PARP-1 ( Ki = 1.4 nM ); PARP-2; PARP-3
Rucaparib (AG014699) is a potential AG14644 N-demethylation metabolite[1].
Rucaparib (0.1, 1, 10, 100 μM; 24 hours) is cytotoxic; in Capan-1 (BRCA2 mutant) cells, its LC50 is 5 μM, while in MX-1 (BRCA1 mutant) cells, it is only 100 nM[2].
Rucaparib causes radiosensitization independent of SSB repair inhibition, as it inhibits NF-κB activation downstream. Without impairing other essential inflammatory functions, rucaparib can target NF-κB that is activated by DNA damage and overcome the toxicity seen with classical NF-κB inhibitors[5].
Rucaparib inhibits PARP-1 activity in permeabilized D283Med cells by 97.1% at a concentration of 1 μM[6].

Rucaparib (AG014699) is a potential AG14644 N-demethylation metabolite[1].
Rucaparib (0.1, 1, 10, 100 μM; 24 hours) is cytotoxic; in Capan-1 (BRCA2 mutant) cells, its LC50 is 5 μM, while in MX-1 (BRCA1 mutant) cells, it is only 100 nM[2].
Rucaparib causes radiosensitization independent of SSB repair inhibition, as it inhibits NF-κB activation downstream. Without impairing other essential inflammatory functions, rucaparib can target NF-κB that is activated by DNA damage and overcome the toxicity seen with classical NF-κB inhibitors[5].
Rucaparib inhibits PARP-1 activity in permeabilized D283Med cells by 97.1% at a concentration of 1 μM[6].

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Rucaparib uptake into SW620 human colorectal cancer cells is carrier-mediated and follows Michaelis-Menten kinetics (Km = 8.4 ± 1.2 μM, Vmax = 469 ± 22 pmol per 10^6 cells per 10 min). It accumulates intracellularly to concentrations more than 10 times higher than the extracellular concentration within 30 minutes.[2]
Following a 30-minute pulse exposure to 400 nM rucaparib, the drug is retained in cells with a biphasic efflux. PARP activity is inhibited by ≥70% for at least 72 hours and remained approximately 35% below baseline control activity at 72 hours post-drug removal in SW620, Capan-1, and MX-1 cell lines.[2]
Rucaparib showed cytotoxic activity in colony formation assays. The LC₅₀ was 5 μM in BRCA2 mutant Capan-1 human pancreatic cancer cells and 100 nM in BRCA1 mutant MX-1 human breast cancer cells following a 24-hour drug exposure.[2]
A carboxylic acid metabolite of rucaparib (4-(8-fluoro-6-oxo-3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-2-yl)benzoic acid) inhibited PARP activity with an IC₅₀ of ~550 nM when added to permeabilized cells but did not inhibit PARP in intact cells, suggesting poor membrane permeability.[2]

Rucaparib (AG014699) and AG14584 greatly heighten the toxicity of temozolomide. The amount of body weight lost when using Temozolomide is greatly increased by Rucaparib (1 mg/kg). A 50% increase in the temozolomide-induced tumor growth delay is observed when rucaparib (0.1 mg/kg) is administered[1].
Rucaparib (10 mg/kg for intraperitoneal injection or 50–150 mg/kg for parenteral administration; five days a week for six weeks) dramatically slows tumor growth and causes one complete tumor regression and two persistent partial tumor regressions[2].
Rucaparib has the strongest antitumor effect with three full regressions at 150 mg/kg p.o. administered once or three times a week for six weeks[2].
Rucaparib improves temozolomide's antitumor activity and shows full and long-lasting tumor regression in NB1691 and SHSY5Y xenografts[6].

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In mice bearing Capan-1 (BRCA2 mutant) xenografts, a single oral dose of rucaparib (150 mg/kg) inhibited tumor PARP activity by >70% at 24 hours and suppression persisted for up to 7 days.[2]
Weekly oral administration of rucaparib (150 mg/kg once per week for 6 weeks) significantly inhibited the growth of Capan-1 xenografts and was as effective as, or more effective than, daily intraperitoneal administration (10 mg/kg daily for 5 days per week for 6 weeks). The weekly schedule resulted in three complete tumor regressions out of ten mice.[2]
In mice bearing MX-1 (BRCA1 mutant) xenografts, rucaparib administration (10 mg/kg i.p. daily five times weekly for 6 weeks, or 50 mg/kg i.p./150 mg/kg p.o. once weekly for 6 weeks) did not significantly delay tumor growth despite >80% PARP inhibition in tumors 24 hours after a single oral dose of 150 mg/kg.[2]

The amount of [32P]NAD+ incorporation-induced inhibition of human full-length recombinant PARP-1 is measured. With a PhosphorImager, the amount of [32P]ADP-ribose added to acid-insoluble material is measured. The nonlinear regression analysis is used to calculate Ki.

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PARP activity was measured in cell and tissue homogenates. Briefly, PARP activity in permeabilized cells or tissue homogenate was maximally stimulated with a double-stranded oligonucleotide in the presence of excess NAD⁺ (350 μM). The amount of poly(ADP-ribose) (PAR) polymer formed was quantified by immunoblot using an anti-PAR antibody, referenced to a PAR standard curve. Activity was expressed as pmol PAR per 10^6 cells or per mg protein.[2]

The MTT assay is used to measure cell proliferation. In 96-well plates, the cells are seeded at a density of 5×10³ cells/ml in a volume of 200 μl/well. The following day, DMSO, BKM120, or rucaparib are added in varying concentrations to the cells. Each well receives 20 μl of MTT (5 mg/ml) after four days. Following an additional 4-hour incubation period at 37 °C, the absorbance at 490 nm is determined. CalcuSyn software is used to analyze data from growth inhibitory experiments in order to ascertain the effect of drug combinations. Next, combination indexes (CI) are computed.

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Rucaparib accumulation studies: Exponentially growing SW620 cells were incubated with [¹⁴C]rucaparib and [³H]sucrose/inulin (to correct for extracellular fluid). At intervals, cells were layered onto silicone oil overlying KOH and centrifuged. The cell pellet (solubilized in KOH) was collected, neutralized, and radioactivity was measured by scintillation counting to determine intracellular drug concentration. Inhibitors of various transporters (ouabain, cytochalasin B, dipyridamole, BCH) or modified buffers were used to study uptake mechanisms.[2]
Cytotoxicity assay (Clonogenic survival): Exponentially growing Capan-1 or MX-1 cells were seeded at low density in multi-well dishes. After attachment, cells were exposed to increasing concentrations of rucaparib (0–50 μM) for 24 hours, then washed and incubated in drug-free medium for 10-14 days. Colonies were stained with crystal violet, counted, and survival relative to untreated control was calculated.[2]

Dissolved in saline; 1 mg/kg; One or four daily by i.p.CD-1 nude mice bearing established D283Med xenografts\nDetermination of Antitumor Activity In vivo[1]
\nFemale athymic nude mice (CD1 nu/nu) used for antitumor studies were maintained and handled in isolators under specific pathogen-free conditions. We implanted SW620 colorectal tumor cells (1 × 107 cells per animal) s.c. into one flank of each mouse, treated the mice (five animals per group) when tumors were palpable (10–12 days after implantation), and monitored tumor growth using two-dimensional caliper measurements. Tumor volume was calculated using the equation a2 × b / 2, where a is the smallest measurement and b is the largest. Data are presented as median relative tumor volumes (RTV), defined as the calculated tumor volume divided by the calculated tumor volume on the initial day of treatment (day 0). Thus, on day 0, the RTV value is 1 and RTV4 is when the tumor is four times as large as its initial value. \n

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\nSingle-Dose Studies. [1]
\nWe administered a single dose of temozolomide p.o. as a suspension in saline at 200 mg/kg either alone or in combination with a single i.p. administration of PARP inhibitor administered at 0.1 [AG14447 and MS-AG14644 (equivalent to 0.078 mg/kg free AG14644 only)], 1.0, and 10 mg/kg (for the mesylate salts equivalent to 0.79 and 7.9 mg/kg free AG14451 and AG14452 and 0.78 and 7.8 free AG14531 and AG14644). Control animals were treated with either normal saline p.o. and i.p or normal saline p.o and PARP inhibitor 10 mg/kg i.p. \n

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\nFive Daily Dosing Studies. [1]
\nWe treated animals with five daily doses of temozolomide administered p.o. as a suspension in saline at 68 mg/kg either alone or in combination with a five daily i.p. administrations of PARP inhibitor at 0.05, 0.15, and 0.5 mg/kg AG14447; 0.15 and 0.5 mg/kg MS-AG14644 (equivalent to 0.12 and 0.39 mg/kg free AG14644); 1.5, 5, and 15 mg/kg AG14361; and 5 mg/kg AG14452. Control animals were treated with either normal saline p.o. and i.p. or normal saline p.o and PARP inhibitor at the higher dose (0.5, 5, or 15 mg/kg, depending on the compound studied) i.p.\n

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\nTissue Distribution[1]
\nWe administered AG14361, AG14452, or AG14447 (10 mg/kg i.p.) to mice (three animals per group) bearing SW620 xenografts (∼10 × 10 mm). After 120 min, the animals were bled by cardiac puncture, under general anesthesia, the tumor was removed and snap frozen on liquid nitrogen. Plasma was removed and stored at −20°C. The concentrations of PARP inhibitor in acetonitrile-treated plasma and homogenized tumor were measured using reverse-phase high-pressure liquid chromatography (isocratic mobile phase: 40% acetonitrile in 0.1% ammonium formate, Hypersil BDS 3 μm 4.6 × 250 mm column, Waters Alliance 2690 high-pressure liquid chromatography; Waters, Elstree, Herts, United Kingdom) by the method of addition.\n

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\nXenograft establishment: Female CD-1 nude mice (10-12 weeks old) were implanted subcutaneously on the flank with 10^7 Capan-1 cells in PBS or 2 x 10^6 MX-1 cells in a 1:1 mix of Matrigel and medium.[2]
\nPharmacokinetics and tissue distribution study: Mice bearing established Capan-1 tumors received a single dose of rucaparib (10 mg/kg intraperitoneally or 50, 100, 150 mg/kg orally). At specified time points (0.5, 4, 24, 48, 72, 168 hours) post-dose, mice were euthanized. Blood was collected for plasma, and tumors and brains were harvested, snap-frozen, and stored. Tissue concentrations of rucaparib and its metabolite were determined by HPLC with fluorescence detection.[2]
\nEfficacy study: Mice bearing palpable Capan-1 or MX-1 tumors (≥5x5 mm) were randomized into treatment groups. They received vehicle or rucaparib according to various schedules (e.g., 10 mg/kg i.p. daily x5/week for 6 weeks; 150 mg/kg p.o. once weekly for 6 weeks). Tumor dimensions were measured regularly, and volume was calculated. Mice were euthanized when tumors reached a predefined size limit or showed signs of stress.[2]

Absorption, Distribution and Excretion
At twice-daily doses ranging from 240 mg to 840 mg, rucapanib exhibits linear pharmacokinetics. At the approved recommended dose, the mean steady-state peak plasma concentration (Cmax) of rucapanib is 1940 ng/mL (54%), and the mean AUC0–12h is 16900 h × ng/mL (54%). The mean cumulative AUC fold is 3.5 to 6.2. At the approved recommended dose, the median time to peak steady-state plasma concentration (Tmax) is 1.9 hours, ranging from 0 to 5.98 hours. The mean absolute bioavailability is 36%, ranging from 30% to 45%. High-fat meals increased Cmax and AUC0–24h by 20% and 38%, respectively. Tmax was delayed by 2.5 hours.
After a single oral administration of radiolabeled rucaparib, 64% of the total radioactivity of rucaparib was unmetabolized. Radioactivity was found in urine and feces at 45% and 95%, respectively.
The mean (coefficient of variation) apparent volume of distribution was 2300 L (21%).
The mean (coefficient of variation) apparent total clearance at steady state was 44.2 L/h (45%).
Metabolism/Metabolites
In vitro studies showed that rucaparib is primarily metabolized by CYP2D6, followed by CYP1A2 and CYP3A4. In addition to CYP-mediated oxidation, rucaparib also undergoes N-demethylation, N-methylation, and glucuronidation. In one study, researchers identified seven rucaparib metabolites in plasma, urine, and feces.
Biological Half-Life
The mean (coefficient of variation) terminal elimination half-life was 26 (39%) hours.

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In tumor-bearing mice, after a single intraperitoneal injection (10 mg/kg), the parent drug rucapanib was only detectable in plasma after 30 minutes. [2]
After a single oral administration (50, 100, 150 mg/kg), the parent drug rucapanib was detectable in plasma for up to 4 hours, and at higher doses, it was detectable in some mice for up to 48 hours. [2]
Rucapanib accumulates in Capan-1 tumors, reaching concentrations up to 10 times higher than plasma concentrations at 4 hours, and remains in tumors longer than in plasma (it was still detectable in most mice 3 days after oral administration). [2]
Brain tissue uptake is low (≤10% of plasma concentration). [2]
The concentration of the carboxylic acid metabolite in plasma is higher than that of the parent drug, but the concentration in tumors is lower than that of the parent drug. [2]

Hepatotoxicity
In large clinical trials of rucaparib, abnormalities in routine liver function tests were common; 74% of patients experienced elevated serum ALT, with 13% having ALT values exceeding 5 times the upper limit of normal (ULN). Although elevated serum enzymes were common during treatment in clinical trials, no reports of hepatitis with jaundice or liver failure were observed. Following the approval and wider use of rucaparib, no published reports of clinically significant liver injury attributed to this drug have been found. Therefore, rucaparib is a common cause of elevated serum enzymes, but it 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 rucaparib during lactation. The manufacturer recommends discontinuing breastfeeding during treatment with rucaparib and for 2 weeks after the last dose.
◉ Effects on breastfed infants
No relevant published information was found as of the revision date.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

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Protein binding
Rucaparib binds to human plasma proteins in vitro at a rate of 70%. Rucaparib preferentially distributes to erythrocytes, with a plasma concentration ratio of 1.8.

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Rucaparib monotherapy at a dose of 50 mg/kg repeated for 6 months at a 5-day cycle has been reported to be non-toxic to mice. [2]
When used in combination with temozolomide (1 mg/kg, once daily for 5 consecutive days), both the effective dose and the maximum tolerated dose of rucaparib were reduced. [2]
In the reported efficacy studies, rucaparib treatment did not result in a significant decrease in body weight in mice under any test protocol. [2]

[1]. Preclinical selection of a novel poly(ADP-ribose) polymerase inhibitor for clinical trial. Mol Cancer Ther, 2007, 6(3), 945-956.

[2]. Tumour cell retention of rucaparib, sustained PARP inhibition and efficacy of weekly as well as daily schedules. Br J Cancer. 2014 Apr 15;110(8):1977-84.

[3]. Inhibition of poly(ADP-ribose) polymerase-1 enhances temozolomide and topotecan activity against childhood neuroblastoma. Clin Cancer Res, 2009, 15(4), 1241-1249.

[4]. Hexose-6-phosphate dehydrogenase blockade reverses prostate cancer drug resistance in xenograft models by glucocorticoid inactivation. Sci Transl Med. 2021 May 26;13(595):eabe8226.

[5]. NF-κB mediates radio-sensitization by the PARP-1 inhibitor, AG-014699. Oncogene, 2012, 31(2), 251-264.

[6]. Rucaparib: A Review in Ovarian Cancer. Target Oncol. 2019 Apr;14(2):237-246.

Pharmacodynamics
Rucaparib is an anticancer drug with cytotoxic effects on cancer cells. Its mechanism of action is through the inhibition of poly(ADP-ribose) polymerase (PARP), an enzyme involved in DNA repair. Rucaparib inhibits PARP-1, PARP-2, and PARP-3. It also interacts with PARP-4, PARP-10, PARP-12, PARP-15, and PARP-16, but to a lesser extent. In mice, rucaparib accumulates and remains in tumors, persistently inhibiting PARP enzyme activity for up to 7 days. Rucaparib inhibits the growth of BRCA1/2 and other DNA repair gene-deficient tumor cell lines. In addition to PARP inhibition, rucaparib also exhibits a non-PARP-dependent cytotoxic mechanism against cancer cells. When used in combination with other chemotherapeutic agents, rucaparib shows synergistic or additive effects both in vitro and in vivo. There is evidence that rucaparib can enhance the sensitivity of cancer cells to chemotherapy. Rucaparib can also induce vasodilation, which may increase tumor perfusion and enhance the accumulation of cytotoxic drugs in cancer cells. Rucaparib is a PARP inhibitor that utilizes the synthetic lethality of homologous recombination-defective tumors, such as those carrying BRCA1/2 mutations. [2] The prolonged PARP inhibition observed in vitro and in vivo is attributed to carrier-mediated accumulation and retention of the drug within cells, rather than simply PARP capture on DNA. [2] This study suggests that intermittent (e.g., once weekly) dosing of rucaparib can achieve antitumor efficacy comparable to daily dosing in preclinical models due to persistent target inhibition, suggesting potential clinical value for alternative dosing regimens. [2] Although in vitro studies showed sensitivity to MX-1 xenograft tumors, efficacy was lacking in this model, which may be related to the slow tumor growth and higher baseline PARP activity in these tumors. [2]

C19H18FN3O

323.37

323.143

C, 70.57; H, 5.61; F, 5.88; N, 12.99; O, 4.95

283173-50-2

1859053-21-6 (camsylate); 459868-92-9 (phosphate); 283173-50-2

9931954

Yellow solid powder

1.3±0.1 g/cm3

625.2±55.0 °C at 760 mmHg

331.9±31.5 °C

0.0±1.8 mmHg at 25°C

1.649

2.85

3

3

3

24

466

0

FC1=C([H])C2C(N([H])C([H])([H])C([H])([H])C3=C(C4C([H])=C([H])C(C([H])([H])N([H])C([H])([H])[H])=C([H])C=4[H])N([H])C(=C1[H])C3=2)=O

HMABYWSNWIZPAG-UHFFFAOYSA-N

InChI=1S/C19H18FN3O/c1-21-10-11-2-4-12(5-3-11)18-14-6-7-22-19(24)15-8-13(20)9-16(23-18)17(14)15/h2-5,8-9,21,23H,6-7,10H2,1H3,(H,22,24)

6-fluoro-2-[4-(methylaminomethyl)phenyl]-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-9-one

AG014699; PF-01367338; AG 14447; AG 014699; PF 01367338; AG-014699,PF01367338; AG-14447; AG14447; Trade name: Rubraca

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (7.73 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 25.0 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: ≥ 2.5 mg/mL (7.73 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 25.0 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: ≥ 2.5 mg/mL (7.73 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 30mg/mL

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