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 significantly amplifies the radiation's impact on a range of human tumor xenografts, including p53 mutant and wild type tumors. Following administration, MK-4827 lowers PAR levels in tumors by 1 hour, a reduction that lasts for up to 24 hours[1]. When compared to single modalities, in vivo treatment with MK-4827 and radiation prolonged survival (p<0.01). The combination group's tumors had significantly higher levels of cleaved caspase-3 and γ-H2AX than the single modality cohorts, further supporting the in vivo superiority of MK-4827 plus radiation[4].

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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.

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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. [3]

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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 [1].

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.

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PARP-1 SPA Assay [3]
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 [3]
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 [3]
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.

A549 and H1299 cells are used to examine the inhibition of PARP using the HT Universal Chemiluminescent PARP Assay Kit. In summary, cells are trypsinized, treated for 15, 30, 60, or 120 minutes with DMSO or 1 μM Niraparib (MK-4827), and then moved to a tube that has been chilled beforehand. The cells are resuspended in cold PARP extraction buffer after being twice rinsed with ice-cold PBS. To break down the cell membrane, the cell suspensions are vortexed periodically while being incubated on ice for 30 minutes. After centrifuging the suspensions, the supernatant is moved to an ice-filled tube that has already been chilled. After being rehydrated with 1X PARP buffer, the histone-coated wells in the 96-well plate are incubated for 30 minutes 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.

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. [3]

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Mice: Treatment groups of five to eight female nude mice each are randomly assigned to receive treatment with MK-4827 once tumors have grown to a diameter of 6.0 mm. After the tumors have grown to a diameter of 8 mm, MK-4827 is administered at a dose of 25 mg/kg twice day or 50 mg/kg once daily for a total of 21 days, after which 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. MK-4827 is given one hour prior to the first radiation dosage of the day on the day when both radiation and the drug are given.

Absorption, Distribution and Excretion
Following a single 300 mg dose of niraparib, the mean (±SD) peak plasma concentration (Cmax) was 804 (±403) ng/mL. Niraparib exposure (Cmax and AUC) increased dose-proportionately with daily doses from 30 mg (0.1 times the approved recommended dose) to 400 mg (1.3 times the approved recommended dose). After 21 days of repeated daily dosing, the cumulative rate of niraparib exposure was approximately 2 times over the 30 to 400 mg dose range. The time to peak concentration (Tmax) was approximately 3 hours. The absolute bioavailability of niraparib was approximately 73%. Food did not appear to affect drug exposure. Niraparib is eliminated via multiple pathways, including hepatic metabolism, hepatobiliary excretion, and renal excretion. Following a single oral dose of 300 mg of radiolabeled niraparib, the mean recovery over 21 days was 47.5% in urine (range: 33.4% to 60.2%) and 38.8% in feces (range: 28.3% to 47.0%). In pooled samples collected over 6 days, 11% and 19% of the dose of unmetabolized niraparib were recovered in urine and feces, respectively. The mean (±SD) apparent volume of distribution (Vd/F) was 1220 (±1114) L. In population pharmacokinetic analysis, the Vd/F of niraparib in cancer patients was 1074 L. In population pharmacokinetic analysis, the apparent total clearance (CL/F) of niraparib in cancer patients was 16.2 L/h. Niraparib is primarily metabolized by carboxylesterases (CEs) to M1, a major inactive metabolite. The M1 metabolite can then undergo glucuronidation mediated by UDP-glucuronyltransferases (UGTs) to generate the M10 metabolite. In mass balance studies, M1 and M10 are the major circulating metabolites. The M1 metabolite can also undergo methylation, monooxygenation, and hydrogenation to generate other minor metabolites.

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

Hepatotoxicity
In pre-registration randomized controlled clinical trials of niraparib, abnormalities in routine liver function tests were common, but most were mild and resolved spontaneously. Elevated serum ALT occurred in 28% of patients (compared to 15% in the control group), but only 1% of patients had ALT levels exceeding 5 times the upper limit of normal (ULN) (compared to 2% in the control group). Although elevated serum enzymes were common during treatment in clinical trials, no cases of hepatitis with jaundice or liver failure were reported. Since niraparib's approval and widespread use, no clinically significant cases of liver injury have been published, but its use and duration are limited. Therefore, niraparib is a known cause of mild serum enzyme elevations, but has not been found to be associated with significant hepatotoxicity. Probability score: E (Unproven but suspected cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the clinical use of niraparib during lactation. Because niraparib binds to plasma proteins at a rate of 83%, its levels in breast milk may be very low. The manufacturer recommends discontinuing breastfeeding during niraparib treatment and for one month after treatment ends.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding
Niraparib binds to human plasma proteins at a rate of 83%.

[1]. Invest New Drugs . 2012 Dec;30(6):2113-20.

[2]. J Biol Chem . 2012 Feb 3;287(6):4198-210.

[3]. J Med Chem . 2009 Nov 26;52(22):7170-85.

[4]. Anticancer Res . 2013 Mar;33(3):755-62.

[5]. Oncotarget . 2014 Jul 15;5(13):5076-86.

Pharmacodynamics
Niraparib exhibits cytotoxicity against tumor cell lines with or without BRCA1/2 deficiency. Tumor growth slowdown was observed in mouse xenograft models of BRCA1/2-deficient human cancer cell lines and in human patient-derived xenograft tumor models with homologous recombination deficiency (HRD), regardless of whether BRCA1/2 was mutant or wild-type. In vitro studies have shown that niraparib inhibits dopamine, norepinephrine, and serotonin transporters, which may explain its off-target cardiovascular effects, such as increased heart rate and blood pressure. We disclose the development of a novel series of 2-phenyl-2H-indazole-7-carboxamide compounds as inhibitors of poly(ADP-ribose) polymerase (PARP) 1 and 2. These compounds were optimized to enhance enzyme and cellular activity, and the resulting PARP inhibitors exhibited antiproliferative activity against BRCA-1 and BRCA-2 deficient cancer cells with high selectivity for BRCA-normal cells. Studies have identified CYP450 1A1 and 1A2-mediated extrahepatic oxidation as a metabolic issue and reported strategies to improve pharmacokinetic properties. These efforts ultimately led to the discovery of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide 56 (MK-4827), a compound with favorable pharmacokinetic properties currently undergoing Phase I clinical trials. This compound exhibits excellent inhibitory activity against PARP 1 and 2, with IC50 values of 3.8 nM and 2.1 nM, respectively. In whole-cell assays, its EC50 value for inhibiting PARP activity was 4 nM, and its CC50 values for inhibiting the proliferation of cancer cells carrying mutant BRCA-1 and BRCA-2 ranged from 10 to 100 nM. Compound 56 is well-tolerated in vivo and has shown efficacy as a single agent in a xenograft model of BRCA-1-deficient cancer. [3]

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Background: Niraparib is an oral poly(ADP-ribose) polymerase (PARP) 1/2 inhibitor that has shown clinical activity in patients with ovarian cancer. We aimed to evaluate the efficacy of niraparib versus placebo as maintenance therapy in patients with platinum-sensitive recurrent ovarian cancer. Methods: In this randomized, double-blind, phase 3 clinical trial, patients were assigned to the niraparib group (300 mg) or the placebo group once daily based on the presence or absence of germline BRCA mutations (gBRCA group and non-gBRCA group) and the type of non-gBRCA mutation, and were randomly assigned in a 2:1 ratio. The primary endpoint was progression-free survival. Results: Of the 553 patients enrolled, 203 were in the gBRCA group (of which 138 were assigned to the niraparib group and 65 to the placebo group) and 350 were in the non-gBRCA group (of which 234 were assigned to the niraparib group and 116 to the placebo group). Compared with the placebo group, patients in the niraparib group had significantly prolonged median progression-free survival (PFS), specifically 21.0 months vs. 5.5 months in the gBRCA cohort (hazard ratio, 0.27; 95% confidence interval [CI], 0.17 to 0.41), 12.9 months vs. 3.8 months in patients with homologous recombination-deficient (HRD) tumors in the non-gBRCA cohort (hazard ratio, 0.38; 95% CI, 0.24 to 0.59), and 9.3 months vs. 3.9 months in patients in the non-gBRCA cohort (hazard ratio, 0.45; 95% CI, 0.34 to 0.61; P values for all three comparisons were < 0.001). The most common grade 3 or 4 adverse events in the niraparib group were thrombocytopenia (33.8%), anemia (25.3%), and neutropenia (19.6%), all of which were managed with dose adjustment. Conclusion: In patients with platinum-sensitive recurrent ovarian cancer, regardless of gBRCA mutations or HRD status, patients treated with niraparib had significantly longer median progression-free survival than those treated with placebo, with moderate myelotoxicity. (Sponsored by Tesaro; ClinicalTrials.gov registration number: NCT01847274).

C26H28N4O4S

492.59

492.183

C, 63.40; H, 5.73; N, 11.37; O, 12.99; S, 6.51

1038915-73-9

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

78357761

Light yellow solid powder

5.943

3

6

4

35

655

1

S(C1C([H])=C([H])C(C([H])([H])[H])=C([H])C=1[H])(=O)(=O)O[H].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]

LCPFHXWLJMNKNC-PFEQFJNWSA-N

InChI=1S/C19H20N4O.C7H8O3S/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;1-6-2-4-7(5-3-6)11(8,9)10/h1,3,5-9,12,14,21H,2,4,10-11H2,(H2,20,24);2-5H,1H3,(H,8,9,10)/t14-;/m1./s1

4-methylbenzenesulfonic acid;2-[4-[(3S)-piperidin-3-yl]phenyl]indazole-7-carboxamide;hydrate

MK-4827 tosylate; MK 4827; MK-4827 tosylate; Niraparib tosylate; MK-4827 (tosylate); MK-4827-tosylate; MK4827 tosylate; MK 4827 tosylate; MK-4827; MK4827; Niraparib; Niraparib HCl; Niraparib hydrochloride; Zejula

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: ≥ 2.5 mg/mL (5.08 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (5.08 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 0.5 mg/mL (1.02 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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