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 )

MK-4827, a novel, orally bioavailable PARP-1 and PARP-2 inhibitor, significantly increased the radiation's impact on a range of human tumor xenografts, including p53 mutant and wild-type tumors. It showed effectiveness as a single agent in a xenograft model of cancer lacking BRCA-1 and was well tolerated in vivo.

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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 (Vd_ss=6.9 L/kg), a long terminal half-life (t_1/2=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].

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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]

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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].

In a buffer containing pH 8.0, 1 mM DTT, 1 mM spermine, 50 mM KCl, 0.01% Nonidet P-40, and 1 mM MgCl₂, the enzyme assay is carried out. The components of the PARP reaction are as follows: 1.5 μM NAD⁺, 150 nM biotinylated NAD⁺, 1 μg/mL activated calf thymus, 0.1 μCi [³H]NAD⁺ (200 000 DPM), and 1.5 nM PARP-1. In white 96-well plates, 50 μL volumes are used for autoreactions that use SPA bead-based detection. Niraparib is one of the compounds that are prepared in 96-well plates using an 11-point serial dilution method, with 5 μL/well in 5% DMSO/H₂O (10× concentrated). The reactions commence with the addition of 35 μL of PARP-1 enzyme in buffer, followed by a 5-minute room temperature incubation period and the addition of 10 μL of NAD⁺ and DNA substrate mixture. These reactions are stopped after three hours at room temperature by adding 50 μL of streptavidin-SPA beads (2.5 mg/mL in 200 mM EDTA, pH 8). They are counted using a TopCount microplate scintillation counter after five minutes. Inhibition curves at different substrate concentrations are used to calculate IC50 values[1].

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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.

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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.

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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.

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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.

The HT Universal Chemiluminescent PARP Assay Kit is used to examine the inhibition of PARP in A549 and H1299 cells. In brief, trypsinization and transfer of the cells to a pre-chilled tube follow a treatment of cells with DMSO or 1 μM niraparib for 15, 30, 60, or 120 minutes. After two cold PBS washes, the cells are resuspended in cold PARP extraction buffer. The cell suspensions undergo a 30-minute ice-soaking period punctuated by periodic vortexing to induce disruption of the cell membrane. When the suspensions are centrifuged, the supernatant is moved to an ice-filled tube that has already been chilled. The 96-well plate's histone-coated wells are rehydrated with 1X PARP buffer and left to incubate for half an hour at room temperature. Remove the PARP buffer, then add 1X PARP buffer, diluted PARP-HSA enzyme, and 20 μg of protein as measured by the Bio-Rad Protein Assay to each well. After 60 minutes of room temperature incubation, the strip wells are twice cleaned with PBS containing 0.1% Triton X-100 and then again with PBS. In the strip wells, diluted Strep-HRP is added, and they are then allowed to sit at room temperature for 60 minutes. Just like before, the wells are cleaned. Chemiluminescent readings are promptly obtained using a plate-reader after equal volumes of PeroxyGlow A and B are mixed and added to the wells[2].

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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.

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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%.

Mice: Niraparib treatment is started when tumors grow to 6.0 mm in diameter in female nude mice (Ncr Nu/Nu). At that point, mice are randomly assigned to treatment groups of five to eight mice each. If the tumors have grown to a diameter of 8 mm, niraparib is administered at a dose of 25 mg/kg twice day or 50 mg/kg once day for a total of 21 days, unless it is stopped after 9 days. When a tumor's diameter reaches 8.0 mm (7.7-8.2 mm), fractionated local tumor irradiation (XRT) is applied. Using a small-animal irradiator with two parallel-opposed 137Cs sources, radiation (2 Gy per fraction) is applied once a day for 14 consecutive days or twice a day for 7 consecutive days to the tumor-bearing leg of mice at a dose rate of 5 Gy/min. In order to create a 3.0 cm diameter radiation field around the tumor and protect the animal's body from radiation exposure, unanesthetized mice are mechanically immobilized in a jig during the radiation process. When receiving both radiation and niraparib on the same day, the medication is given one hour prior to the first radiation dosage.

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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]

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.

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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.

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Biological Half-Life
Following multiple daily doses of 300 mg of niraparib, the mean half-life (t_1/2) is 36 hours.

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).

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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.

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Protein Binding
Niraparib is 83% bound to human plasma proteins.

[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.

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.

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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]

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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]

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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]

C19H21CLN4O

356.85

356.14

C, 63.95; H, 5.93; Cl, 9.93; N, 15.70; O, 4.48

1038915-64-8

1038915-64-8 (HCl); 1038915-73-9; 1613220-15-7 (tosylate hydrate);1038915-60-4; 1476777-06-6 (Niraparib metabolite M1); 1038915-58-0 (Niraparib R-enantiomer)

44550597

Light yellow solid powder

1.343 g/cm3

463.619ºC at 760 mmHg

234.188ºC

3.804

3

3

3

25

449

1

NC(C1=CC=CC2=CN(C3=CC=C([C@H]4CNCCC4)C=C3)N=C21)=O.Cl

YXYDNYFWAFBCAN-PFEQFJNWSA-N

InChI=1S/C19H20N4O.ClH/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);1H/t14-;/m1./s1

2-[4-[(3S)-piperidin-3-yl]phenyl]indazole-7-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: ≥ 2.08 mg/mL (5.83 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.83 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.83 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.

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Solubility in Formulation 4: 100 mg/mL (280.23 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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