PARP2 ( Ki = 0.87 nM ); PARP1 ( Ki = 1.2 nM )

BMN 673 is readily orally bioavailable, with more than 40% absolute oral bioavailability in rats when dosed in carboxylmethyl cellulose. Oral administration of BMN 673 elicited remarkable antitumor activity in vivo; xenografted tumors that carry defects in DNA repair due to BRCA mutations or PTEN deficiency were profoundly sensitive to oral BMN 673 treatment at well-tolerated doses in mice. Synergistic or additive antitumor effects were also found when BMN 673 was combined with temozolomide, SN38, or platinum drugs. Conclusion: BMN 673 is currently in early-phase clinical development and represents a promising PARP1/2 inhibitor with potentially advantageous features in its drug class.[1]

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Combinatorial clinical trials of PARP inhibitors with immunotherapies are ongoing, yet the immunomodulatory effects of PARP inhibition have been incompletely studied. Here, we sought to dissect the mechanisms underlying PARP inhibitor-induced changes in the tumor microenvironment of BRCA1-deficient triple-negative breast cancer (TNBC). We demonstrate that the PARP inhibitor olaparib induces CD8+ T-cell infiltration and activation in vivo, and that CD8+ T-cell depletion severely compromises antitumor efficacy. Olaparib-induced T-cell recruitment is mediated through activation of the cGAS/STING pathway in tumor cells with paracrine activation of dendritic cells and is more pronounced in HR-deficient compared with HR-proficient TNBC cells and in vivo models. CRISPR-mediated knockout of STING in cancer cells prevents proinflammatory signaling and is sufficient to abolish olaparib-induced T-cell infiltration in vivo. These findings elucidate an additional mechanism of action of PARP inhibitors and provide a rationale for combining PARP inhibition with immunotherapies for the treatment of TNBC. SIGNIFICANCE: This work demonstrates cross-talk between PARP inhibition and the tumor microenvironment related to STING/TBK1/IRF3 pathway activation in cancer cells that governs CD8+ T-cell recruitment and antitumor efficacy. The data provide insight into the mechanism of action of PARP inhibitors in BRCA-associated breast cancer.[3]

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BMN 673 exhibits >50% oralbioavailability and pharmacokinetic characteristics that permit single-daily dosing in rat pharmacokinetic studies. Daily oral dosing of BMN 673 significantly and dose-dependently increases the antitumor effects of cytotoxic therapies in MX-1 xenograft tumor model studies.[2]

In order to determine the PARP inhibitor Ki, enzyme assays were carried out in 96-well FlashPlate using 0.5 U PARP1 enzyme, 0.25x activated DNA, 0.2 mCi [3H] NAD, and 5 mmol/L cold NAD (Sigma) in a final volume of 50 mL reaction buffer that contained 10% glycerol (v/v), 25 mmol/L HEPES, 12.5 mmol/L MgCl2, 50 mmol/L KCl, 1 mmol/L dithiothreitol (DTT), and 0.01% NP-40 (v/v), and pH 7.6. NAD was added to the PARP reaction mixture, either with or without inhibitors, to start the reaction, and it was then incubated for one minute at room temperature. The reaction was then stopped by adding 50 microliter of ice-cold 20% trichloroacetic acid (TCA) to each well. After the plate was sealed and shaken for an additional 120 minutes at room temperature, centrifugation was performed. Top-Count was used to determine the radioactive signal bound to the FlashPlate. The Michaelis-Menten equation was used to calculate PARP1 Km at different substrate concentrations (ranging from 1 to 100 mmol/L NAD). Using the formula Ki ¼ IC50/[1þ (substrate)/Km], compound Ki was computed from the enzyme inhibition curve. Using the same assay protocol, Km for the PARP2 enzyme and compound Ki were found. However, instead of using 30 ng of PARP2, 0.25x activated DNA, 0.2 mCi [3H] NAD, and 20 mmol/L cold NAD, the reaction was run for 30 minutes at room temperature.

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PARP enzyme assays[1]
The ability of a test compound to inhibit PARP-1 enzyme activity was assessed using Trevigen’s PARP Assay Kit following manufacturer’s instruction. IC50 values were calculated using GraphPad Prism5 software. For PARP inhibitor Ki determination, enzyme assays were carried out in 96-well FlashPlate with 0.5 unit PARP1 enzyme, 0.25x activated DNA (Trevigen), 0.2 μCi [3H] NAD and 5 μM cold NAD in a final volume of 50 μL reaction buffer containing 10%glycerol(v/v), 25 mM Hepes, 12.5 mM MgCl2, 50 mM KCl, 1 mM DTT and 0.01% NP-40(v/v), pH 7.6. Reactions were initiated by adding NAD to the PARP reaction mixture with or without inhibitors and incubated for 1 min at room temperature. 50 μL of ice-cold 20% TCA was then added to each well to stop the reaction. The plate was sealed and shaken for a further 120 min at RT, followed by centrifugation. Radioactive signal bound to the FlashPlate was determined using TopCount. PARP1 Km was determined using Michaelis–Menten equation from various substrate concentrations (1-100 μM NAD). Compound Ki was calculated from enzyme inhibition curve according to the formula: Ki = IC50/(1+[substrate]/Km). Km for PARP2 enzyme and compound Ki were determined with the same assay protocol except 30 ng PARP2, 0.25x activated DNA, 0.2 μCi [3H] NAD and 20 μM cold NAD were used in the reaction for 30min at room temperature.

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Biacore binding assay[1]
Recombinant human PARP1 (rhPARP1) catalytic domain (residues 662 – 1011) with N-terminal 6XHis-tag was generated in house and used in binding assay for PARP inhibitor interaction using Biacore T200 (GE Healthcare). rhPARP1 was immobilized on a CM5 sensor chip by amine coupling method. Briefly, one flow cell of a CM5 chip was first activated by a 7-min injection at 10 μL/min of freshly prepared 50 mM NHS: 200 mM EDC (1:1) at rate of 10 μL/min. Then rhPARP1 (100 μg/mL, in 10 mM MES pH 6.5) was injected onto the flow cell for 60-sec at 10 μL/min. The remaining active coupling sites were blocked with a 7-min injection of 1M ethanolamine at 10 μL/min. The immobilization buffer contains 10 mM Hepes pH 7.4, 150 mM NaCl, 0.05% Surfectant P20, 5 mM MgCl2, and 0.5 mM TCEP (tris(2-carboxyethyl)phosphine). The immobilization level was ~7600 RU. For binding kinetics measurement, PARP inhibitors at increasing concentrations (12.5, 25, 50, 100, 200 nM) were injected over the chip surface for 60 sec per injection. The exposure was followed by a dissociation phase of 3600 sec in running buffer (immobilization buffer + 1% DMSO) after the last injection. The flow rate was 50 μL/min. After sensorgrams were corrected for signals from a reference flow, kinetics was calculated with Biacore T200 evaluation software ver.1.0.

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Intracellular PAR formation assay[1]
Cellular PAR synthesis assay assesses the ability of a test compound to inhibit polymerization of PAR. LoVo human colorectal tumor cells grown in 96-well microtiter plates overnight were pre-treated with increasing concentrations of PARP inhibitors for 30 min before H2O2 was added at a final concentration of 50 mM. After a 5-min treatment at room temperature, cells were fixed for 10 minutes with pre-chilled methanol/acetone(7:3) at −20 °C. Fixed cells were incubated with anti-PAR monoclonal antibody for 60 min, followed by incubation with FITC coupled goat anti-mouse IgG (diluted 1:100) and 1 μg/mL DAPI for 60 min. FITC signal was normalized with DAPI signal, and EC50 values were calculated using GraphPad Prism.

A panel of 11 SCLC cell lines (IC50=1.7 to 15 nmol/L), all of which fall within clinically feasible ranges, demonstrate Talazoparib (BMN 673; MDV3800) 's strong inhibitory action. Furthermore, PI3K pathway activity and DNA repair protein expression are correlated with Talazoparib (BMN 673; MDV3800)  sensitivity.

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Confocal microscopy[1]
Cells were seeded on coverslips placed in 6-well plates and after 24 hours treated with several concentrations of olaparib or Talazoparib (BMN 673; MDV3800). 24 hours after treatment the cells were fixed in 10% formalin (3.7% PFA) for 1 hour. Cells were permeabilized with 0.2% Triton X-100 in PBS for 20 minutes, treated with 50 μL DNase I (diluted 1/10 in PBS) for 1 hour at 37°C and then blocked with IFF (PBS + 1% BSA and 2% FBS followed by filter sterilization) for 1 hour. The coverslips were then incubated with rabbit anti-γH2Ax primary (Millipore) and mouse anti-RAD51 primary (both 1:1000 in 50μL IFF) overnight at 4°C. The next day cells were incubated with anti-mouse Alexafluor 546 secondary and anti-rabbit Alexafluor 488 secondary (both 1:1000 in 50μL IFF) for one hour. Cells were then washed in PBS containing DAPI 1:10.000 for 10 minutes and attached on glass plates using Vectashield and nail polish. A minimum of four pictures were made of each coverslip using the Leica confocal microscope, and cells were subsequently counted. At least 100 cells were assessed per coverslip, being positive for γH2Ax if they had more than 5 foci per nucleus. The percentage of positive cells was plotted.

Xenograft experiments[1]
Female athymic nu/nu mice (8-10 week old) were used for all in vivo xenograft studies. Mice were quarantined for at least 1 week before experimental manipulation. Exponentially growing cells (LNcap, MDA-MB-468) or in vivo passaged tumor fragments (MX-1) were implanted subcutaneously at the right flank of nude mice. When tumors reached an average volume of ~150 mm3, mice were randomized into various treatment groups (6-8 mice/group) in each study. Mice were visually observed daily and tumors were measured twice weekly by calliper to determine tumor volume using the formula [length/2] × [width2]. Group median tumor volume (mm3) was graphed over time to monitor tumor growth. In single agent studies, olaparib (100mg/kg), Talazoparib (BMN 673; MDV3800)  (various doses as indicated), or vehicle (10% DMAc, 6% Solutol and 84% PBS) was administered by oral gavage (p.o.), once daily or Talazoparib (BMN 673; MDV3800)  (0.165 mg/kg) twice daily for 28 consecutive days. Mice were continuously monitored for 10 more days after last day of dosing. In cisplatin combination study, Talazoparib (BMN 673; MDV3800) , olaparib, or vehicle was administered p.o. once daily for 8 days starting on day 1. Cisplatin at a dosage of 6 mg/kg or its vehicle (saline) was administered intra-peritoneally (i.p) as a single injection on day 3, 30 minutes after PARP inhibitor was administered. Combination with carboplatin was conducted in a similar way in MX-1 model in which Talazoparib (BMN 673; MDV3800)  was administered p.o. once daily for either 8 days or 5 days and carboplatin was injected i.p. at single dose of 35 mg/kg, 30 min after Talazoparib (BMN 673; MDV3800)  on day 3.[1]

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PAR assay in vivo[1]
MX-1 tumor xenografts were prepared as described in methods. When tumors reached an average volume of ~150 mm3, olaparib (100 mg/kg), Talazoparib (BMN 673; MDV3800)  (1 mg/kg) or vehicle was administered in a single p.o. dosing. Tumors were harvested at 2, 8 and 24 hours after drug dosing, snap frozen in liquid N2. Tumor tissue was then homogenized in PBS on ice and extracted with lysis buffer (25mM Tris pH 8.0, 150mM NaCl, 5mM EDTA, 2mM EGTA, 25mM NaF, 2mM Na3VO4, 1mM Pefabloc, 1% Triton X-100, and protease inhibitor cocktail) containing 1% SDS. Levels of PAR in the tumor lysates were determined by ELISA using PARP in vivo PD Assay II kit.

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0.33 and 0.1 mg/kg; Oral gavage and twice daily for 28 consecutive days.

Nude mice bearing established subcutaneous MX-1 tumor xenografts.

Absorption, Distribution and Excretion
After administration of talazoparib 1 mg orally once daily, the mean [% coefficient of variation (CV%)] AUC and maximum observed plasma concentration (Cmax) of talazoparib at steady-state was 208 (37%) ng x hr/mL and 16.4 (32%) ng/mL, respectively. The mean (CV%) steady-state Ctrough was 3.53 (61%) ng/mL. Steady state was reached within two to three weeks of therapy. The Tmax ranges from one to two hours. A high-fat, high-calorie food increased the mean Cmax by 46% and the median Tmax from one to four hours, without affecting the AUC.
The major route of elimination is renal excretion. Approximately 68.7% of the total administered radiolabeled dose of talazoparib was recovered in urine, where 54.6% of that dose was in the form of an unchanged drug. About 19.7% of the drug was recovered in feces, with 13.6% of the dose is unchanged.
The mean apparent volume of distribution of talazoparib is 420 L.
The mean apparent oral clearance is 6.45 L/h. The inter-subject variability is 31%.

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Metabolism / Metabolites
Talazoparib undergoes minimal hepatic metabolism. The metabolic pathways include mono-oxidation, dehydrogenation, cysteine conjugation of mono-desfluoro talazoparib, and glucuronide conjugation.

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Biological Half-Life
The mean terminal plasma half-life (±standard deviation) is 90 (±58) hours in patients with cancer.

Hepatotoxicity
Elevations in serum aminotransferase levels are common during talazoparib therapy occurring in 33% of patients, but rising above 5 times the upper limit of the normal range in only 1%. The elevations are generally transient and not associated with symptoms or jaundice. Furthermore, similar rates of aminotransferase elevations were reported in control, comparator arms. Talazoparib has had limited clinical use but has not been linked to instances of acute liver injury with symptoms or jaundice. Because of the limited clinical experience with using talazoparib and other PARP inhibitors, their potential for causing liver injury is not well defined.
Likelihood score: E* (unproved but suspected 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 talazoparib during breastfeeding. Because talazoparib is 74% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during talazoparib 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
_In vitro_, the protein binding of talazoparib is 74% and is independent of talazoparib concentration.

[1]. Clin Cancer Res. 2013, 19(18):5003-15.

[2]. Cancer Research, 2010, 70 (8 Suppl), Abstract nr 3514.

[3]. Cancer Discov . 2019 Jun;9(6):722-737.

Pharmacodynamics
Talazoparib is a cytotoxic and anti-tumour agent. _In vitro_, talazoparib caused cytotoxicity in cancer cell lines that harboured defects in DNA repair genes, including BRCA1 and BRCA2. Talazoparib mediated anti-tumour effects on patient-derived xenograft breast cancer models bearing mutated BRCA1 or mutated BRCA2 or wild type BRCA1 and BRCA2.

C19H14F2N6O

380.35

380.119

C, 60.00; H, 3.71; F, 9.99; N, 22.10; O, 4.21

1207456-01-6

1207456-00-5; 1207456-01-6; 1207454-56-5 (racemic); 1373431-65-2

135565082

White solid powder

1.6±0.1 g/cm3

1.775

1.91

2

7

2

28

654

2

FC1=C([H])C2C(N([H])N=C3C=2C(=C1[H])N([H])[C@]([H])(C1C([H])=C([H])C(=C([H])C=1[H])F)[C@@]3([H])C1=NC([H])=NN1C([H])([H])[H])=O

HWGQMRYQVZSGDQ-HZPDHXFCSA-N

InChI=1S/C19H14F2N6O/c1-27-18(22-8-23-27)15-16(9-2-4-10(20)5-3-9)24-13-7-11(21)6-12-14(13)17(15)25-26-19(12)28/h2-8,15-16,24H,1H3,(H,26,28)/t15-,16-/m1/s1

(11S,12R)-7-fluoro-11-(4-fluorophenyl)-12-(2-methyl-1,2,4-triazol-3-yl)-2,3,10-triazatricyclo[7.3.1.05,13]trideca-1,5(13),6,8-tetraen-4-one

BMN 673; BMN673; MDV-3800; MDV 3800; 1207456-01-6; Talazoparib (BMN 673); Talzenna; (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-8,9-dihydro-2H-pyrido[4,3,2-de]phthalazin-3(7H)-one; MDV3800; BMN-673; LT673; LT 673; LT-673; Talazoparib; trade name: Talzenna

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: 5 mg/mL (13.15 mM) in 10% DMAC 6% Solutol HS-15 84% PBS (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.57 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 3: 1.25 mg/mL (3.29 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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: ≥ 0.5 mg/mL (1.31 mM) (saturation unknown) in 2% DMSO + 40% PEG300 + 5% Tween80 + 53% 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 5: ≥ 0.25 mg/mL (0.66 mM) (saturation unknown) in 1% DMSO + 99% Saline (add these co-solvents sequentially from left to right, and one by one),clear solution.

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