H, 4.55; Cl, 7.63; N, 6.03; O, 27.53

127-33-3

Demeclocycline hydrochloride;64-73-3; 127-33-3; 64-73-3 (HCl); 13215-10-6 (hydrate); 17146-81-5 (calcium)

54680690

Green to dark green solid powder

1.8±0.1 g/cm3

684.5±55.0 °C at 760 mmHg

367.8±31.5 °C

0.0±2.2 mmHg at 25°C

1.761

0.57

6

9

2

32

961

5

NC(C1C(=O)[C@@H](N(C)C)[C@@H]2C[C@@H]3[C@H](O)C4=C(C=CC(O)=C4C(O)=C3C(=O)[C@]2(O)C=1O)Cl)=O

GUXHBMASAHGULD-SEYHBJAFSA-N

InChI=1S/C21H21ClN2O8/c1-24(2)14-7-5-6-10(16(27)12-9(25)4-3-8(22)11(12)15(6)26)18(29)21(7,32)19(30)13(17(14)28)20(23)31/h3-4,6-7,14-15,25-27,30,32H,5H2,1-2H3,(H2,23,31)/t6-,7-,14-,15-,21-/m0/s1

(4S,4aS,5aS,6S,12aR)-7-chloro-4-(dimethylamino)-1,6,10,11,12a-pentahydroxy-3,12-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide

Demeclocycline; Demethylchlortetracycline; 127-33-3; DMCT; Ledermycin; Declostatin; Ledermycin; Bioterciclin; Deganol; Deteclo; Ledermycin; Declomycin; Demeclociclina; Demeclocyclinum; DMCTC; Detravis; Meciclin; Mexocine; Clortetrin

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 : ~25 mg/mL (~53.78 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.69 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 12.5 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: ≥ 1.25 mg/mL (2.69 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 12.5 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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 (Please use freshly prepared in vivo formulations for optimal results.)