Niraparib (formerly known as MK4827; MK-4827; Zejula) is a potent, orally bioavailable and selective inhibitor of PARP1/2 with anticancer activity. It inhibits PARP1/2, with IC50 values for PARP 1 and 2 of 3.8 nM and 2.1 nM, respectively. On March 27, 2017, the US FDA approved niraparib for the maintenance of patients who are responding either completely or partially to platinum-based chemotherapy and have recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer. In cancer cells exhibiting mutated BRCA-1 and BRCA-2, niraparib exhibited significant efficacy, with an IC50 within the 10−100 nM range. It has a >330-fold selectivity against Tank1, V-PARP, and PARP3. MK-4827 inhibited PARP activity in a whole cell assay, with an EC50 of 4 nM. Moreover, MK-4827 slowed the growth of cancer cells harboring mutated BRCA-1 and BRCA-2, with CC50 values between 10 and 100 nM. On a range of human tumor xenografts, including p53 wild type and p53 mutant tumors, MK-4827 significantly increased the impact of radiation.

Physicochemical Properties

Molecular Formula	C19H20N4O
Molecular Weight	320.39
Exact Mass	320.163
Elemental Analysis	C, 71.23; H, 6.29; N, 17.49; O, 4.99
CAS #	1038915-60-4
Related CAS #	1038915-60-4; 1038915-73-9; 1038915-64-8 (HCl); 1613220-15-7 (tosylate hydrate); 1476777-06-6 (Niraparib metabolite M1); 1038915-58-0 (Niraparib R-enantiomer)
PubChem CID	24958200
Appearance	Light yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	463.6±45.0 °C at 760 mmHg
Flash Point	234.2±28.7 °C
Vapour Pressure	0.0±1.1 mmHg at 25°C
Index of Refraction	1.705
LogP	2.85
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	3
Heavy Atom Count	24
Complexity	449
Defined Atom Stereocenter Count	1
SMILES	O=C(C1=C([H])C([H])=C([H])C2C1=NN(C=2[H])C1C([H])=C([H])C(=C([H])C=1[H])[C@@]1([H])C([H])([H])N([H])C([H])([H])C([H])([H])C1([H])[H])N([H])[H]
InChi Key	PCHKPVIQAHNQLW-CQSZACIVSA-N
InChi Code	InChI=1S/C19H20N4O/c20-19(24)17-5-1-3-15-12-23(22-18(15)17)16-8-6-13(7-9-16)14-4-2-10-21-11-14/h1,3,5-9,12,14,21H,2,4,10-11H2,(H2,20,24)/t14-/m1/s1
Chemical Name	2-[4-[(3S)-piperidin-3-yl]phenyl]indazole-7-carboxamide
Synonyms	MK-4827; 1038915-60-4; (S)-2-(4-(piperidin-3-yl)phenyl)-2H-indazole-7-carboxamide; UNII-HMC2H89N35; MK 4827 (Base); Niraparib [USAN]; MK 4827; Niraparib free base; Zejula; MK4827;
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	PARP-2 ( IC50 = 2.1 nM ); PARP-1 ( IC50 = 3.8 nM ); V-PARP ( IC50 = 330 nM ); TANK-1 ( IC50 = 570 nM ); PARP-3 ( IC50 = 1300 nM )
ln Vitro	Niraparib (MK-4827) blocks PARP activity with EC50=4 nM and EC90=45 nM in a whole cell assay. MK-4827 has a CC50 in the 10-100 nM range, which inhibits the growth of cancer cells containing mutant BRCA-1 and BRCA-2. MK-4827 shows good inhibition of PARP 1 and 2 in a whole cell assay (IC50=3.8 and 2.1 nM, respectively)[1]. In order to verify that Niraparib (MK-4827) suppresses PARP in these cell lines, PARP enzymatic activity was measured using a chemiluminescent assay after A549 and H1299 cells were treated with 1 μM MK-4827 for varied durations. The findings demonstrate that niraparib (MK-4827) inhibits PARP within 15 minutes of treatment, with the A549 cells showing an 85% inhibition at 1 hour and the H1299 cells showing an approximately 55% inhibition at 1 hour[2].
ln Vivo	Niraparib (MK-4827) exhibits good tolerability and efficacy when used as a single agent in a xenograft model of cancer lacking BRCA-1. In a BRCA-1 deficient cancer xenograft model, niraparib (MK-4827) shows efficacy when used as a single agent and is well tolerated in vivo. With a plasma clearance of 28 (mL/min)/kg, a very high volume of distribution (Vdss=6.9 L/kg), a long terminal half-life (t1/2=3.4 h), and exceptional bioavailability (F=65%), niraparib (MK-4827) exhibits acceptable pharmacokinetics in rats[1]. In both situations, niraparib (MK-4827) improves the p53 mutant Calu-6 tumor's radiation response; a single daily dosage of 50 mg/kg is more beneficial than two doses of 25 mg/kg[3]. The in vivo efficacy of niraparib (MK-4827) was demonstrated preclinically in a BRCA-1 mutant MDA-MB-436 xenograft model (Figure 4), and 2 × 106 cells were injected subcutaneously in the right flank of 6-week-old nude CD1 female mice. When tumors reached an average volume of 150 mm3, mice were randomized to form homogeneous groups and treated with niraparib (MK-4827), dosing orally at either 100 mg/kg q.d. or 50 mg/kg b.i.d. Tumor regression was observed with both dosing regimes, and both were well tolerated, with no mortality. Less than 10% body weight loss was seen during the experiment. [1] The poly-(ADP-ribose) polymerase (PARP) inhibitor, MK-4827, is a novel potent, orally bioavailable PARP-1 and PARP-2 inhibitor currently in phase I clinical trials for cancer treatment. No preclinical data currently exist on the combination of MK-4827 with radiotherapy. The current study examined combined treatment efficacy of MK-4827 and fractionated radiotherapy using a variety of human tumor xenografts of differing p53 status: Calu-6 (p53 null), A549 (p53 wild-type [wt]) and H-460 (p53 wt) lung cancers and triple negative MDA-MB-231 human breast carcinoma. To mimic clinical application of radiotherapy, fractionated radiation (2 Gy per fraction) schedules given once or twice daily for 1 to 2 weeks combined with MK-4827, 50 mg/kg once daily or 25 mg/kg twice daily, were used. MK-4827 was found to be highly and similarly effective in both radiation schedules but maximum radiation enhancement was observed when MK-4827 was given at a dose of 50 mg/kg once daily (EF = 2.2). MK-4827 radiosensitized all four tumors studied regardless of their p53 status. MK-4827 reduced PAR levels in tumors by 1 h after administration which persisted for up to 24 h. This long period of PARP inhibition potentially adds to the flexibility of design of future clinical trials. Thus, MK-4827 shows high potential to improve the efficacy of radiotherapy [3].
Enzyme Assay	PARP-1 SPA Assay [1] Enzyme assay was conducted in buffer containing 25 mM Tris, pH 8.0, 1 mM DTT, 1 mM spermine, 50 mM KCl, 0.01% Nonidet P-40, and 1 mM MgCl2. PARP reactions contained 0.1 μCi [3H]NAD+ (200 000 DPM), 1.5 μM NAD+, 150 nM biotinylated NAD+, 1 μg/mL activated calf thymus, and 1−5 nM PARP-1. Autoreactions utilizing SPA bead-based detection were carried out in 50 μL volumes in white 96-well plates. Compounds were prepared in 11-point serial dilution in 96-well plate, 5 μL/well in 5% DMSO/H2O (10× concentrated). Reactions were initiated by adding first 35 μL of PARP-1 enzyme in buffer and incubating for 5 min at room temperature and then 10 μL of NAD+ and DNA substrate mixture. After 3 h at room temperature, these reactions were terminated by the addition of 50 μL of streptavidin-SPA beads (2.5 mg/mL in 200 mM EDTA, pH 8). After 5 min, they were counted using a TopCount microplate scintillation counter. IC50 data was determined from inhibition curves at various substrate concentrations. PARP Isoform TCA Assays [1] The enzymatic reaction was conducted in the presence of 25 mM Tris-HCl pH 8.0, 1 mM MgCl2, 50 mM KCl, 1 mM spermine, 0.01% Nonidet P-40, and 1 mM DTT. PARP reactions contained 0.1 μCi [3H]NAD (200 000 DPM), 1.5 μM NAD+, 1 μg/mL activated calf thymus, and 0.2−1 nM human PARP-1 enzyme. Assays were carried out in 50 μL volumes in white 96-well polypropylene microplate. A 96-well plate was prepared with serial dilutions over 10 points over a 0.1−50 nM concentration range 5% DMSO/H2O, 5 μL. Reactions were initiated by adding first 35 μL of PARP-1 enzyme in buffer and incubating for 5 min at room temperature, then 10 μL of NAD+ and DNA substrate mixture. After 2 h incubation at room temperature, the reaction was stopped by the addition of TCA (50 μL/well, 20% in 20 mM NaPPi solution) and incubated for 10 min over ice. The resulting precipitate was filtered on a Unifilter GF/B microplate and washed four times with 2.5% TCA. After addition of 50 μL/well of scintillation liquid the amount of radioactivity incorporated into the PAR polymers was determined using a TopCount microplate scintillation counter. IC50 data were determined from inhibition curves at various substrate concentrations. The protocols for the other PARP family members are very similar with subtle changes as described in the Supporting Information. PARylation Assay [1] HeLa cells were seeded into a 96-well Viewplate black microplate at an initial concentration of 10 000 cells/well in culture medium (100 μL of DMEM containing 10% FCS, 0.1 mg/mL penicillin−streptomycin, and 2 mM l-glutamine). The plates were incubated for 4 h at 37 °C under 5% CO2 atmosphere, and then compounds were added with serial dilutions over nine points over a 0.3−100 nM concentration range in 5% DMSO/H2O, 10 μL/well. The plate was then incubated for 18 h at 37 °C in 5% CO2, and then DNA damage was provoked by addition of 5 μL of H2O2 solution in H2O (final concentration 200 μM). As a negative control, cells untreated with H2O2 were used. The plate was kept at 37 °C for 5 min. Then the medium was gently removed by plate inversion, and the cells were fixed by addition of ice-cold MeOH (100 μL/well) and kept at −20 °C for 20 min. After removal of the fixative by plate inversion and washing 10 times with PBS (300 μL), the detection buffer (100 μL/well, containing PBS, Tween (0.05%), and BSA (1 mg/mL)) together with the primary PAR mAb (1:2000), the secondary antimouse Alexa Fluor 488 antibody (1:3000), and nuclear dye Draq5 (Alexis Bos 889001R200, 5 μM) were added. Following 3 h incubation at room temperature in the dark, removal of the solution, and washing 10 times with PBS (300 μL), the plate was read on an InCell1000. Monitoring for PAR polymer was by detection of Alexa488 at Ex. S 475_20X, Em. HQ 535_50, exposure time of 600 ms, and identification of the nuclei was by tracking Draq5 with Ex. HQ 620_60X, Em. HQ 700_75M, exposure time of 300 ms. The % PAR-positive cells was calculated by measuring the ratio between the numbers of PAR-positive nuclei over the total number of Draq5-labeled nuclei. The IC50 was determined on the basis of the residual enzyme activity in the presence of increasing PARPi concentration. In a whole cell assay, MK-4827 inhibits PARP activity with EC(50) = 4 nM and prevents the growth of cancer cells expressing mutant BRCA-1 and BRCA-2 with CC(50) in the 10-100 nM range. It also exhibits excellent inhibition of PARP 1 and 2 with IC(50) = 3.8 and 2.1 nM, respectively.
Cell Assay	Proliferation Assay in BRCA-1 Silenced and Wild Type HeLa Cells [1] HeLa BRCA1-silenced cells were generated by transducing HeLa cells at an MOI of 100 with a lentivirus containing an H1-derived expression cassette for a shRNA against BRCA-1 and an expression cassette for GFP (GFP under the control of EF1-a promoter). Silencing of BRCA1 was more than 80% as assessed by Taqman analysis. Control BRCA wild type HeLa cells were generated by transducing them with a lentivirus expressing GFP only. Proliferation assays were conducted in 96-well black viewplates, and 300 cells/well (250 cell/well for BRCA-1 wt) in culture medium, 190 μL/well (DMEM containing 10% FCS, 0.1 mg/mL penicillin−streptomycin, and 2 mM l-glutamine), were plated and incubated for 4 h at 37 °C under 5% CO2 atmosphere. Inhibitors were then added with serial dilutions, 10 μL/well to obtain the desired final compound concentration in 0.5% DMSO. The cells were then incubated for 7 days at 37 °C in 5% CO2 after which time viability was assessed. Briefly, with CellTiter-Blue (Promega) solution prediluted 1:10 in medium, 100 μL/well was added and the cells left for 45 min at 37 °C under 5% CO2 and then a further 15 min at room temperature in the dark. The number of living cells was determined by reading the plate at fluorimeter, excitation at 550 nm and emission at 590 nm. Cell growth was expressed as the percentage growth with respect to vehicle treated cells. The concentration required to inhibit cell growth by 50% (CC50) was determined. The protocols for the other cell lines are very similar and are described in the Supporting Information. In 96-well black viewplates, proliferation assays were carried out. 300 cells/well (250 cells/well for BRCA-1 wt) in culture medium, 190 μL/well (DMEM containing 10% FCS, 0.1 mg/mL penicillin-streptomycin, and 2 mM L-glutamine), were plated, and the cells were incubated for 4 hours at 37°C in an atmosphere of 5% CO2. Next, in 0.5% DMSO, inhibitors were added in serial dilutions of 10 μL/well until the final compound concentration was as desired. The viability of the cells was then evaluated after they had been incubated for 7 days at 37°C in 5% CO2. In brief, after adding 100 μL/well of prediluted 1:10 CellTiter-Blue solution to the medium, the cells were incubated for 45 minutes at 37°C with 5% CO2 and then for an additional 15 minutes at room temperature in the dark. By reading the plate at a fluorimeter, excitation at 550 nm, and emission at 590 nm, the number of living cells was ascertained. The percentage growth of the cells in comparison to the vehicle-treated cells was used to express the cell growth. It was established what concentration (CC50) was needed to stop cell growth by 50%.
Animal Protocol	The MDA-MB-436 human breast cancer cells (ATCC) were grown in RPMI 1640 medium with l-glutamine supplemented with 10% FCS, penicillin (100 U/mL), and streptomycin (100 μg/mL) in standard adherent culture conditions at 37 °C and 5% CO2. For establishment of xenograft tumors, cells were harvested from subconfluent cultures using EDTA/trypsin, washed in serum free-medium, and injected (2 × 106 cells) subcutaneously in the right flank of 6-week-old nude CD1 female mice in 100 μL total volume of 1:1 mix of cell suspension in serum-free media and RGF-Matrigel. When tumor reached an average volume of 150 mm3, mice were randomized to form homogeneous groups and treatment started, dosing orally. Mice were dosed orally in water (10 mL/kg) with 100 mg/kg q.d. or 50 mg/kg b.i.d. for 33 days, with tumor growth and body weight measurements done at least once a week. [1] Formulated in 0.5% Methocel in deionized water; 25 mg/kg twice daily or 50 mg/kg once daily; Oral gavage Female nude mice
ADME/Pharmacokinetics	Absorption, Distribution and Excretion Following a single-dose administration of 300 mg niraparib, the mean (±SD) peak plasma concentration (Cmax) was 804 (±403) ng/mL. The exposure (Cmax and AUC) of niraparib increased in a dose-proportional manner with daily doses ranging from 30 mg (0.1 times the approved recommended dose) to 400 mg (1.3 times the approved recommended dose). The accumulation ratio of niraparib exposure following 21 days of repeated daily doses was approximately 2-fold for doses ranging from 30 to 400 mg. The Tmax is about three hours. The absolute bioavailability of niraparib is approximately 73%. Food does not appear to affect drug exposure. Niraparib is eliminated via multiple pathways, including liver metabolism, hepatobiliary excretion, and renal elimination. Following administration of a single oral 300-mg dose of radio-labeled niraparib, the average percent recovery of the administered dose over 21 days was 47.5% (range: 33.4% to 60.2%) in urine and 38.8% (range: 28.3% to 47.0%) in feces. In pooled samples collected over 6 days, unchanged niraparib accounted for 11% and 19% of the administered dose recovered in urine and feces, respectively. The average (±SD) apparent volume of distribution (Vd/F) was 1,220 (±1,114) L. In a population pharmacokinetic analysis, the Vd/F of niraparib was 1,074 L in patients with cancer. In a population pharmacokinetic analysis, the apparent total clearance (CL/F) of niraparib was 16.2 L/h in patients with cancer. Metabolism / Metabolites Niraparib is primarily metabolized by carboxylesterases (CEs) to form M1, which is a major inactive metabolite. The M1 metabolite can subsequently undergo glucuronidation mediated by UDP-glucuronosyltransferases (UGTs) to form the M10 metabolite. In a mass balance study, M1 and M10 were the major circulating metabolites. The M1 metabolite can also undergo methylation, monooxygenation, and hydrogenation to form other minor metabolites. Biological Half-Life Following multiple daily doses of 300 mg of niraparib, the mean half-life (t1/2) is 36 hours.
Toxicity/Toxicokinetics	Hepatotoxicity In preregistration, randomized controlled clinical trials of niraparib, abnormalities in routine liver tests were common, but were mostly mild and self-limited in course. Serum ALT elevations occurred in 28% of patients (vs 15% of controls), but values were above 5 times the upper limit of normal (ULN) in only 1% (vs 2% of controls). Despite the frequency of serum enzyme elevations during therapy in clinical trials, there were no reports of hepatitis with jaundice or liver failure. Subsequent to its approval and more wide scale use, there have been no published reports of clinically apparent liver injury attributed to niraparib, but the extent and duration of its use have been limited. Thus, niraparib is a known cause of mild serum enzyme elevations but has not been linked to significant hepatotoxicity. Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on the clinical use of niraparib during breastfeeding. Because niraparib is 83% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during niraparib therapy and for 1 month following therapy. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding Niraparib is 83% bound to human plasma proteins.
References	[1]. Discovery of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide (MK-4827): a novel oral poly(ADP-ribose)polymerase (PARP) inhibitor efficacious in BRCA-1 and -2 mutant tumors. J Med Chem. 2009 Nov 26;52(22):7170-85. [2]. Niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase inhibitor, radiosensitizes human lung and breast cancer cells. Oncotarget. 2014 Jul 15;5(13):5076-86. [3]. MK-4827, a PARP-1/-2 inhibitor, strongly enhances response of human lung and breast cancer xenografts to radiation. Invest New Drugs. 2012 Dec;30(6):2113-20. [4]. Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer. N Engl J Med. 2016 Dec 1;375(22):2154-2164.
Additional Infomation	Pharmacodynamics Niraparib mediated cytotoxic effects in tumour cell lines with or without deficiencies in _BRCA1/2_. Decreased tumour growth was observed in both mouse xenograft models of human cancer cell lines with deficiencies in _BRCA1/2_ and human patient-derived xenograft tumour models with homologous recombination deficiency (HRD) that had either mutated or wild-type _BRCA1/2_. _In vitro_ studies suggest that niraparib inhibits dopamine, norepinephrine, and serotonin transporters, which may explain its off-target cardiovascular effects such as increased pulse rate and blood pressure. We disclose the development of a novel series of 2-phenyl-2H-indazole-7-carboxamides as poly(ADP-ribose)polymerase (PARP) 1 and 2 inhibitors. This series was optimized to improve enzyme and cellular activity, and the resulting PARP inhibitors display antiproliferation activities against BRCA-1 and BRCA-2 deficient cancer cells, with high selectivity over BRCA proficient cells. Extrahepatic oxidation by CYP450 1A1 and 1A2 was identified as a metabolic concern, and strategies to improve pharmacokinetic properties are reported. These efforts culminated in the identification of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide 56 (MK-4827), which displays good pharmacokinetic properties and is currently in phase I clinical trials. This compound displays excellent PARP 1 and 2 inhibition with IC50 = 3.8 and 2.1 nM, respectively, and in a whole cell assay, it inhibited PARP activity with EC50 = 4 nM and inhibited proliferation of cancer cells with mutant BRCA-1 and BRCA-2 with CC50 in the 10−100 nM range. Compound 56 was well tolerated in vivo and demonstrated efficacy as a single agent in a xenograft model of BRCA-1 deficient cancer.[1] The aim of this study was to assess niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase (PARP) inhibitor, for its ability to radiosensitize human tumor cells. Human tumor cells derived from lung, breast and prostate cancers were tested for radiosensitization by niraparib using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of niraparib to alter the repair of radiation-induced DNA double strand breaks (DSBs) was determined using detection of γ-H2AX foci and RAD51 foci. Clonogenic survival analyses indicated that micromolar concentrations of niraparib radiosensitized tumor cell lines derived from lung, breast, and prostate cancers independently of their p53 status but not cell lines derived from normal tissues. Niraparib also sensitized tumor cells to H2O2 and converted H2O2-induced single strand breaks (SSBs) into DSBs during DNA replication. These results indicate that human tumor cells are significantly radiosensitized by the potent and selective PARP-1 inhibitor, niraparib, in the in vitro setting. The mechanism of this effect appears to involve a conversion of sublethal SSBs into lethal DSBs during DNA replication due to the inhibition of base excision repair by the drug. Taken together, our findings strongly support the clinical evaluation of niraparib in combination with radiation. [2] Background: Niraparib is an oral poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) 1/2 inhibitor that has shown clinical activity in patients with ovarian cancer. We sought to evaluate the efficacy of niraparib versus placebo as maintenance treatment for patients with platinum-sensitive, recurrent ovarian cancer. Methods: In this randomized, double-blind, phase 3 trial, patients were categorized according to the presence or absence of a germline BRCA mutation (gBRCA cohort and non-gBRCA cohort) and the type of non-gBRCA mutation and were randomly assigned in a 2:1 ratio to receive niraparib (300 mg) or placebo once daily. The primary end point was progression-free survival. Results: Of 553 enrolled patients, 203 were in the gBRCA cohort (with 138 assigned to niraparib and 65 to placebo), and 350 patients were in the non-gBRCA cohort (with 234 assigned to niraparib and 116 to placebo). Patients in the niraparib group had a significantly longer median duration of progression-free survival than did those in the placebo group, including 21.0 vs. 5.5 months in the gBRCA cohort (hazard ratio, 0.27; 95% confidence interval [CI], 0.17 to 0.41), as compared with 12.9 months vs. 3.8 months in the non-gBRCA cohort for patients who had tumors with homologous recombination deficiency (HRD) (hazard ratio, 0.38; 95% CI, 0.24 to 0.59) and 9.3 months vs. 3.9 months in the overall non-gBRCA cohort (hazard ratio, 0.45; 95% CI, 0.34 to 0.61; P<0.001 for all three comparisons). The most common grade 3 or 4 adverse events that were reported in the niraparib group were thrombocytopenia (in 33.8%), anemia (in 25.3%), and neutropenia (in 19.6%), which were managed with dose modifications. Conclusions: Among patients with platinum-sensitive, recurrent ovarian cancer, the median duration of progression-free survival was significantly longer among those receiving niraparib than among those receiving placebo, regardless of the presence or absence of gBRCA mutations or HRD status, with moderate bone marrow toxicity. (Funded by Tesaro; ClinicalTrials.gov number, NCT01847274). [4]

Solubility Data

Solubility (In Vitro)

DMSO: 25~64 mg/mL (78.0~199.8 mM) Water: <1 mg/mL Ethanol: ~64 mg/mL (~199.8 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.49 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (6.49 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 20.8 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (6.49 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.1212 mL	15.6060 mL	31.2120 mL
	5 mM	0.6242 mL	3.1212 mL	6.2424 mL
	10 mM	0.3121 mL	1.5606 mL	3.1212 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.