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
Olaparib is rapidly absorbed after oral administration. Following a single 300 mg dose of olaparib, the mean (CV%) Cmax was 5.4 μg/mL (32%), and the AUC was 39.2 μg·h/mL (44%). Following two daily doses of 300 mg, the steady-state Cmax and AUC were 7.6 μg/mL (35%) and 49.2 μg·h/mL (44%), respectively. Tmax was 1.5 hours. High-fat, high-calorie diets may delay Tmax but do not significantly alter the absorption of olaparib. Following a single dose of radiolabeled olaparib, 86% of the administered radioactivity was recovered over a 7-day collection period, primarily as metabolites. Approximately 44% of the drug is excreted in the urine, and 42% is excreted in the feces. Following oral administration of radiolabeled olaparib to female patients, 15% and 6% of the radioactive material in urine and feces, respectively, were unchanged olaparib. The mean (± standard deviation) apparent volume of distribution after a single oral dose of 300 mg olaparib was 158 ± 136 L. The mean apparent plasma clearance after a single oral dose in cancer patients was 4.55 L/h. Olaparib is metabolized in vitro by cytochrome P450 (CYP) 3A4/5. Following oral administration of radiolabeled olaparib to female patients, 70% of the circulating radioactive material in plasma was unchanged olaparib. Olaparib undergoes oxidation followed by glucuronide or sulfate conjugation. In the human body, olaparib also undergoes hydrolysis, hydroxylation, and dehydrogenation. Although up to 37 olaparib metabolites were detected in plasma, urine, and feces, most of them accounted for less than 1% of the total administered dose and have not been fully characterized. The major circulating metabolites are an open-ring piperazine-3-ol moiety and two monooxygenated metabolites. The pharmacodynamic activities of these metabolites are unknown.

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Biological Half-Life
The mean terminal half-life after a single oral administration in cancer patients is 6.10 hours.

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Pharmacokinetic Characteristics[1]
To determine whether either compound 46 or 47/olaparib has a suitable pharmacokinetic profile sufficient to be a candidate drug with high oral bioavailability for clinical use, we first evaluated the oral bioavailability and pharmacokinetic stability of the two compounds in rats (Table 7). Although the compounds are structurally similar and exhibit very similar pharmacokinetic parameters after intravenous administration in rats, the oral exposure of cyclopropyl compound 47/olaparib was significantly higher than that of compound 46, consistent with the mouse data. Therefore, compound 47 was advanced to dogs for further pharmacokinetic analysis. Table 7 highlights the pharmacokinetic characteristics of compound 47 in dogs after intravenous administration of 2.5 mg/kg and oral administration of 10 mg/kg via methylcellulose. The data showed that the compound maintained excellent bioavailability similar to that in rats, but with a lower relative clearance, equivalent to approximately 16% of hepatic blood flow (compared to 68% in rats). Therefore, these pharmacokinetic characteristics suggest that the PARP inhibitor can maintain treatment-relevant concentrations in this species for at least 4 hours. Cyclopropamide analog 47 exhibits favorable exposure levels, potency, and physicochemical properties, therefore this compound was advanced to further analyses of cellular potency and in vivo efficacy to determine its potential clinical candidate status.

Hepatotoxicity
In large clinical trials of olaparib, abnormalities in routine liver function tests were uncommon, with elevated serum transaminases observed in only 4% of patients, and levels exceeding 5 times the upper limit of normal (ULN) in only 1% or fewer. No reports of hepatitis with jaundice or liver failure have been observed in trials of olaparib for patients with various advanced solid tumors. Since olaparib's approval and widespread use, no clinically significant cases of liver injury have been reported. Probability score: E (unlikely to be the 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 olaparib during lactation. Because olaparib binds to plasma proteins at a rate of 82%, its levels in breast milk are likely to be low. The manufacturer recommends discontinuing breastfeeding during olaparib treatment and for one month after the last dose.
◉ 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
Olaparib's protein binding rate is approximately 82% in vitro. In purified protein solutions, olaparib binds approximately 56% to albumin and 29% to α-1 acid glycoprotein.

[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 an N-acylpiperazine compound formed by the condensation of the carboxyl group of 2-fluoro-5-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzoic acid with the free amino group of N-(cyclopropylcarbonyl)piperazine; it is used to treat advanced ovarian cancer. It possesses antitumor activity and is an EC 2.4.2.30 (NAD(+) ADP-ribosyltransferase) inhibitor and apoptosis inducer. It belongs to the N-acylpiperazine, cyclopropane, monofluorobenzene, and phthalazine classes of compounds. Olaparib is a selective and potent inhibitor of poly(ADP-ribose) polymerase (PARP) 1 and PARP 2. PARP inhibitors are a novel class of anticancer therapies whose mechanism of action involves inducing cell death by exploiting DNA repair defects in cancer cells carrying BRCA mutations. Olaparib is used to treat various BRCA-related tumors, including ovarian cancer, breast cancer, pancreatic cancer, and prostate cancer. It was first approved by the US FDA and EU in December 2014, and by Health Canada in April 2016. Olaparib is a poly(ADP-ribose) polymerase inhibitor. Its mechanism of action is as a poly(ADP-ribose) polymerase inhibitor. Olaparib is a small-molecule poly(ADP-ribose) polymerase inhibitor used to treat refractory and advanced ovarian cancer. The incidence of transient elevations in serum transaminases during olaparib treatment is low, and no clinically significant cases of liver injury have been found. Olaparib is a small-molecule ribonucleotide poly(ADP-ribose) polymerase (PARP) inhibitor with potential chemosensitizing, radiosensitizing, and antitumor activities. Olaparib selectively binds to and inhibits PARP, thereby inhibiting PARP-mediated single-strand DNA break repair; PARP inhibition may enhance the cytotoxicity of DNA-damaging drugs and may reverse chemosensitivity and radioresistance in tumor cells. PARP catalyzes post-translational ADP ribosylation of nucleoproteins and can be activated by single-strand DNA breaks. Drug Indications Ovarian Cancer Olaparib is indicated for maintenance therapy in adult patients with advanced epithelial ovarian, fallopian tube, or primary peritoneal cancer who have achieved complete or partial remission to first-line platinum-based chemotherapy and carry pathogenic or suspected pathogenic germline or somatic BRCA mutations. Olaparib in combination with bevacizumab is indicated for maintenance therapy in adult patients with advanced epithelial ovarian, fallopian tube, or primary peritoneal cancer who have achieved complete or partial remission to first-line platinum-based chemotherapy and whose cancer is associated with a homologous recombination deficiency (HRD) positive status, defined as pathogenic or suspected pathogenic BRCA mutations and/or genomic instability. Olaparib is indicated for maintenance therapy in adult patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer who have achieved complete or partial remission to platinum-based chemotherapy. Breast Cancer Olaparib is indicated for adjuvant therapy in adult patients with high-risk early-stage breast cancer who have received neoadjuvant or adjuvant chemotherapy, carry pathogenic or suspected pathogenic gBRCA m mutations, and are HER2-negative. Olaparib is also indicated for the treatment of adult patients with metastatic breast cancer who have received neoadjuvant, adjuvant, or metastatic chemotherapy, carry pathogenic or suspected pathogenic gBRCA m mutations, and are HER2-negative. Hormone receptor (HR)-positive breast cancer patients should have received endocrine therapy or be considered unsuitable for endocrine therapy. Pancreatic Cancer Olaparib is indicated for maintenance therapy in adult patients with metastatic pancreatic adenocarcinoma carrying pathogenic or suspected pathogenic gBRCA m mutations who have not progressed after at least 16 weeks of first-line platinum-based chemotherapy. Prostate Cancer Olaparib is indicated for the treatment of adult patients with metastatic castration-resistant prostate cancer (mCRPC) whose disease has progressed after treatment with hormonal drugs such as enzalutamide or abiraterone and who harbor pathogenic or suspected pathogenic germline or somatic homologous recombination repair (HRR) gene mutations. It can also be used in combination with abiraterone and prednisone or prednisolone for the treatment of adult patients with metastatic castration-resistant prostate cancer (mCRPC) harboring pathogenic or suspected pathogenic BRCA mutations (BRCAm). Lynparza can be used as monotherapy for: maintenance therapy in adult patients with advanced (FIGO stage III and IV) BRCA1/2-mutated (germline and/or somatic mutations) high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer who have achieved complete or partial remission after first-line platinum-based chemotherapy; and maintenance therapy in adult patients with platinum-sensitive recurrent high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer who have achieved complete or partial remission after platinum-based chemotherapy. Lynparza can also be used in combination with bevacizumab for: maintenance therapy in adult patients with advanced (FIGO stage III and IV) high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer who have achieved complete or partial remission after first-line platinum-based chemotherapy combined with bevacizumab, whose cancer is associated with homologous recombination deficiency (HRD) positive status, defined by BRCA1/2 mutations and/or genomic instability (see Section 5.1). The indications for Lynparza in breast cancer include: monotherapy or adjuvant therapy in combination with endocrine therapy for adult patients with HER2-negative, high-risk early breast cancer who harbor germline BRCA1/2 mutations and have previously received neoadjuvant or adjuvant chemotherapy (see Sections 4.2 and 5.1). It is also indicated for monotherapy in adult patients with HER2-negative, locally advanced or metastatic breast cancer who harbor germline BRCA1/2 mutations. Patients should have previously received (neo)adjuvant or metastatic therapy with anthracyclines and taxanes, unless they are not suitable for these treatments (see Section 5.1). Patients with hormone receptor (HR)-positive breast cancer should also have experienced disease progression during or after previous endocrine therapy, or be deemed unsuitable for endocrine therapy. For pancreatic adenocarcinoma: Lynparza is indicated for monotherapy maintenance therapy in adult patients with metastatic pancreatic adenocarcinoma who harbor germline BRCA1/2 mutations and have not experienced disease progression after at least 16 weeks of platinum-based chemotherapy in a first-line chemotherapy regimen. Lynparza is indicated for prostate cancer: as monotherapy in adult patients with metastatic castration-resistant prostate cancer (mCRPC) whose disease has progressed after prior treatment, including with novel hormonal agents, and who carry BRCA1/2 mutations (germline and/or somatic mutations). In combination with abiraterone and prednisone or prednisolone, it can be used to treat adult patients with mCRPC who are ineligible for chemotherapy (see Section 5.1). It can also be used to treat all malignancies (excluding central nervous system tumors, hematopoietic and lymphoid tissue tumors). Mechanism of Action: Poly(ADP-ribose) polymerases (PARPs) are a class of multifunctional enzymes comprising 17 members. They are involved in important cellular functions such as DNA transcription and DNA repair. PARPs can recognize and repair cellular DNA damage, such as single-strand breaks (SSBs) and double-strand breaks (DSBs). Different DNA repair pathways can repair these DNA damages, including the base excision repair (BER) pathway for SSB repair and the BRCA-dependent homologous recombination pathway for DSB repair. Olaparib is a PARP inhibitor: while it acts on PARP1, PARP2, and PARP3, olaparib is a more selective competitive inhibitor that inhibits NAD+ at the catalytic sites of PARP1 and PARP2. Inhibition of the BER pathway by olaparib leads to the accumulation of unrepaired SSBs, which in turn form DSBs, the most toxic form of DNA damage. Although BRCA-dependent homologous recombination can repair DSBs in normal cells, this repair pathway is defective in cells carrying BRCA1/2 mutations, such as some tumor cells. Inhibition of PARP in cancer cells carrying BRCA mutations leads to genomic instability and apoptosis. This end result, also known as synthetic lethality, refers to the fact that the two defects—PARP activity inhibition and loss of homologous recombination (HR)-mediated DNA double-strand break (DSB) repair—are harmless individually, but together lead to detrimental consequences. In vitro studies have shown that olaparib-induced cytotoxicity may involve inhibition of PARP enzyme activity and increased PARP-DNA complex formation, leading to DNA damage and cancer cell death. Activation of poly(ADP-ribose) polymerase (PARP) is an immediate cellular response to metabolic, chemical, or ionizing radiation-induced DNA damage, representing a novel therapeutic target for cancer. This article reports a series of novel substituted 4-benzyl-2H-phthalazine-1-one compounds exhibiting high inhibitory activity against both PARP-1 and PARP-2, as well as cytotoxic activity. This series of optimized compounds also demonstrates favorable pharmacokinetic profiles, oral bioavailability, and in vivo activity in the SW620 colorectal cancer xenograft model. 4-[3-(4-cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2H-phthalazine-1-one (KU-0059436, AZD2281)47 is a nanomolar-level PARP-1 and PARP-2 inhibitor with independent inhibitory activity against BRCA1-deficient breast cancer cell lines. Compound 47 is currently in clinical development for the treatment of BRCA1 and BRCA2-deficient cancers. [1]

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Ataxia-telangiectasia mutant gene (ATM) is frequently inactivated in lymphatic malignancies such as chronic lymphocytic leukemia (CLL), T-pre-PLL, and mantle cell lymphoma (MCL) and is associated with defective apoptotic responses to alkylating agents and purine analogues. ATM mutant cells exhibit impaired DNA double-strand break repair. Inhibition of poly(ADP-ribose) polymerase (PARP) makes DNA double-strand break repair essential, thereby selectively enhancing the cytotoxic sensitivity of ATM-deficient tumor cells. We investigated the in vitro sensitivity of five ATM-mutant lymphoblastic cell lines (LCL), one ATM-mutant MCL cell line, one ATM-knockdown PGA CLL cell line, and nine induced-proliferation ATM-deficient primary CLL cells to the poly(ADP-ribose) polymerase inhibitor olaparib (AZD2281) and observed its differential cytotoxic effect compared to its ATM wild-type counterparts. Pharmacological inhibition and knockdown of ATM confirmed that this effect is ATM-dependent, mediated by mitotic catastrophe, and independent of apoptosis. In a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse xenograft model of ATM-mutant MCL cell lines, in vivo olaparib treatment significantly reduced tumor burden and prolonged survival. Adding olaparib enhances the sensitivity of ATM-deficient tumor cells to DNA-damaging agents. We believe that olaparib may be a suitable drug for the treatment of refractory ATM-mutant lymphoma. [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.)