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 )

Niraparib (MK-4827) tosylate hydrate inhibits PARP activity with EC50=4 nM and EC90=45 nM in a whole cell assay. In the 10-100 nM range, Niraparib tosylate hydrate inhibits the growth of cancer cells expressing mutant BRCA-1 and BRCA-2 with CC50. In a whole cell assay, niraparib tosylate hydrate exhibits good inhibition of PARP 1 and 2 (IC50=3.8 and 2.1 nM, respectively)[1].
Niraparib tosylate hydrate suppresses PARP within 15 minutes of treatment, with the A549 cells showing 85% inhibition at 1 hour and the H1299 cells showing about 55% inhibition at 1 hour[2].

Niraparib (MK-4827) tosylate hydrate is well tolerated and effective when used alone in a xenograft model of BRCA-1 deficient cancer[1].
Niraparib (MK-4827) tosylate hydrate is effective as a single agent in a xenograft model of cancer lacking BRCA-1 and is well tolerated in vivo[1].
Niraparib (MK-4827) tosylate hydrate is defined by very high volume of distribution (Vd_ss=6.9 L/kg), long terminal half-life (t_1/2=3.4 h), superb bioavailability (F=65%), and acceptable pharmacokinetics in rats with plasma clearance of 28 (mL/min)/kg[1].
Niraparib (MK-4827) tosylate hydrate increases the p53 mutant Calu-6 tumor's radiation response in both situations, with a single daily dosage of 50 mg/kg working better than two doses of 25 mg/kg[3].

---

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].

---

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].

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.

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.

---

The assays for proliferation were carried out in 96-well black viewplates. 300 cells/well (250 cells/well for BRCA-1 wt) were plated in 190 μL/well of culture medium (DMEM containing 10% FCS, 0.1 mg/mL penicillin-streptomycin, and 2 mM L-glutamine), which was then incubated for four hours at 37°C in an atmosphere of 5% CO2. After that, inhibitors were added in 10-μL/well serial dilutions to achieve the target final compound concentration in 0.5% DMSO. Following a 7-day incubation period at 37°C with 5% CO2, the viability of the cells was evaluated. In summary, 100 μL/well of prediluted 1:10 CellTiter-Blue solution was added, and 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 the 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 cell growth. It was established what concentration (CC50) would stop cell growth 50% of the time.

Female nude mice
25 mg/kg twice daily or 50 mg/kg once daily
oral administration

---

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 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.

---

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.

---

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

[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.

Niraparib tosylate monohydrate is a highly bioavailable oral hydrated form of niraparib. Niraparib is an inhibitor of poly(ADP-ribose) polymerase (PARP) types 1 and 2 (PARP-1 and PARP-2) with antitumor activity. After administration, niraparib binds to and inhibits the activity of PARP-1 and PARP-2, thereby inhibiting PARP-1 and PARP-2-mediated DNA repair, increasing the accumulation of DNA strand breaks, promoting genomic instability, and ultimately leading to apoptosis. The PARP protein family catalyzes post-translational ADP-ribosylation of nucleoproteins and is activated by single-stranded DNA (ssDNA) breaks.
See also: Niraparib (with active moiety)... See more...

---

Pharmacodynamics
Niraparib has cytotoxic effects on both BRCA1/2-deficient and normal tumor cell lines. Slowed tumor growth was observed in mouse xenograft models of BRCA1/2-deficient human cancer cell lines and in xenograft tumor models derived from human patients with homologous recombination deficiency (HRD) and BRCA1/2 mutations or wild-type cells. In vitro studies showed that niraparib inhibits dopamine, norepinephrine, and serotonin transporters, which may explain its non-targeted 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) polymerases (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. We identified extrahepatic oxidation of CYP450 1A1 and 1A2 as a metabolic problem and reported strategies to improve pharmacokinetic properties. These efforts ultimately identified 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide 56 (MK-4827), which has good pharmacokinetic properties and is currently undergoing a phase I clinical trial. The compound showed excellent inhibitory activity against PARP 1 and PARP 2, with IC50 values of 3.8 nM and 2.1 nM, respectively. In whole-cell assays, the compound showed an EC50 value of 4 nM for inhibiting PARP activity and a CC50 value of 10-100 nM for inhibiting the proliferation of cancer cells carrying mutant BRCA-1 and BRCA-2. Compound 56 was well tolerated in vivo and showed efficacy as a monotherapy in a xenograft model of BRCA-1 deficient cancer. [1]

---

This study aimed to evaluate the ability of the novel poly(ADP-ribose) polymerase (PARP) inhibitor niraparib (MK-4827) to enhance the radiosensitivity of human tumor cells. Clonogenic survival assays were used to assess the radiosensitivity of niraparib to human tumor cells derived from lung, breast, and prostate cancer. The assays included p53 wild-type and p53-deficient cell lines. The ability of niraparib to alter radiation-induced DNA double-strand break (DSB) repair was determined by detecting γ-H2AX and RAD51 foci. Clonogenic survival analysis showed that micromolar concentrations of niraparib enhanced the radiosensitivity of tumor cell lines derived from lung, breast, and prostate cancer, and this effect was independent of p53 status, but had no effect on cell lines derived from normal tissues. Niraparib also enhanced the sensitivity of tumor cells to H₂O₂ and converted H₂O₂-induced single-strand breaks (SSBs) into double-strand breaks (DSBs) during DNA replication. These results demonstrate that, in vitro, the potent and selective PARP-1 inhibitor niraparib significantly enhances the radiosensitivity of human tumor cells. This mechanism of action appears to be related to the drug's inhibition of base excision repair, thereby converting sublethal SSBs into lethal DSBs during DNA replication. In summary, our findings strongly support the clinical evaluation of niraparib in combination with radiotherapy. [2]

---

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 randomly assigned in a 2:1 ratio to receive either niraparib (300 mg) or placebo 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. The primary endpoint was progression-free survival. Results: Of the 553 enrolled patients, 203 were in the gBRCA cohort (138 of whom were assigned to the niraparib group and 65 to the placebo group) and 350 were in the non-gBRCA cohort (234 of whom 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 by dose adjustment. Conclusion: In patients with platinum-sensitive recurrent ovarian cancer, regardless of the presence of gBRCA mutations or HRD status, patients treated with niraparib had significantly longer median progression-free survival than those treated with placebo, and myelotoxicity was moderate. (Funded by Tesaro; ClinicalTrials.gov registration number: NCT01847274). [4]

C26H30N4O5S

510.6052

510.193

C, 61.16; H, 5.92; N, 10.97; O, 15.67; S, 6.28

1613220-15-7

1038915-64-8 (HCl); 1038915-73-9; 613220-15-7 (tosylate hydrate); 1038915-60-4; 1038915-58-0

74763937

White to off-white solid powder

4

7

4

36

655

1

S(C1C=CC(C)=CC=1)(=O)(=O)O.O.C1(C(N)=O)=CC=CC2=CN(C3=CC=C([C@H]4CNCCC4)C=C3)N=C12

ACNPUCQQZDAPJH-FMOMHUKBSA-N

InChI=1S/C19H20N4O.C7H8O3S.H2O/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);1H2/t14-;;/m1../s1

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

MK-4827; Niraparib tosylate monohydrate; 1613220-15-7; Niraparib tosylate hydrate; Niraparibtosylate; 195Q483UZD;MK-4827; MK4827; MK4827-tosylate; Niraparib tosylate

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)

DMSO: ~64 mg/mL (~199.8 mM)
Ethanol: ~64 mg/mL

5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O: 2.5mg/ml (7.80mM) (Please use freshly prepared in vivo formulations for optimal results.)