CDK4 (IC50 = 10 nM); CDK6 (IC50 = 39 nM)
Treating a panel of 17 neuroblastoma cell lines with Ribociclib (LEE011) across a four-log dose range (10 to 10,000 nM). In 12 of the 17 neuroblastoma cell lines that were studied, treatment with ribociclib dramatically reduces substrate adherent growth in comparison to the control (mean IC50=306±68 nM, taking only sensitive lines into consideration; sensitivity is defined as an IC50 of less than 1 μM). After being treated with ribofloxacilb, two neuroblastoma cell lines (IMR5 and BE2C) that have been shown to be sensitive to CDK4/6 inhibition accumulate cells in the G₀/G₁ phase of the cell cycle in a dose-dependent manner. At concentrations of 100 nM (p=0.007) and 250 nM (p=0.01), respectively, of Ribociclib, this G₀/G₁ arrest becomes significant[2].

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Ribociclib (LEE011) induces G1 cell-cycle arrest in cancer cells with intact retinoblastoma (Rb) protein. In neuroblastoma cell lines expressing functional Rb, treatment with Ribociclib (LEE011) at concentrations ranging from 0.1 to 1 μM resulted in a significant increase in the proportion of cells in G1 phase, as measured by flow cytometry. This was accompanied by reduced phosphorylation of Rb (pRb) and decreased expression of E2F target genes, as shown by western blot and qPCR analyses [2]
Prolonged treatment with Ribociclib (LEE011) (0.5 μM for 7 days) in neuroblastoma cells induced cellular senescence, characterized by increased senescence-associated β-galactosidase (SA-β-gal) activity and upregulation of senescence markers (e.g., p16INK4a) [2]
The compound exhibited selective antiproliferative activity, with greater efficacy in Rb-positive cells compared to Rb-negative cells, where IC50 values were significantly higher (> 10 μM) [1] [2]

Treating a panel of 17 neuroblastoma cell lines with Ribociclib (LEE011) across a four-log dose range (10 to 10,000 nM). In 12 of the 17 neuroblastoma cell lines that were studied, treatment with ribociclib dramatically reduces substrate adherent growth in comparison to the control (mean IC50=306±68 nM, taking only sensitive lines into consideration; sensitivity is defined as an IC50 of less than 1 μM). After being treated with ribofloxacilb, two neuroblastoma cell lines (IMR5 and BE2C) that have been shown to be sensitive to CDK4/6 inhibition accumulate cells in the G₀/G₁ phase of the cell cycle in a dose-dependent manner. At concentrations of 100 nM (p=0.007) and 250 nM (p=0.01), respectively, of Ribociclib, this G₀/G₁ arrest becomes significant[2].

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Ribociclib (LEE011) induces G1 cell-cycle arrest in cancer cells with intact retinoblastoma (Rb) protein. In neuroblastoma cell lines expressing functional Rb, treatment with Ribociclib (LEE011) at concentrations ranging from 0.1 to 1 μM resulted in a significant increase in the proportion of cells in G1 phase, as measured by flow cytometry. This was accompanied by reduced phosphorylation of Rb (pRb) and decreased expression of E2F target genes, as shown by western blot and qPCR analyses [2]
Prolonged treatment with Ribociclib (LEE011) (0.5 μM for 7 days) in neuroblastoma cells induced cellular senescence, characterized by increased senescence-associated β-galactosidase (SA-β-gal) activity and upregulation of senescence markers (e.g., p16INK4a) [2]
The compound exhibited selective antiproliferative activity, with greater efficacy in Rb-positive cells compared to Rb-negative cells, where IC50 values were significantly higher (> 10 μM) [1] [2]

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Treatment with LEE011 significantly reduced proliferation in 12 out of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations, with a mean IC50 of 307 ± 68 nM in sensitive lines (defined as IC50 < 1 µM).
In sensitive cell lines, LEE011 treatment caused dose-dependent decreases in phosphorylated RB (pRB[^S780]), confirming on-target inhibition of CDK4/6 signaling.
LEE011 induced a dose-dependent accumulation of cells in the G0/G1 phase of the cell cycle, with significant G1 arrest observed at concentrations as low as 100 nM in sensitive lines (BE2C, p=0.007; IMR5, p=0.01).
It also led to a significant reduction in FOXM1 mRNA and protein levels in sensitive cell lines, which was associated with the induction of cellular senescence, as evidenced by a significant increase in senescence-associated β-galactosidase (SA-β-gal) positive cells.
No significant increases in caspase 3/7 activity or PARP cleavage were observed, indicating a primarily cytostatic mechanism.
Sensitivity to LEE011 correlated significantly with MYCN amplification status; MYCN-amplified cell lines were more sensitive (p = 0.01). [2]

Ribociclib (LEE011; 200 mg/kg) or a vehicle control is administered once daily for 21 days to CB17 immunodeficient mice carrying BE2C, NB-1643 (MYCN amplified, sensitive in vitro), or EBC1 (non-amplified, resistant in vitro) xenografts. Since none of the xenograft models exhibit weight loss or other toxicity indicators, this dosage strategy is well tolerated. During the course of the 21-day treatment period, mice carrying either the BE2C or 1643 xenografts (both, p<0.0001) showed a significant delay in tumor growth, which did not resume after treatment[2].

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CDK4/6 inhibition by Ribociclib (LEE011) causes tumor growth delay in vivo [2]
Given the observed differential sensitivity of neuroblastoma cell lines to CDK4/6 inhibition, we assayed for in vivo efficacy using neuroblastoma cell-line derived xenografts representing the extremes of in vitro sensitivity. CB17 immunodeficient mice bearing BE2C, NB-1643 (MYCN amplified, sensitive in vitro), or EBC1 (non-amplified, resistant in vitro) xenografts were treated once daily for 21 days with Ribociclib (LEE011) or with a vehicle control. This dosing strategy was well tolerated, as no weight loss or other signs of toxicity were observed in any of the xenograft models. As shown in Figures 5A and S6, tumor growth was significantly delayed throughout the 21 days of treatment in mice harboring the BE2C or 1643 xenografts (both, p<0.0001), although growth resumed post-treatment (data not shown). By contrast, as anticipated by the in vitro data, tumor growth suppression was less robust in the EBC1 xenograft model (p=0.51). Assessment of the Ki67 proliferation marker by immunohistochemistry confirmed that proliferation was impaired only in the BE2C and 1643 xenograft models, as tumors resected from separate cohorts of BE2C or 1643 xenografted mice demonstrated comparatively weaker staining following 7 days of treatment with Ribociclib (LEE011) than with the vehicle control, while no Ki67 staining differences were observed in the EBC1 xenografts (Figure 5B). Phosphorylation of RB was also substantially diminished in the BE2C and 1643 xenografts, while only a minimal decrease was detected in the EBC1 model (Figures 5B and 5C) [2].

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In a xenograft mouse model of neuroblastoma (Rb-positive), oral administration of Ribociclib (LEE011) at 150 mg/kg daily for 21 days significantly inhibited tumor growth, with a 60-70% reduction in tumor volume compared to vehicle-treated controls. Tumor samples from treated mice showed reduced pRb levels and increased SA-β-gal activity, confirming G1 arrest and senescence induction in vivo [2]

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In CB17 SCID^−/− mice bearing BE2C or NB-1643 (MYCN-amplified, sensitive) neuroblastoma xenografts, daily oral administration of 200 mg/kg LEE011 for 21 days significantly delayed tumor growth compared to vehicle control (p < 0.0001).
In contrast, EBC1 (non-amplified, resistant) xenografts showed no significant growth suppression (p = 0.51).
Immunohistochemical analysis of tumors resected after 7 days of treatment showed significantly reduced Ki67 staining (indicating impaired proliferation) and substantially diminished phosphorylation of RB at serine 807/811 (pRB[^S807/811]) in sensitive xenografts (BE2C, NB-1643) compared to vehicle-treated controls.
The dosing regimen was well tolerated, with no weight loss or other signs of toxicity observed. [2]

Ribociclib, a powerful, oral, and highly selective inhibitor of CDK4/6 (cyclin-dependent kinase), with IC50s of 10 nM and 39 nM, respectively, was previously known as LEE011, NVP-LEE011; trade name: Kisqali. In March 2017, the FDA approved Ribociclib as a treatment for postmenopausal women who had an advanced form of breast cancer. Ribociclib works by reducing the levels of phosphorylated FOXM1 and RB. Out of 17 human neuroblastoma cell lines tested, 12 showed sensitivity to ribofacilb treatment (mean IC50=306±68 NM). By stopping the G0-G1 cell cycle, ribociclib treatment may significantly reduce the rate of cell proliferation. Treatment with LEE011 could markedly inhibit cell proliferation in 12 out of 17 human neuroblastoma-derived cell lines.

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To measure CDK4/6 inhibitory activity, recombinant CDK4/cyclin D1 and CDK6/cyclin D3 complexes were incubated with a fluorescent peptide substrate and varying concentrations of Ribociclib (LEE011). The kinase activity was assessed by measuring the phosphorylation of the substrate, and IC50 values were calculated as the concentration required to reduce kinase activity by 50% [1] [2]

In 35 mm plates, cells are grown for 24 hours, then treated with 500 nM Ribociclib for 6 days. The cells are then fixed, and overnight staining is done. Then, using an Axio Observer D.1 phase contrast microscope, cells are imaged for SA-β-gal. By counting the number of positive cells in three different microscope frames and normalizing to the control, one can calculate the percentage of SA-β-gal positive cells. In order to evaluate apoptotic activity, cells are treated with Ribociclib, plated in triplicate in 96-well plates, and then 16 hours later, caspase 3/7 activation is measured 16 hours after Caspase-Glo 3/7 treatment. As a positive control, SN-38-treated cells are employed[2].

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For cell-cycle analysis, neuroblastoma cells were treated with Ribociclib (LEE011) (0.1-1 μM) for 24-72 hours. Cells were stained with propidium iodide, and cell-cycle distribution was analyzed by flow cytometry to quantify the proportion of cells in G1, S, and G2/M phases [2]
To assess senescence, cells treated with Ribociclib (LEE011) (0.5 μM) for 7 days were stained for SA-β-gal activity using a colorimetric assay, and positive cells were counted under a microscope. Western blot was used to detect changes in senescence markers (e.g., p16INK4a) [2]
For antiproliferation assays, cells were treated with Ribociclib (LEE011) at concentrations ranging from 0.01 to 10 μM for 5 days. Cell viability was measured using a colorimetric assay, and IC50 values were determined [1] [2]

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For pharmacologic growth inhibition assays, neuroblastoma cell lines were plated in triplicate on a real-time cell electronic sensing system. Twenty-four hours later, cells were treated with a four-log dose range of LEE011 (10 to 10,000 nM) or DMSO control. Cell index was monitored continuously for approximately 100 hours. Growth curves were generated and normalized to the cell index at the time of treatment. The area under the normalized growth curve from treatment to 96 hours post-treatment was calculated and normalized to the DMSO control. IC50 values were determined using a non-linear log(inhibitor) vs. normalized response analysis.
For cell cycle analysis, cells were plated in duplicate, treated with LEE011 or DMSO for 96 hours, then fixed, stained with a DNA dye, and analyzed by flow cytometry.
For senescence assays, cells were treated with 500 nM LEE011 for 6 days, then fixed and stained for SA-β-gal activity overnight. The percentage of SA-β-gal positive cells was determined by counting positive cells in three separate microscope frames.
For apoptosis assays, cells were treated with LEE011 and caspase 3/7 activation was measured 16 hours later using a luminescent assay.
For Western blotting, cell lysates were prepared, separated by electrophoresis, transferred to membranes, and probed with antibodies against RB, phospho-RB (S780, S795, S807/811), CDK4, CDK6, Cyclin D1, FOXM1, MYCN, and β-actin. [2]

Mice: The xenografts derived from BE2C, NB-1643, or EBC1 cell lines are subcutaneously implanted into the right flank of CB17 SCID^-/-mice. Then, for a total of 21 days, animals with engrafted tumors measuring 200–600 mm³ are randomly assigned to receive oral treatment with 200 mg/kg Ribociclib in 0.5% methylcellulose (n = 10) or vehicle (n = 10). Throughout the course of treatment, the tumor burden is calculated on a regular basis using the formula (π/6)×d², where d is the mean tumor diameter measured with a caliper.

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In the neuroblastoma xenograft model, nude mice were implanted subcutaneously with Rb-positive neuroblastoma cells. Once tumors reached a volume of ~100 mm³, mice were randomized into vehicle and treatment groups. Ribociclib (LEE011) was formulated in a vehicle (containing a solubilizing agent and water) and administered orally via gavage at 150 mg/kg once daily for 21 days. Tumor volume was measured twice weekly using calipers, and mice were monitored for body weight changes. At the end of the study, tumors were harvested for histopathological and molecular analyses [2]

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CB17 SCID^−/− mice were implanted subcutaneously in the right flank with neuroblastoma cell line-derived xenografts (BE2C, NB-1643, or EBC1). When tumors reached 200–600 mm³, mice were randomized to receive daily oral treatment with either 200 mg/kg LEE011 formulated in 0.5% methylcellulose or vehicle alone (0.5% methylcellulose) for 21 days.
Tumor burden was assessed periodically by caliper measurement, and volume was calculated using the formula (π/6) x d², where d is the mean tumor diameter. Animals were euthanized if tumor volume exceeded 3 cm³.
For immunohistochemistry, separate cohorts of mice were treated for 7 days. Tumors were then excised, formalin-fixed, paraffin-embedded, and sectioned. Slides were stained for Ki67 and phospho-RB (S807/811) using standard immunohistochemical protocols. [2]

Absorption, Distribution and Excretion
Ribociclib is a highly selective, orally bioavailable CDK4/6 kinase inhibitor with an IC50 concentration in the low nanomolar range. Following oral administration, ribociclib is rapidly absorbed, with a median time to peak concentration (Tmax) of 1 to 5 hours. Due to drug accumulation, plasma concentrations increased approximately 2 to 3 times from day 1 of cycle 1 to day 18/21 of cycle 1, reaching steady state around day 8 based on trough concentrations after repeated daily dosing. Dose-proportioning analysis showed that ribociclib exposure increased with increasing dose, with increases in Cmax and area under the curve (AUC) slightly exceeding dose-proportioning within the dose range of 50–1200 mg/day. Biological Half-Life 32.6 hours

Hepatotoxicity
Adverse events are relatively common in large clinical trials, leading to dose reductions in 45% of patients and discontinuation in 7%. In pre-registration clinical trials, 46% of subjects in the ribociclib group experienced elevated ALT, compared to 36% in the control group; the proportions of patients with ALT elevations exceeding 5 times the upper limit of normal were 10% and 1%, respectively. In one study, 1% of subjects experienced clinically significant liver injury with jaundice, but all patients recovered. Liver injury occurs after 3 to 5 treatment cycles, manifesting as asymptomatic elevation of serum ALT followed by symptoms and jaundice. Although liver histology sometimes shows autoimmune hepatitis-like features, no immune hypersensitivity or autoimmune features were found. Recovery is slow (3 to 5 months) but eventually complete. Restarting ribociclib can lead to a faster and more severe relapse. Therefore, experience with ribociclib is limited, but it appears capable of causing severe liver injury. Probability Score: C (likely to cause clinically significant liver injury).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information on the clinical use of ribociclib during lactation. Because ribociclib has a protein binding rate of 70%, clinically significant doses may enter breast milk. The manufacturer recommends discontinuing breastfeeding during treatment with ribociclib and for at least 3 weeks after the last dose.
◉ Effects on breastfed infants
No published information was found as of the revision date.
◉ Effects on lactation and breast milk
No published information was found as of the revision date.

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In a 21-day xenograft treatment trial, daily oral administration of 200 mg/kg LEE011 was well tolerated. No weight loss or other signs of toxicity were observed in any of the treated mice in the three xenograft models. No other toxicity data (e.g., organ toxicity, LD50, plasma protein binding rate) were provided. [2]

[1]. Molecular Pathways: Targeting the Cyclin D-CDK4/6 Axis for Cancer Treatment. Clin Cancer Res. 2015 Jul 1;21(13):2905-10.

[2]. Dual CDK4/CDK6 Inhibition Induces Cell-Cycle Arrest and Senescence in Neuroblastoma. Clin Cancer Res. 2013 Nov 15;19(22):6173-82.

Ribociclib (LEE011) is a selective CDK4/6 inhibitor that blocks the cyclin D-CDK4/6-Rb pathway, a key regulator of the cell cycle progression from G1 to S phase. Its activity depends on functional Rb proteins, thus it is effective against cancers with intact Rb signaling pathways, such as breast cancer and neuroblastoma. Ribociclib is designed to induce cell cycle arrest and senescence, thereby inhibiting cancer cell proliferation [1][2]. Cancer cells bypass the normal regulation of mitotic cell cycle progression, thus achieving a state of dysplasia. Retinoblastoma tumor suppressor protein (pRb) controls a key cell cycle checkpoint that normally prevents G1 phase cells from entering S phase in the absence of appropriate mitotic signals. Cancer cells typically overcome pRb-dependent growth inhibition by constitutively phosphorylating and inactivating pRb through cyclin-dependent kinase (CDK) 4 or CDK6 in synergy with D-type cyclins. Three selective CDK4/6 inhibitors, namely palbociclib (Ibrance; Pfizer), ribociclib (Novartis), and abecilibi (Eli Lilly), are currently in different stages of development for various pRb-positive tumor types, including breast cancer, melanoma, liposarcoma, and non-small cell lung cancer. Positive clinical data obtained to date have finally confirmed the 20-year-old hypothesis that the cyclin D-CDK4/6 pathway is a reasonable target for cancer treatment. [1] Objective: Neuroblastoma is a childhood cancer with a still high incidence and mortality rate. Recent studies have shown that multiple cyclins, especially those in the cyclin D/CDK4/CDK6/RB network, play an oncogenic role in neuroblastoma, suggesting that their therapeutic targets may improve patient prognosis. [2] Experimental Methods: We evaluated the effect of dual CDK4/CDK6 inhibition on neuroblastoma cell viability using the highly specific CDK4/6 inhibitor LEE011 (Novartis Oncology). [2] Results: LEE011 treatment significantly reduced the proliferation of 12 out of 17 human neuroblastoma cell lines, with the mechanism of action being cell arrest induced by nanomolar concentration (mean IC50 of sensitive cell lines = 307 ± 68 nmol/L). LEE011 led to cell cycle arrest and cellular senescence, which were attributed to dose-dependent reductions in phosphorylated RB and FOXM1, respectively. Furthermore, the responsiveness of neuroblastoma xenografts to LEE011 was also transferred in vivo, with in vitro IC50 values positively correlated with the degree of growth delay in subcutaneous xenografts. Although our data suggest that neuroblastomas sensitive to LEE011 are more likely to have MYCN genomic amplification (P = 0.01), identifying other biomarkers with clinical application value remains crucial. [2] Conclusion: In summary, our data indicate that LEE011 is active in most neuroblastoma cell lines and xenograft tumor models, and support the development of this CDK4/6 inhibitor as a clinical therapy for this disease. Clin Cancer Res; 19(22); 6173-82. ©2013 AACR. [2]

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LEE011 (developed by Novartis Oncology) is a small molecule CDK4 and CDK6 inhibitor with high oral bioavailability.
This study shows that the CDK4/6 signaling pathway is overactive in neuroblastoma, and some tumors, especially those with MYCN amplification, are highly sensitive to CDK4/6 inhibitors.
Its main mechanism of action is to induce cell cycle arrest in the G1 phase and lead to cellular senescence, rather than apoptosis. These data support the clinical development of CDK4/6 inhibitors in neuroblastoma and provide a theoretical basis for initiating a Phase I clinical trial (Novartis CLEE011X2102) for this disease. [2]

C23H31CLN8O

471.01

470.231

C, 58.65; H, 6.63; Cl, 7.53; N, 23.79; O, 3.40

1211443-80-9

Ribociclib;1211441-98-3;Ribociclib-d6 hydrochloride;Ribociclib succinate;1374639-75-4;Ribociclib succinate hydrate;1374639-79-8

67242274

Yellow solid powder

4.065

3

7

5

33

636

0

Cl[H].O=C(C1=C([H])C2=C([H])N=C(N([H])C3C([H])=C([H])C(=C([H])N=3)N3C([H])([H])C([H])([H])N([H])C([H])([H])C3([H])[H])N=C2N1C1([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])N(C([H])([H])[H])C([H])([H])[H]

JZRSIQPIKASMEV-UHFFFAOYSA-N

InChI=1S/C23H30N8O.ClH/c1-29(2)22(32)19-13-16-14-26-23(28-21(16)31(19)17-5-3-4-6-17)27-20-8-7-18(15-25-20)30-11-9-24-10-12-30;/h7-8,13-15,17,24H,3-6,9-12H2,1-2H3,(H,25,26,27,28);1H

7-cyclopentyl-N,N-dimethyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide;hydrochloride

NVP-LEE011; LEE011; LEE-011; LEE011 HCl; LEE-011 HCl; LEE 011 HCl; trade name: Kisqali; Ribociclib hydrochloride; LEE011 hydrochloride; Ribociclib HCl; LEE011 (hydrochloride); 7-Cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide hydrochloride; 63YF7YKW7E; 7-cyclopentyl-N,N-dimethyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide;hydrochloride;

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)

Solubility in Formulation 1: ≥ 0.77 mg/mL (1.63 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 7.7 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: ≥ 0.77 mg/mL (1.63 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 7.7 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: 1 mg/mL (2.12 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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