Adenylyl cyclase ( IC50 = 41 nM ); Adenylyl cyclase ( EC50 = 0.5 μM )
Adenylyl Cyclase (AC) isoforms (AC1: EC50 = 0.5 μM; AC2: EC50 = 0.3 μM; AC3: EC50 = 0.4 μM; AC5: EC50 = 1.2 μM; AC6: EC50 = 0.8 μM) [4][5]
- No significant binding to G protein-coupled receptors, kinases, or ion channels (Ki > 100 μM) [4]

In vitro activity: Forskolin increases cAMP levels in preparations of membranes, cells, or tissues. In addition to activating AC, forskolin also interacts with ion channels and glucose transporters, among other proteins. With the exception of AC9, which is somewhat less effective than the other nine transmembrane isoforms of AC, forskolin can activate nine of them. This property makes it possible to identify and measure high-affinity binding sites, or G-proteins (Gs)–AC complexes. Forskolin-stimulated cAMP generation in cells is facilitated by GPCR-mediated s activation because s-Forskolin potentiates AC activity.[1] Forskolin stimulates adenylate cyclase activity without interacting with cell surface receptors. The potentiation of cAMP by forskolin consequently suppresses the release of histamine and the degranulation of mast cells and basophils, reduces intraocular pressure and blood pressure, stops platelet aggregation, increases thyroid hormone secretion, vasodilation, bronchodilation, and fat cell lipolysis. Regardless of the production of cAMP, forskolin inhibits the binding of platelet-activating factor (PAF). This effect may be due to forskolin's direct interaction with PAF or to forskolin's interference with PAF's ability to bind to receptor sites. Forskolin suppresses the transport of glucose in erythrocytes, adipocytes, platelets, and other cells. It also seems to have an impact on a number of membrane transport proteins. Forskolin is used to treat with glaucoma. [2]

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Forskolin directly activates adenylyl cyclase (AC) isoforms, increasing intracellular cyclic adenosine monophosphate (cAMP) levels in a dose-dependent manner [4][5]
- In rat cortical neurons, Forskolin (0.1-10 μM) elevated cAMP concentrations by 2.5-8.3 fold, activating protein kinase A (PKA) and phosphorylating cAMP response element-binding protein (CREB) at Ser133 [1][4]
- In human umbilical vein endothelial cells (HUVECs), Forskolin (1-5 μM) induced nitric oxide (NO) production by 3.2 fold via PKA-mediated phosphorylation of endothelial nitric oxide synthase (eNOS) [4]
- Against glioblastoma cell lines (U87, U251), Forskolin (5-20 μM) inhibited cell proliferation with IC50 values of 12.3 μM and 15.7 μM, inducing G1 phase arrest and decreasing cyclin D1 expression [4]
- In smooth muscle cells (rat aortic), Forskolin (0.5-5 μM) relaxed cell contractions by increasing cAMP, reducing intracellular calcium levels by 40-60% [4][5]
- Forskolin (1-10 μM) enhanced neurite outgrowth in PC12 cells by 2.1-3.5 fold, mediated by PKA/CREB signaling [1]

The Forskolin (Fsk)-treated Mrp4-/- mice shows an increased number of Ki67-positive and cleaved caspase 3-positive ECs, a significant decrease in the amount of pericyte coverage, and a reduced number of empty sleeves. In pups exposed to hyperoxia (75% oxygen) from P7 to P12, the Mrp4-/- mice shows a significant increase in the unvascularized retinal area[3]. The average blood glucose in the healthy rat group is 102.12±1.94 mg/dL, 101.25±3.56 for control group and 103±2.08 in forskolin group. The data shows that glucose levels at the end of the study are lower in forskolin group, with a significant difference according to the statistical tests applied (p=0.03)[6].

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In spontaneously hypertensive rats (SHR), intravenous administration of Forskolin (0.5-2 mg/kg) dose-dependently reduced systolic blood pressure by 15-30 mmHg, with peak effect at 15 minutes post-administration [4]
- In mice models of anxiety (elevated plus maze test), Forskolin (5-20 mg/kg, p.o.) increased open arm exploration time by 40-70%, indicating anxiolytic effects [4]
- In rat models of focal cerebral ischemia, Forskolin (1 mg/kg, i.v., administered 1 hour post-occlusion) reduced infarct volume by 35% and improved neurological deficit scores at 24 hours [1]
- In diabetic rats, oral Forskolin (10 mg/kg/day for 4 weeks) improved insulin sensitivity, reducing fasting blood glucose by 28% and increasing glucose uptake in skeletal muscle [2]

In Vitro Kinase Assays[5]
For Jak3 kinase assays, Fsk-treated MT-2 cells were lysed, clarified, and immunoprecipitated using Jak3 antibody as described above. Kinase reactions were carried out as described previously at 30 °C for 20 min. For PKA kinase assays, untreated MT-2 cells were lysed, and Jak3 was immunoprecipitated and bound to PAS beads as described previously. Immunoprecipitated Jak3 was washed with kinase buffer (50 mm Hepes-NaOH (pH 7.4), 10 mm MgCl2, 0.5 mm EGTA, 0.5 mm DTT, 20 μg/ml aprotinin, 10 μg/ml leupeptin, 1 μg/ml pepstatin A) and incubated with 200 μm ATP and purified protein kinase A catalytic subunit (PKAc) as indicated in the figure legends. Kinase reactions were carried out at 32 °C for 30 min followed by vigorous washing of the beads with cold kinase wash buffer as described previously. For [γ-32P]ATP radiolabeled kinase assays using recombinant Jak3, Hek293 cells were transfected with wild type (WT) Jak3 or kinase-dead Jak3 K855A using Lipofectamine 2000 according to the manufacturer's instructions. Cells were lysed and immunoprecipitated with Jak3 antibody (described above). Jak3-bound PAS beads were washed three times in cold lysis buffer (described above) followed by kinase buffer. Kinase reactions were initiated by adding 10 μCi [γ-32P]ATP, 10 μm unlabeled ATP, and 1 μg of purified PKAc to Jak3-bound PAS bead reaction mixtures. Kinase reactions were performed at 32 °C for 30 min. Jak3-bound PAS beads were washed three times in radioimmunoassay buffer (10 mm Tris-HCl, pH 7.4, 75 mm NaCl, 20 mm EDTA, 10 mm EGTA, 20 mm Na4P2O7, 50 mm NaF, 20 mm 2-glycerolphosphate, 1 mm p-nitrophenylphosphate, 0.1% Triton X-100) and one time in kinase wash buffer (described above). The reactions were stopped by adding 2× SDS-PAGE sample buffer followed by SDS-PAGE. Coomassie stainable Jak3 bands were excised from the PVDF membrane and subjected to phosphoamino acid analysis.

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Adenylyl Cyclase (AC) activation assay: Recombinant human AC isoforms (AC1/AC2/AC3/AC5/AC6) were incubated with ATP, magnesium chloride, and Forskolin (0.01-20 μM) at 37°C for 30 minutes. Intracellular cAMP production was quantified by ELISA to calculate EC50 values [4][5]
- PKA activity assay: Rat cortical neuron lysates were incubated with PKA substrate peptide, ATP, and Forskolin-induced cAMP (0.1-10 μM equivalent) at 30°C for 45 minutes. Phosphorylated substrate was detected by colorimetric assay to assess PKA activation [1][4]
- eNOS phosphorylation assay: HUVEC lysates were treated with Forskolin (1-5 μM) for 30 minutes, then incubated with eNOS antibody and kinase buffer. Phosphorylated eNOS (Ser1177) was quantified by Western blot densitometry [4]

In 96-well plates, 5×10 4 cells of either MT-2 or Quiescent Kit 225 are seeded into each well. Afterwards, cells are pretreated for one hour at concentrations of 1, 5, 10, 25, 50, and 100 μM of forskolin or 1% DMSO (vehicle). Following 20 hours of culture at 37°C and IL-2 stimulation, the cells are harvested. A 20-hour treatment of 1% DMSO is given to control cells. [ 3 H]thymidine is pulsed into the cells at a concentration of 0.5 μCi/200 μL during the last 4 hours of incubation. Using liquid scintillation counting, cells are collected onto fiberglass filters for analysis.

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cAMP elevation assay: Rat cortical neurons or HUVECs were seeded in 96-well plates and cultured for 24-48 hours. Forskolin (0.1-20 μM) was added, and cells were incubated for 1-4 hours. Cells were lysed, and cAMP levels were measured by competitive ELISA [1][4][5]
- Glioblastoma antiproliferation assay: U87/U251 cells were plated in 96-well plates and treated with Forskolin (1-50 μM) for 72 hours. Cell viability was determined by MTT assay, and IC50 values were calculated. Cell cycle distribution was analyzed by flow cytometry after propidium iodide staining [4]
- Neurite outgrowth assay: PC12 cells were seeded on collagen-coated plates and treated with Forskolin (0.1-10 μM) for 7 days. Neurite length was measured by phase-contrast microscopy, and outgrowth rate was calculated relative to control [1]
- Calcium imaging assay: Rat aortic smooth muscle cells were loaded with calcium-sensitive dye and treated with Forskolin (0.5-5 μM). Intracellular calcium fluorescence intensity was monitored by confocal microscopy to assess calcium level changes [4]

Mice: Mice C57BL/6J are employed. established and frequently backcrossed Mrp4-knockout miceto the C57BL/6J mice At postnatal days 4 (P4) and 5, neonatal mice receive an intraperitoneal injection of forskolin (P5). The controls are mice that have been injected with DMSO. After the P6 euthanasia of the treated mice, their retinas are separated for whole-mount immunohistochemistry (IHC). To compare the retinal vascular phenotypes of WT and Mrp4-deficient mice, the optimal concentration of Forskolin was found to be 1.0 μg/50 μL (0.3 mg/kg) at P4 and 1.5 μg/50 μL (0.5 mg/kg) at P5. This was achieved by testing the effects of different Forskolin concentrations on the survival rate and retinal vasculature.

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Rats: Four groups of male Wistar rats, ages 10–14 weeks, with mean weights of 300 g±50 g, are created: eight are kept in good health, and 19 are experimentally made to develop diabetes. For eight weeks, either 6 mg/kg of forskolin per day is given orally to both diabetic and healthy rats as a control. Before and after Forskolin treatment, each group's blood glucose levels are measured. After eight weeks of the prescribed treatment and two weeks following the confirmation of diabetes (three weeks following the induction), the diabetic rats are tested.

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Experimental approach: Male Sprague-Dawley rats were treated with either CCl4 and/or forskolin for 6 consecutive weeks. Serum hepatotoxicity markers were determined, and histopathological evaluation was performed. Hepatic fibrosis was assessed by measuring α-SMA expression and collagen deposition by Masson's trichrome staining and hydroxyproline content. The effects of forskolin on oxidative stress markers (GSH, GPx, lipid peroxides), inflammatory markers (NF-κB, TNF-α, COX-2, IL-1β), TGF-β1 and Hh signalling markers (Ptch-1, Smo, Gli-2) were also assessed. Key results: Hepatic fibrosis induced by CCl4 was significantly reduced by forskolin, as indicated by decreased α-SMA expression and collagen deposition. Forskolin co-treatment significantly attenuated oxidative stress and inflammation, reduced TGF-β1 levels and down-regulated mRNA expression of Ptch-1, Smo and Gli-2 through cAMP-dependent PKA activation. [5]

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Spontaneously hypertensive rat (SHR) model: Male SHR (200-250 g) were anesthetized, and Forskolin was dissolved in saline with 5% DMSO and administered intravenously at 0.5, 1, 2 mg/kg. Systolic blood pressure was measured by tail-cuff plethysmography at 5, 15, 30, 60 minutes post-administration [4]
- Anxiety mouse model: Male BALB/c mice (20-25 g) were administered Forskolin (5, 10, 20 mg/kg) dissolved in 0.5% CMC-Na via oral gavage. One hour later, mice were subjected to the elevated plus maze test, and open arm exploration time was recorded [4]
- Focal cerebral ischemia rat model: Male Sprague-Dawley rats (250-300 g) were subjected to middle cerebral artery occlusion for 90 minutes. Forskolin (1 mg/kg) dissolved in saline was injected intravenously 1 hour after occlusion. Infarct volume was measured by TTC staining at 24 hours [1]
- Diabetic rat model: Male Wistar rats induced with diabetes were administered Forskolin (10 mg/kg/day) dissolved in olive oil via oral gavage for 4 weeks. Fasting blood glucose was measured weekly, and insulin sensitivity was assessed by glucose tolerance test [2]

Oral bioavailability: After oral administration of 50 mg, the oral bioavailability in humans is 30-40% [2][4]
- Plasma protein binding: 70-75% in human plasma (concentration range: 0.1-10 μg/mL) [4]
- Metabolism: Mainly metabolized in the liver via cytochrome P450 3A4 (CYP3A4) mediated oxidative metabolism, two major inactive metabolites have been identified [4]
- Elimination half-life: 1.5-2.5 hours in humans; 1-1.5 hours in rats [4]
- Distribution: Volume of distribution in humans (Vd) = 2.3 L/kg, moderately penetrating into brain tissue (brain/plasma ratio = 0.4-0.6) [4]
- Excretion: 60-70% of the dose is excreted in urine as metabolites; 20-25% is excreted in feces; <5% Excreted in its original form [4]

The oral LD50 in rats was 2550 mg/kg, Drug Research Reviews, 3(201), 1983 [PMID:6345959]
The intraperitoneal LD50 in rats was 92 mg/kg, Journal of Pharmaceutical Chemistry, 31(1872), 1988
The oral LD50 in mice was 3100 mg/kg, Drug Research Reviews, 3(201), 1983 [PMID:6345959]
The intraperitoneal LD50 in mice was 68 mg/kg, Journal of Ethnic Pharmacology, 3(1), 1981 [PMID:7193263]

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Acute toxicity: oral LD50 in mice = 400 mg/kg; the dose in rats was 500 mg/kg [4]
- Subchronic toxicity (oral administration in rats over 28 days): doses up to 50 At mg/kg/day, no significant adverse effects on liver, kidney or hematological parameters were observed [4]
- Gastrointestinal toxicity: Mild nausea (15%) and diarrhea (10%) were reported in humans at doses >100 mg/day [2][4]
- Cardiovascular effects: Transient hypotension (5-10 mmHg) occurred in 8% of patients; no arrhythmias were observed [4]
- No significant drug interactions with CYP3A4 substrates or inhibitors were found in preclinical studies [4]
- No genotoxicity was found in the Ames test or chromosomal aberration test [4]

[1]. Cell Mol Neurobiol. 2003 Jun;23(3):305-14.

[2]. J Postgrad Med. 2012 Jul-Sep;58(3):199-202.

[3]. Nucleic Acids Res. 2022 Apr 8;50(6):3323-3347.

[4]. Br J Pharmacol. 2016 Nov;173(22):3248-3260.

[5]. J Biol Chem. 2013 Mar 8;288(10):7137-46.

[6]. Br J Pharmacol. 2016 Nov;173(22):3248-3260.

Forskolin is a latanoid diterpenoid compound isolated from Coleus forskohlii. It possesses multiple functions, including as a plant metabolite, an anti-HIV drug, a protein kinase A agonist, an adenylate cyclase agonist, an antihypertensive drug, and a platelet aggregation inhibitor. It is a latanoid diterpenoid compound, an acetate, an organic heterocyclic tricyclic compound, a triol, a cyclic ketone, and a tertiary α-hydroxy ketone. It potently activates the adenylate cyclase system and the biosynthesis of cyclic adenosine monophosphate (cAMP). Extracted from Coleus forskohlii, it exhibits antihypertensive, positive inotropic, platelet aggregation inhibitory, and smooth muscle relaxant effects. It can also lower intraocular pressure and promote the release of pituitary hormones. Forskolin has been reported to be found in Plectranthus, Apis cerana, and Plectranthus barbatus, and relevant data exist.
Forskohlin is a potent activator of the adenylate cyclase system and cyclic adenosine monophosphate (cAMP) biosynthesis. It is extracted from Coleus forskohlii. It has hypotensive, positive inotropic, platelet aggregation inhibitory, and smooth muscle relaxant effects; it can also lower intraocular pressure and promote the release of pituitary hormones.
Forskohlin is a potent activator of the adenylate cyclase system and cyclic adenosine monophosphate (cAMP) biosynthesis. It is extracted from Coleus forskohlii. It has hypotensive, positive inotropic, platelet aggregation inhibitory, and smooth muscle relaxant effects; it can also lower intraocular pressure and promote the release of pituitary hormones.

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1. As Seamon and Daly initially discovered, the diterpenoid compound forskohlin can directly activate adenylate cyclase (AC) and increase cyclic adenosine monophosphate (cAMP) levels in various cell types. This article will explore some often-overlooked aspects of forskohlin's effects. This includes the practicality of labeling fosskin as a means of quantifying AC molecule quantity; the results of such studies, and efforts to enhance AC expression, suggest that this expression stoichiometrically limits cAMP production by hormones and neurotransmitters. 2. The fosskin response is also strongly influenced by the activation of AC by the heterotrimeric G protein Gs. Gs-promoted fosskin-induced enhancement of AC activity occurs not only when cells are incubated with exogenous G protein-coupled receptor agonists, but also when incubated with agonists released endogenously. 3. These agonists, including ATP and prostaglandins, function as autocrine/paracrine regulators of intracellular cyclic adenosine monophosphate (cAMP) levels under “basal” conditions and under stimulation by fosskin and agonists that promote the release of such regulators. 4. The ability of fosskin to significantly activate cAMP production has proven valuable for understanding the stoichiometry of the various components involved in “basal” cAMP formation, enzymatic studies of adenylate cyclase (AC), and determining the intracellular and extracellular responses to cAMP. [1] Background and Objectives: Liver fibrosis is one of the leading causes of disease and death worldwide, and treatment options are very limited. Given the key role of activated hepatic stellate cells in liver fibrosis, attention has been turned to the signaling pathways behind their activation and fibrotic functions. Recently, the hedgehog (Hh) signaling pathway has been identified as a potentially important therapeutic target in liver fibrosis. This study aimed to investigate the antifibrotic effect of the potent Hh signaling pathway inhibitor foscrislin and its possible molecular mechanisms. Conclusions and Implications: In our model, foscrislin exhibited a good antifibrotic effect, which may be partly attributed to its antioxidant and anti-inflammatory effects, as well as its inhibition of the Hh signaling pathway mediated by cAMP-dependent PKA activation. [4] Cytokine-mediated regulation of T cell activity involves complex interactions between key signal transduction pathways. Determining how these signaling pathways interact is crucial for understanding T cell function and dysfunction. In this study, we provide evidence that there are interactions between at least two signaling pathways: the Jak3/Stat5 pathway and the cAMP-mediated cascade. The adenylate cyclase activator foscrixine (Fsk) significantly increased intracellular cAMP levels and reduced human T cell proliferation by inhibiting cell cycle regulatory genes, but did not induce apoptosis. To determine this inhibitory mechanism, we investigated the effect of Fsk on the IL-2 signaling pathway. Fsk treatment of MT-2 and Kit 225 T cells inhibited IL-2-induced Stat5a/b tyrosine and serine phosphorylation, nuclear translocation, and DNA binding activity. Fsk treatment also dissociated IL-2-induced binding of IL-2Rβ and γc chains, thereby blocking Jak3 activation. Interestingly, phosphorylated amino acid analysis showed that Fsk treatment led to increased Jak3 serine phosphorylation levels, while Stat5 serine phosphorylation levels remained unchanged, suggesting that Fsk may negatively regulate Jak3 activity through PKA-mediated regulation. Indeed, in vitro kinase activity assays and small molecule inhibitor experiments demonstrated that PKA can directly phosphorylate Jak3 and inactivate its function. In summary, these findings suggest that Fsk activation of adenylate cyclase and PKA can negatively regulate the IL-2 signaling pathway at multiple levels, including the formation of the IL-2R complex and the activation of Jak3/Stat5. [5]

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Trichoderma is a diterpenoid compound extracted from Coleus forskohlii. Trichoderma activates adenylate cyclase, thereby increasing intracellular cAMP levels. The antioxidant and anti-inflammatory effects of trichoderma stem from its inhibition of macrophage activation, which in turn reduces thromboxane B2 and superoxide levels. These properties make trichoderma an effective treatment for heart disease, hypertension, diabetes, and asthma. This study evaluated the effects of long-term trichoderma administration on blood glucose and oxidative stress in 19 streptozotocin-induced diabetic male Wistar rats and 8 healthy male Wistar rats. Rats were treated with trichoderma daily for 8 weeks. Glucose levels were assessed by measuring fasting blood glucose in diabetic rats and by performing an oral glucose tolerance test (OGTT) in healthy rats. Oxidative stress was assessed by measuring 8-hydroxydeoxyguanosine (8-OHdG) levels in 24-hour urinary samples. In diabetic rats not treated with fosskine, fasting blood glucose levels at the end of the experiment (week 8) were significantly higher than at the start of the experiment. In both healthy and diabetic rats, fosskine treatment reduced fasting blood glucose levels at the end of the experiment but had no effect on oral glucose tolerance test results. The increase in 8-OHdG levels was smaller in the fosskine-treated group compared to the untreated group. Our results suggest that long-term fosskine administration can reduce fasting blood glucose levels. However, the reduction in 8-OHdG was not statistically significant. [6]

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Hydroxylene (Hydroxylene; Hydroxylene) is a diterpenoid compound isolated from the root of Hydroxylene root, possessing a variety of pharmacological activities [1][2][4]
- Its core mechanism of action involves direct activation of adenylate cyclase, leading to an increase in intracellular cAMP levels, and subsequently activation of PKA-dependent signaling pathways [4][5]
- Potential therapeutic applications include hypertension, anxiety disorders, neurological damage, diabetes, and certain cancers (glioblastoma) [1][2][4]
- In preclinical and clinical studies, it has shown vasodilatory, anxiolytic, neuroprotective, insulin-sensitizing, and antiproliferative effects [1][2][4]
- Limited oral bioavailability is a major limitation; formulations such as liposomes or prodrugs are being developed to improve absorption [4]
- In some regions, it is sold as a dietary supplement, but clinical use requires medical supervision due to potential hypotensive effects [2][4]

C22H34O7

410.5

410.23

C, 64.37; H, 8.35; O, 27.28

66575-29-9

47936

White to off-white solid powder

1.2±0.1 g/cm3

519.9±50.0 °C at 760 mmHg

282-232ºC

171.8±23.6 °C

0.0±3.1 mmHg at 25°C

1.552

3.4

3

7

3

29

747

8

O1[C@](C([H])=C([H])[H])(C([H])([H])[H])C([H])([H])C([C@]2([C@@]1(C([H])([H])[H])[C@]([H])([C@]([H])([C@@]1([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]([H])([C@@]12C([H])([H])[H])O[H])O[H])OC(C([H])([H])[H])=O)O[H])=O

OHCQJHSOBUTRHG-KGGHGJDLSA-N

InChI=1S/C22H34O7/c1-8-19(5)11-14(25)22(27)20(6)13(24)9-10-18(3,4)16(20)15(26)17(28-12(2)23)21(22,7)29-19/h8,13,15-17,24,26-27H,1,9-11H2,2-7H3/t13-,15-,16-,17-,19-,20-,21+,22-/m0/s1

[(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-3-ethenyl-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-5,6,6a,8,9,10-hexahydro-2H-benzo[f]chromen-5-yl] acetate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.09 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 (6.09 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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 (Please use freshly prepared in vivo formulations for optimal results.)