PARP-2 ( IC50 = 1 nM ); PARP-1 ( IC50 = 5 nM ); tankyrase-1 ( IC50 = 1.5 μM ); Autophagy; Mitophagy

Olaparib would combat mutations in either BRCA1 or BRCA2. Tankyrase-1 does not affect olaparib (IC50 >1 μM). At concentrations ranging from 30 to 100 nM, olaparib was able to inhibit the PARP-1 activity in SW620 cells. Compared to BRCA1- and BRCA2-proficient cell lines (Hs578T, MDA-MB-231, and T47D), olaparib is more sensitive to BRCA1-deficient cell lines (MDA-MB-463 and HCC1937).[1] Olaparib is highly susceptible to KB2P cells because PARP inhibition suppresses base excision repair, which could cause single-strand breaks to become double-strand breaks during DNA replication and trigger recombination pathways that are BRCA2-dependent.[2]

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In Vitro Cellular Potency [1]
The cellular potency of 47/Olaparib was evaluated in a number of in vitro models. Initial studies identified 47 as an effective agent to potentiate the cell killing by alkylating agent MMS. Following the establishment of a growth-inhibition curve for MMS on SW620 cells, the addition of the PARP-1 inhibitor at nM concentrations dose dependently increased the effectiveness of MMS (Figure 2a). The level of potentiation was seen to plateau around 100 nM concentration, which indicates that the maximal cellular activity for 47 in combination with MMS lies within this range. We confirmed this by directly measuring PARP-1 inhibitory activity in cells by using a PAR formation assay in which 47 was applied to SW620 cell lysates at a similar concentration range and identified the IC50 for PARP-1 inhibition to be around 6 nM and the total ablation of PARP-1 activity to be at concentrations of 30−100 nM (Figure 2b).

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Results: Genetic, transcriptional, and functional analyses confirmed the successful isolation of BRCA2-deficient and BRCA2-proficient mouse mammary tumor cell lines. Treatment of these cell lines with 11 different anticancer drugs or with gamma-irradiation showed that Olaparib/AZD2281, a novel and specific PARP inhibitor, caused the strongest differential growth inhibition of BRCA2-deficient versus BRCA2-proficient mammary tumor cells. Finally, drug combination studies showed synergistic cytotoxicity of AZD2281 and cisplatin against BRCA2-deficient cells but not against BRCA2-proficient control cells.
Conclusion: We have successfully established the first set of BRCA2-deficient mammary tumor cell lines, which form an important addition to the existing preclinical models for BRCA-mutated breast cancer. The exquisite sensitivity of these cells to the PARP inhibitor OlaparibAZD2281, alone or in combination with cisplatin, provides strong support for AZD2281 as a novel targeted therapeutic against BRCA-deficient cancers.

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Olaparib selectively targets ATM mutant lymphoid cells, including proliferating primary CLL cells. Sensitivity to Olaparib is mediated by absence of ATM activity and by cell proliferation. Olaparib sensitivity of ATM dysfunctional cells is related to the accumulation of DNA damage and is apoptosis independent. Researchers investigated in vitro sensitivity to the poly (ADP-ribose) polymerase inhibitor olaparib (AZD2281) of 5 ATM mutant lymphoblastoid cell lines (LCL), an ATM mutant MCL cell line, an ATM knockdown PGA CLL cell line, and 9 ATM-deficient primary CLLs induced to cycle and observed differential killing compared with ATM wildtype counterparts. Pharmacologic inhibition of ATM and ATM knockdown confirmed the effect was ATM-dependent and mediated through mitotic catastrophe independently of apoptosis.

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Olaparib sensitizes ATM mutant cells to conventional cytotoxic agents [4]
Finally, to address the ability of PARP inhibition to increase the effect of standard CLL treatments as well as other chemotherapy agents, we tested the ability of olaparib to sensitize ATM mutant cells to the purine analogue fludarabine, the alkylating agents 4HC and bendamustine, the histone deacetylase inhibitor VPA, and IR (supplemental Table 2; Figure 6A). When treated with the agents alone, Granta-519 cells were resistant to bendamustine and 4HC, yet were sensitive to VPA. Olaparib pretreatment was able to significantly sensitize cells to all the agents tested (Figure 6A). The greatest synergism was observed between olaparib and VPA, whereas olaparib and IR revealed only moderate synergism (supplemental Table 2). The effect of 4HC and olaparib was largely additive (supplemental Table 2), although moderate synergism was observed at the 4HC dose of 0.1µM (Figure 6A). Finally, the impact of olaparib on fludarabine and bendamustine cytotoxicity was generally additive, although synergistic activity could be detected at some doses of fludarabine (supplemental Table 2). Western blot analysis demonstrated enhanced cleavage of PARP1 and caspases 3 and 7 when Granta-519 cells were exposed to either 4HC, VPA, or IR in combination with 1µM olaparib (Figure 6B), indicating that drug-induced apoptosis was increased in the presence of olaparib.

Olaparib (10 mg/kg, p.o.) greatly inhibits tumor growth in SW620 xenografts when combined with temozolomide.[1] Olaparib (50 mg/kg i.p. daily) responds well to Brca1-/-;p53-/- mammary tumors, but not to HR-deficient Ecad-/-;p53-/- mammary tumors. In mice bearing tumors, olaparib even does not exhibit dose-limiting toxicity. [3] Olaparib has been used to treat BRCA-mutated tumors, including cancers of the breast, prostate, and ovary. Additionally, Olaparib selectively inhibits tumor cells deficient in ATM (Ataxia Telangiectasia Mutated), suggesting that it may be a useful treatment for ATM mutant lymphoid tumors.[4]

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In Vivo Efficacy [1]
On the basis of our in vitro cell data, the ability of compound 47/Olaparib to potentiate the antitumor activity of the methylating chemotherapeutic agent, TMZ, was evaluated in an SW620 tumor model. Animals bearing SW620 xenografted tumors were treated with compound 47/Olaparib (10 mg/kg, po) in combination with TMZ (50 mg/kg, po) once daily for 5 consecutive days, after which the tumors were left to grow out. A considerable inhibition of tumor volumes as compared with that of the TMZ alone group was observed for the TMZ plus compound 47 combination (mean values given as relative tumor volumes (RTV), Figure 4). This equated to over 80% tumor growth inhibition throughout the entire terminal phase of the study between TMZ treatment and the combination. The time to reach 2× RTVs for TMZ and combination treatment was 44 and 70 days, respectively (59% increase in latency). Higher RTV values such as 4× RTV (two doublings) could not be comparably assessed because tumors that were treated with the combination treatment did not attain this size during the duration of the study, which clearly demonstrates the pronounced potentiation of TMZ activity by compound 47 (Figure 4). TMZ was well tolerated with a maximum mean body weight loss of 6% on day 7 (3 days after dosing concluded) and full recovery within a week. Likewise, when administered in combination, the PARP inhibitor did not exacerbate the systemic toxicity of TMZ, with a maximum mean body weight loss of 9% on day 6 with full recovery of body weight within 3 days and with no mortalities (>20% weight loss), which signifies that the combination therapy was well tolerated under this dosing regimen.

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To evaluate PARP1 inhibition in a realistic in vivo setting, we tested the PARP inhibitor OlaparibAZD2281 in a genetically engineered mouse model (GEMM) for BRCA1-associated breast cancer. Treatment of tumor-bearing mice with AZD2281 inhibited tumor growth without signs of toxicity, resulting in strongly increased survival. Long-term treatment with AZD2281 in this model did result in the development of drug resistance, caused by up-regulation of Abcb1a/b genes encoding P-glycoprotein efflux pumps. This resistance to AZD2281 could be reversed by coadministration of the P-glycoprotein inhibitor tariquidar. Combination of AZD2281 with cisplatin or carboplatin increased the recurrence-free and overall survival, suggesting that AZD2281 potentiates the effect of these DNA-damaging agents. Our results demonstrate in vivo efficacy of AZD2281 against BRCA1-deficient breast cancer and illustrate how GEMMs of cancer can be used for preclinical evaluation of novel therapeutics and for testing ways to overcome or circumvent therapy resistance.[3]

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ATM-mutant lymphoid tumor cells are sensitive to Olaparib in vivo [4]
To investigate the in vivo impact of olaparib, we generated murine xenograft models of the ATM mutant MCL cell line, Granta-519. To determine whether infiltration and engraftment of the tumor cell line had already taken place before initiation of olaparib treatment in the different animal cohorts, 3 representative mice from each cohort were analyzed on the day that treatment was to begin (14 days after intravenous or 5 days after subcutaneous injection of cells). The presence of tumor cells at the level of at least 1% of all cells, which is considered to be engraftment, was observed by FACS analysis in the bone marrow and spleen both at 5 days (subcutaneous) and 14 days (intravenous) after injection (Figure 5A). Furthermore, using immunocytochemistry and anti–human CD5, Pax-5, and Ki-67 antibodies, we confirmed significant infiltration of proliferating human B-lymphoid tumor cells in both the spleen and bone marrow at both time points before treatment initiation (Figure 5A).

Subsequently, the degree of tumor load was compared in the lymphoid organs of 23 NOD/SCID Granta-519 cell–injected mice 5 weeks after intravenous injection of cells and 14 days after treatment with olaparib. However, early in the experiment, 7 mice died of graft-unrelated causes, leaving 16 mice, which were treated with either olaparib (n = 8) or vehicle alone (n = 8). Analysis of the percentage of human CD45 staining by FACS analysis (Figure 5B) revealed a significant reduction in the percentage of Granta-519 cells in the bone marrow and a trend toward reduced tumor cell load in the spleen of mice treated with olaparib compared with those receiving vehicle alone (Figure 5B). We next assessed the effect of olaparib on the growth of subcutaneous tumors generated by the localized injection of ATM mutant Granta-519 cells into mice and found a significant positive correlation between olaparib treatment and reduced tumor size (Figure 5C). Finally, the overall survival of mice engrafted with Granta-519 cells was significantly increased by olaparib treatment compared with vehicle alone (Figure 5D).

The assay assessed Olaparib's capacity to suppress PARP-1 enzyme activity. An alternative method of measuring PARP-2 activity inhibition involves binding down the recombinant PARP-2 protein in a 96-well plate with white walls using an antibody specific to PARP-2. Measurements of PARP-2 activity are made after ³H-NAD⁺ DNA additions. Scintillant is added after washing in order to quantify ³H-incorporated ribosylations. An AlphaScreen assay for tankyrase-1 is created, involving the incubation of HIS-tagged recombinant TANK-1 protein in a 384-well ProxiPlate assay with biotinylated NAD⁺. A proximity signal is produced by adding alpha beads to bind the HIS and biotin tags; the loss of this signal is directly correlated with TANK-1 activity inhibition. At least three replications of each experiment are conducted.

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In Vitro Isolated Enzyme Assay [1]
This assay determined the ability of test compounds to inhibit PARP-1 enzyme activity. The method that was used was as reported. We measured PARP-2 activity inhibition by using a variation of the PARP-1 assay in which PARP-2 protein (recombinant) was bound down by a PARP-2 specific antibody in a 96-well white-walled plate. PARP-2 activity was measured following 3H−NAD+ DNA additions. After washing, scintillant was added to measure 3H-incorporated ribosylations. For tankyrase-1, an AlphaScreen assay was developed in which HIS-tagged recombinant TANK-1 protein was incubated with biotinylated NAD+ in a 384-well ProxiPlate assay. Alpha beads were added to bind the HIS and biotin tags to create a proximity signal, whereas the inhibition of TANK-1 activity was directly proportional to the loss of this signal. All experiments were repeated at least three times.

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Ex Vivo PARP Activity Assay [1]
SW620 whole-cell protein extracts were prepared by incubation in extraction buffer (1× PBS, 1% NP-40, protease inhibitor cocktail, 200 μM DTT) for 10 min at 4 °C. PARP activity was determined by the quantification of the amount of PAR formation after ex vivo activation. PARP activation reactions were performed by the use of 65 ng of SW620 whole-cell extract in a reaction mix (50 mM Tris pH 8, 4 mM MgCl2, 200 μM DTT, 200 μM NAD+, 20 ng/μL DNA) at 30 °C for 5 min. The amount of PAR that formed in each reaction was then quantified by the use of the Meso Scale Discovery assay platform. Data were calculated from triplicate experiments as the mean percentage of PARP activity relative to vehicle controls ±SE and IC50, which were calculated by the use of XL-FIT 4 software.

The potentiation factor, or PF50 value, is determined by dividing the IC50 of the alkylating agent methylmethane sulfonate (MMS) used in the control growth by the IC50 of the MMS plus PARP inhibitor. Olaparib is tested for MMS screening at a fixed 200 nM concentration using HeLa B cells. The concentrations of olaparib that are tested on the colorectal cell line SW620 are 1, 3, 10, 100, and 300 nM. Sulforhodamine B (SRB) assay is used to measure cell growth.

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In Vitro Cell PF50 Assay [1]
The PF50 value is the potentiation factor, which is calculated as the ratio of the IC50 of the control growth with alkylating agent methylmethane sulfonate (MMS) divided by the IC50 of the MMS combined with the PARP inhibitor. HeLa B cells were used, and the test compounds were tested at a fixed 200 nM concentration for screening with MMS. For the testing of compound 47/Olaparib on the SW620 colorectal cell line (Figure 2), the concentrations that were used were 1, 3, 10, 100 and 300 nM. Cell growth was assessed by the use of the sulforhodamine B (SRB) assay.

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Cell Lines and Culture [1]
SW620 colon, A2780 ovarian, HCC1937, Hs578T, MDA-MB-231, MDA-MB-436, and T47D breast cancer cell lines were obtained from either ATCC or ECACC repositories. All cell lines were grown as monolayers in RPMI1640 medium that was supplemented with 10% v/v FBS, 100 μg/mL penicillin, and 100 μg/mL streptomycin.

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Cell Line Cytotoxicity Assays [1]
The effect of KU-0059436 on the cell survival of breast cancer cell lines was determined by the use of clonogenic assays, as previously described. Briefly, cells were seeded in six-well plates and were left to attach overnight. Vehicle control (DMSO) or increasing concentrations of KU-0059436 (up to 4 μM) were added to the cells, and the mixture was left for 7−14 days, depending on the cell type, before surviving colonies were counted. Data were calculated from triplicate wells as the mean percentage of cell survival relative to vehicle controls ±SE and IC50, which were calculated by the use of XL-FIT 4 software.

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Potentiation of MMS Cytotoxicity by 47/Olaparib Determined by the Use of Sulforhodamine B Cell Growth Assays [1]
SW620 cells were seeded in 96-well plates and were left to attach overnight. Cells were preincubated with vehicle control (DMSO) or with a single concentration of KU-0059436 (1, 3, 10, 30, 100 or 300 nM) for 1 h before the addition of increasing concentrations of MMS. Cells were incubated in the presence of each drug combination for 4 days before cell growth was quantified by the use of an SRB assay. Data were calculated from triplicate wells as the mean percentage of cell growth relative to KU-0059436-only wells, and ±SE and IC50 were calculated by the use of XL-FIT 4 software. SW620 cells showed <24% growth inhibition (>76% cell growth) when only KU-0059436 was used at concentrations below 300 nM (data not shown).

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Experimental design: We established and thoroughly characterized a panel of clonal cell lines from independent BRCA2-deficient mouse mammary tumors and BRCA2-proficient control tumors. Subsequently, we assessed sensitivity of these lines to conventional cytotoxic drugs and the novel PARP inhibitor AZD2281. Finally, in vitro combination studies were done to investigate interaction between AZD2281 and cisplatin.[2]

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Induction of primary CLL cell proliferation using CD40L/IL-4 [4]
Primary CLL leukemia cells obtained from the peripheral blood were typically arrested at gap 1/gap 0 (G1/G0) of the cell cycle. To stimulate and sustain proliferation of these cells, we compared 5 different mitogenic stimuli (see supplemental Figure 3) and found the CD40L/IL-4 culture system the most effective and reproducible. Briefly, after adherence of irradiated (50 Gy) CD40L-expressing murine fibroblasts at 3 × 105 cells/well, 1-1.5 × 106 primary CLL cells were seeded into each well with 10 ng/mL IL-4 in a total volume of 2 mL RPMI containing 10% fetal calf serum nd incubated at 37°C for 3-4 days. At this point, survival assays were initiated. As bromodeoxyuridine (BrdU) staining revealed that CLL proliferation could only be sustained for 7-11 days in culture (supplemental Figure 3), primary CLL cells initiated to cycle over 3-4 days were then treated with 0-10µM Olaparib for an additional 7 days. For consistency, therefore, in all survival assays, all cell types were exposed to olaparib for only 7 days.

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Cell survival assays[4]
Suspensions of lymphoid cells were exposed to increasing concentrations of Olaparib for up to 7 days in triplicate experiments and counted 3 times using a hemocytometer; the surviving fraction was then calculated. In experiments using a single olaparib dose, 3µM was used irrespective of cell type, as this produced a survival response on the second part of the curve beyond the initial sharp reduction and ensured a maximal differ-ential between normal and ATM-deficient cells. It also reflected the maximum clinically achievable dose, making the cellular effects at this dose clinically important.

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Combination Olaparib/cytotoxic treatment[4]
Granta-519 cells seeded in triplicate at 1 × 105 cells/mL in a 200µL volume were pretreated with Olaparib (dose range 0-10µM) for 2 days. Subsequently, increasing doses of 4-hydroxycyclophosphamide (4HC; 0-0.25µM; NIOMECH), fludarabine (0-0.5µM), valproic acid (VPA; 0-10mM), bendamustine (0-12.5µM), and IR (0-5 Gy) were added to the olaparib-containing culture for an additional 5 days. This time frame enabled consistency in the duration of olaparib treatment (7 days total) and allowed sufficient time for the cytotoxic effects of the conventional agents to occur before calculation of the surviving fraction of cells. Cell viability was measured using the CellTiter-Glo luminescent cell viability assay according to the manufacturer's instructions. Luminescence was quantified using a Wallac Victor2 1420 multilabel counter. Synergism was determined using Calcusyn Version 2.1 for Windows software.

Mice: Four treatment groups (n = 5) are randomly assigned to mice with tumors measuring 220-250 mm³: Vehicle control (10% DMSO in PBS/10% 2-hydroxy-propyl-β-cyclodextrin daily for 5 days by oral gavage), Olaparib (50 mg/kg daily for 5 days by oral gavage), 10 Gy fractionated radiotherapy (2 Gy daily for 5 days), and Olaparib and 10 Gy (5×2 Gy) fractionated radiotherapy (with olaparib given 30 min prior to each daily 2 Gy dose of radiation) are the options available. Measurements of tumor volume are made every day until they reach 1000 mm³. For each group of tumors, the number of days needed for each tumor to quadruple in size from the beginning of the treatment is calculated (relative tumor volume×4; RTV4).

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Mouse Xenografts [1]
Tagged mice were inoculated sc with 5 × 106 cells in 0.1 mL of PBS to the right flank. Tumors were measured thrice weekly, and tumor volumes were estimated from the formula [length/2] × [width2]. For xenograft chemopotentiation studies, established tumors in each animal were individually normalized to their size at the start of the experiment, and the data were calculated as the change in tumor volume relative to the day 0 volume by the use of the relative tumor volume (RTV) formula, RTV = TVx/TV0, where TVx is the tumor volume on any day and TV0 is the tumor volume at the initiation of dosing (i.e., day 0). In the tumor growth curves, the mean represents a full complement of animals in the treatment groups; below this threshold, we ceased plotting.

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Dosing Regimen [1]
When the mean tumor volume reached 100 mm3, tumor-bearing mice were randomized into treatment groups with eight animals in each group being dosed orally once daily for 5 consecutive days (po, q.d., ×5); for the combination therapies, the PARP inhibitor was administered 45 min before TMZ. Compound 47/Olaparib was formulated in solution, and TMZ was formulated as a homogeneous suspension in corn oil; both dosing solutions were freshly made each day. Mice in the no-treatment group received both vehicles on a mg/kg basis. Mice were weighed daily during the dosing phase to calculate the day’s dose and any signs of body weight loss accurately (20% weight loss led to the animal being euthanized). Statistical analyses were calculated on the data only when a full complement of animals was present in the treatment groups (i.e., day 13 for the vehicle and 47 monotherapy groups and day 45 for the TMZ and combination comparisons). From the Jonckheere−Terpstra trend test, we concluded that at day 45 there was a statistically significant effect of increasing the dose of 47 (10 mg/kg, data shown only in Figure 3) when used in combination with 50 mg/kg TMZ as compared with that of TMZ alone (p < 0.0001). This was confirmed by the use of Wilcoxon rank-sum tests, which compared the combination treatment groups versus TMZ alone to demonstrate statistically significant differences between TMZ monotherapy and TMZ in combination with 47 at 10 mg/kg (p = 0.007) at day 45.

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Pharmacokinetics [1]
We carried out pharmacokinetics determinations in mouse, rat, and dog. All doses of the compounds were given as a single dose either intravenously (iv) or orally (po; see dose level, as described in Tables 6 and 7). For the iv studies, the compounds were formulated in a mixture of 10% DMSO/10% cyclodextrin in PBS. For the oral studies, the compounds were also predominantly formulated as a solution in 10% DMSO/10% cyclodextrin in PBS except for compound 47, where a suspension in methylcellulose/PBS was used for oral dosing in dogs.

Absorption, Distribution and Excretion
Following oral administration, olaparib is rapidly absorbed. After administration of a single 300 mg dose of olaparib, the mean (CV%) Cmax was 5.4 μg/mL (32%) and AUC was 39.2 μg x h/mL (44%). The steady state Cmax and AUC following a dose of 300 mg twice daily was 7.6 μg/mL (35%) and 49.2 μg x h/mL (44%), respectively. Tmax is 1.5 hours. A high-fat and high-calorie meal may delay Tmax, but does not significantly alter the extent of olaparib absorption.
Following a single dose of radiolabeled olaparib, 86% of the dosed radioactivity was recovered within a seven-day collection period, mostly in the form of metabolites. About 44% of the drug was excreted via the urine and 42% of the dose was excreted via the feces. Following an oral dose of radiolabeled olaparib to female patients, the unchanged drug accounted for 15% and 6% of the radioactivity in urine and feces, respectively.
The mean (± standard deviation) apparent volume of distribution of olaparib is 158 ± 136 L following a single 300 mg dose.
Following a single oral dose in patients with cancer, the mean apparent plasma clearance was 4.55 L/h.

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Metabolism / Metabolites
Olaparib is metabolized by cytochrome P450 (CYP) 3A4/5 _in vitro_. Following an oral dose of radiolabeled olaparib to female patients, unchanged olaparib accounted for 70% of the circulating radioactivity in plasma. Olaparib undergoes oxidation reactions as well as subsequent glucuronide or sulfate conjugation. In humans, olaparib can also undergo hydrolysis, hydroxylation, and dehydrogenation. While up to 37 metabolites of olaparib were detected in plasma, urine, and feces, the majority of metabolites represent less than 1% of the total administered dose and they have not been fully characterized. The major circulating metabolites are a ring-opened piperazin-3-ol moiety and two mono-oxygenated metabolites. The pharmacodynamic activity of the metabolites is unknown.

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Biological Half-Life
Following a single oral dose in patients with cancer, the mean terminal half-life was 6.10 hours.

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Pharmacokinetic Profiles [1]
To establish whether one of these compounds (46 or 47/Olaparib) had the right PK profile to warrant its selection as an orally bioavailable candidate for clinical use, we initially evaluated both compounds for oral bioavailability and pharmacokinetic stability in rats (Table 7). Despite these compounds being structurally comparable and profiling very similarly in terms of their rat iv PK values, cyclopropyl compound 47/Olaparib showed a significantly greater oral exposure than did compound 46, which confirms the mouse data. Accordingly, compound 47 was advanced to further pharmacokinetic analysis in dogs. Table 7 highlights the PK profile of 47 in dog following its dosing iv at 2.5 mg/kg and po at 10 mg/kg in a methylcellulose vehicle as a suspension formulation. The data show that this compound retains an excellent level of bioavailability that is similar to that seen in rat but has a lower relative clearance that equates to approximately 16% hepatic blood flow (68% in rat). As such, this pharmacokinetic profile provided therapeutically relevant levels of PARP inhibitor over at least 4 h in this species. Cyclopropylamide analog 47 was shown to possess good exposure together with attributes of potency and physicochemistry such that this compound was advanced to further analysis of its cellular potency and in vivo efficacy to identify it as a potential clinical candidate.

Hepatotoxicity
In large clinical trials of olaparib, abnormalities in routine liver tests were uncommon with serum aminotransferase elevations occurring in 4% of patients and values above 5 times the upper limit of normal (ULN) in 1% or less. In trials of olaparib in patients with various advanced solid tumors there were no reports of hepatitis with jaundice or liver failure. Subsequent to its approval and more widescale use, there have been no published reports of clinically apparent liver injury attributed to olaparib.
Likelihood score: E (unlikely 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 olaparib during breastfeeding. Because olaparib is 82% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during olaparib therapy and for one month after the last dose.
◉ 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
The protein binding of olaparib is approximately 82% _in vitro_. In solutions of purified proteins, the olaparib fraction bound to albumin was approximately 56% and the fraction bound to alpha-1 acid glycoprotein was 29%.

[1]. J Med Chem . 2008 Oct 23;51(20):6581-91.

[2]. Clin Cancer Res . 2008 Jun 15;14(12):3916-25.

[3]. Proc Natl Acad Sci U S A . 2008 Nov 4;105(44):17079-84.

[3]. Blood . 2010 Nov 25;116(22):4578-87.

Olaparib is a member of the class of N-acylpiperazines obtained by formal condensation of the carboxy group of 2-fluoro-5-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzoic acid with the free amino group of N-(cyclpropylcarbonyl)piperazine; used to treat advanced ovarian cancer. It has a role as an antineoplastic agent, an EC 2.4.2.30 (NAD(+) ADP-ribosyltransferase) inhibitor and an apoptosis inducer. It is a N-acylpiperazine, a member of cyclopropanes, a member of monofluorobenzenes and a member of phthalazines.
Olaparib is a selective and potent inhibitor of poly (ADP-ribose) polymerase (PARP) enzymes, PARP1 and PARP2. PARP inhibitors represent a novel class of anti-cancer therapy and they work by taking advantage of a defect in DNA repair in cancer cells with BRCA mutations and inducing cell death. Olaparib is used to treat a number of BRCA-associated tumours, including ovarian cancer, breast cancer, pancreatic cancer, and prostate cancer. It was first approved by the FDA and EU in December 2014, and by Health Canada in April 2016.
Olaparib is a Poly(ADP-Ribose) Polymerase Inhibitor. The mechanism of action of olaparib is as a Poly(ADP-Ribose) Polymerase Inhibitor.
Olaparib is a small molecule inhibitor of poly ADP-ribose polymerase and is used as an antineoplastic agent in the therapy of refractory and advanced ovarian carcinoma. Olaparib therapy is associated with a low rate of transient elevations in serum aminotransferase during therapy and has not been linked to instances of clinically apparent liver injury.
Olaparib is a small molecule inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) with potential chemosensitizing, radiosensitizing, and antineoplastic activities. Olaparib selectively binds to and inhibits PARP, inhibiting PARP-mediated repair of single strand DNA breaks; PARP inhibition may enhance the cytotoxicity of DNA-damaging agents and may reverse tumor cell chemoresistance and radioresistance. PARP catalyzes post-translational ADP-ribosylation of nuclear proteins and can be activated by single-stranded DNA breaks.

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Drug Indication
**Ovarian cancer** Olaparib is indicated for the maintenance treatment of adults with deleterious or suspected deleterious germline or somatic BRCA-mutated advanced epithelial ovarian, fallopian tube or primary peritoneal cancer who are in complete or partial response to first-line platinum-based chemotherapy. Olaparib is indicated in combination with [bevacizumab] for the maintenance treatment of adults with advanced epithelial ovarian, fallopian tube or primary peritoneal cancer who are in complete or partial response to first-line platinum-based chemotherapy and whose cancer is associated with homologous recombination deficiency (HRD)-positive status defined by either: a deleterious or suspected deleterious BRCA mutation, and/or genomic instability. Olaparib is indicated for the maintenance treatment of adult patients with recurrent epithelial ovarian, fallopian tube or primary peritoneal cancer, who are in complete or partial response to platinum-based chemotherapy. **Breast cancer** Olaparib is indicated for the adjuvant treatment of adult patients with deleterious or suspected deleterious g_BRCA_m human epidermal growth factor receptor 2 (HER2)-negative high risk early breast cancer who have been treated with neoadjuvant or adjuvant chemotherapy. Olaparib is indicated for the treatment of adult patients with deleterious or suspected deleterious g_BRCA_m, HER2-negative metastatic breast cancer, who have been treated with chemotherapy in the neoadjuvant, adjuvant, or metastatic setting. Patients with hormone receptor (HR) positive breast cancer should have been treated with a prior endocrine therapy or be considered inappropriate for endocrine therapy. **Pancreatic cancer** Olaparib is indicated for the maintenance treatment of adult patients with deleterious or suspected deleterious gBRCAm metastatic pancreatic adenocarcinoma whose disease has not progressed on at least 16 weeks of a first-line platinum-based chemotherapy regimen. **Prostate cancer** Olaparib is indicated for the treatment of adult patients with deleterious or suspected deleterious germline or somatic homologous recombination repair (HRR) gene-mutated metastatic castration-resistant prostate cancer (mCRPC) who have progressed following prior treatment with a hormone agent, such as [enzalutamide] or [abiraterone]. It is also indicated in combination with [abiraterone] and [prednisone] or [prednisolone] for the treatment of adult patients with deleterious or suspected deleterious BRCA-mutated (BRCAm) metastatic castration-resistant prostate cancer (mCRPC).
Ovarian cancer Lynparza is indicated as monotherapy for the: maintenance treatment of adult patients with advanced (FIGO stages III and IV) BRCA1/2-mutated (germline and/or somatic) high-grade epithelial ovarian, fallopian tube or primary peritoneal cancer who are in response (complete or partial) following completion of first-line platinum-based chemotherapy. maintenance treatment of adult patients with platinum sensitive relapsed high grade epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in response (complete or partial) to platinum based chemotherapy. Lynparza in combination with bevacizumab is indicated for the: maintenance treatment of adult patients with advanced (FIGO stages III and IV) high-grade epithelial ovarian, fallopian tube or primary peritoneal cancer who are in response (complete or partial) following completion of first-line platinum-based chemotherapy in combination with bevacizumab and whose cancer is associated with homologous recombination deficiency (HRD) positive status defined by either a BRCA1/2 mutation and/or genomic instability (see section 5. 1). Breast cancer Lynparza is indicated as: monotherapy or in combination with endocrine therapy for the adjuvant treatment of adult patients with germline BRCA1/2-mutations who have HER2-negative, high risk early breast cancer previously treated with neoadjuvant or adjuvant chemotherapy (see sections 4. 2 and 5. 1). monotherapy for the treatment of adult patients with germline BRCA1/2-mutations, who have HER2 negative locally advanced or metastatic breast cancer . Patients should have previously been treated with an anthracycline and a taxane in the (neo)adjuvant or metastatic setting unless patients were not suitable for these treatments (see section 5. 1). Patients with hormone receptor (HR)-positive breast cancer should also have progressed on or after prior endocrine therapy, or be considered unsuitable for endocrine therapy. Adenocarcinoma of the pancreasLynparza is indicated as: monotherapy for the maintenance treatment of adult patients with germline BRCA1/2-mutations who have metastatic adenocarcinoma of the pancreas and have not progressed after a minimum of 16 weeks of platinum treatment within a first-line chemotherapy regimen. Prostate cancer Lynparza is indicated as: monotherapy for the treatment of adult patients with metastatic castration-resistant prostate cancer (mCRPC) and BRCA1/2-mutations (germline and/or somatic) who have progressed following prior therapy that included a new hormonal agent. in combination with abiraterone and prednisone or prednisolone for the treatment of adult patients with mCRPC in whom chemotherapy is not clinically indicated (see section 5. 1).
Treatment of all conditions included in the category of malignant neoplasms (except central nervous system tumours, haematopoietic and lymphoid tissue neoplasms)

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Mechanism of Action
Poly(ADP-ribose) polymerases (PARPs) are multifunctional enzymes comprising 17 members. They are involved in essential cellular functions, such as DNA transcription and DNA repair. PARPs recognize and repair cellular DNA damage, such as single-strand breaks (SSBs) and double-strand breaks (DSBs). Different DNA repair pathways exist to repair these DNA damages, including the base excision repair (BER) pathway for SSBs and BRCA-dependent homologous recombination for DSBs. Olaparib is a PARP inhibitor: while it acts on PARP1, PARP2, and PARP3, olaparib is a more selective competitive inhibitor of NAD⁺ at the catalytic site of PARP1 and PARP2. Inhibition of the BER pathway by olaparib leads to the accumulation of unrepaired SSBs, which leads to the formation of DSBs, which is the most toxic form of DNA damage. While BRCA-dependent homologous recombination can repair DSBs in normal cells, this repair pathway is defective in cells with BRCA1/2 mutations, such as certain tumour cells. Inhibition of PARP in cancer cells with BRCA mutations leads to genomic instability and apoptotic cell death. This end result is also referred to as synthetic lethality, a phenomenon where the combination of two defects - inhibition of PARP activity and loss of DSB repair by HR - that are otherwise benign when alone, lead to detrimental results. _In vitro_ studies have shown that olaparib-induced cytotoxicity may involve inhibition of PARP enzymatic activity and increased formation of PARP-DNA complexes, resulting in DNA damage and cancer cell death.

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Poly(ADP-ribose) polymerase activation is an immediate cellular response to metabolic-, chemical-, or ionizing radiation-induced DNA damage and represents a new target for cancer therapy. In this article, we disclose a novel series of substituted 4-benzyl-2 H-phthalazin-1-ones that possess high inhibitory enzyme and cellular potency for both PARP-1 and PARP-2. Optimized compounds from the series also demonstrate good pharmacokinetic profiles, oral bioavailability, and activity in vivo in an SW620 colorectal cancer xenograft model. 4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2 H-phthalazin-1-one (KU-0059436, AZD2281) 47 is a single digit nanomolar inhibitor of both PARP-1 and PARP-2 that shows standalone activity against BRCA1-deficient breast cancer cell lines. Compound 47 is currently undergoing clinical development for the treatment of BRCA1- and BRCA2-defective cancers.[1]

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The Ataxia Telangiectasia Mutated (ATM) gene is frequently inactivated in lymphoid malignancies such as chronic lymphocytic leukemia (CLL), T-prolymphocytic leukemia (T-PLL), and mantle cell lymphoma (MCL) and is associated with defective apoptosis in response to alkylating agents and purine analogues. ATM mutant cells exhibit impaired DNA double strand break repair. Poly (ADP-ribose) polymerase (PARP) inhibition that imposes the requirement for DNA double strand break repair should selectively sensitize ATM-deficient tumor cells to killing. We investigated in vitro sensitivity to the poly (ADP-ribose) polymerase inhibitor olaparib (AZD2281) of 5 ATM mutant lymphoblastoid cell lines (LCL), an ATM mutant MCL cell line, an ATM knockdown PGA CLL cell line, and 9 ATM-deficient primary CLLs induced to cycle and observed differential killing compared with ATM wildtype counterparts. Pharmacologic inhibition of ATM and ATM knockdown confirmed the effect was ATM-dependent and mediated through mitotic catastrophe independently of apoptosis. A nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft model of an ATM mutant MCL cell line demonstrated significantly reduced tumor load and an increased survival of animals after olaparib treatment in vivo. Addition of olaparib sensitized ATM null tumor cells to DNA-damaging agents. We suggest that olaparib would be an appropriate agent for treating refractory ATM mutant lymphoid tumors.[4]

C24H23FN4O3

434.46

434.175

C, 66.35; H, 5.34; F, 4.37; N, 12.90; O, 11.05

763113-22-0

23725625

White solid powder

1.4±0.1 g/cm3

1.702

1.9

1

5

4

32

790

0

FC1C([H])=C([H])C(C([H])([H])C2C3=C([H])C([H])=C([H])C([H])=C3C(N([H])N=2)=O)=C([H])C=1C(N1C([H])([H])C([H])([H])N(C([H])([H])C1([H])[H])C(C1([H])C([H])([H])C1([H])[H])=O)=O

FDLYAMZZIXQODN-UHFFFAOYSA-N

InChI=1S/C24H23FN4O3/c25-20-8-5-15(14-21-17-3-1-2-4-18(17)22(30)27-26-21)13-19(20)24(32)29-11-9-28(10-12-29)23(31)16-6-7-16/h1-5,8,13,16H,6-7,9-12,14H2,(H,27,30)

4-[[3-[4-(cyclopropanecarbonyl)piperazine-1-carbonyl]-4-fluorophenyl]methyl]-2H-phthalazin-1-one

AZD2281; Ku-0059436; AZD2281; 763113-22-0; Lynparza; KU-0059,436; 1-(Cyclopropylcarbonyl)-4-[5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoyl]piperazine; AZD-2281; AZD 2281; KU59436; KU-59436; KU 59436; KU0059436; KU-0059436; KU 0059436; Olaparib; trade name Lynparza

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: 10 mg/mL (23.02 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 100.0 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: ≥ 5 mg/mL (11.51 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 3: ≥ 5 mg/mL (11.51 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 4: ≥ 2.5 mg/mL (5.75 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 5: ≥ 2.5 mg/mL (5.75 mM) (saturation unknown) in 10% DMF 40% PEG300 + 5% Tween80 + 45% 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 6: ≥ 2.5 mg/mL (5.75 mM) (saturation unknown) in 10% DMF 90% (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 7: ≥ 2.5 mg/mL (5.75 mM) (saturation unknown) in 10% DMF 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.

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Solubility in Formulation 8: ≥ 2.08 mg/mL (4.79 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 9: ≥ 0.5 mg/mL (1.15 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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Solubility in Formulation 10: 20 mg/mL (46.03 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.)