Niraparib: A Poly(ADP-ribose) Polymerase (PARP) Inhibitor for the

Drug Annotation pubs.acs.org/jmc

Niraparib: A Poly(ADP-ribose) Polymerase (PARP) Inhibitor for the Treatment of Tumors with Defective Homologous Recombination Philip Jones,†,§ Keith Wilcoxen,‡ Michael Rowley,*,†,∥ and Carlo Toniatti†,§ †

Istituto di Ricerche di Biologia Molecolare, Via Pontina km 30600, 00040 Pomezia, Italy TESARO, Inc., 1000 Winter Street, Waltham, Massachusetts 02451, United States

‡

ABSTRACT: Poly(ADP-ribose) polymerases (PARPs) are involved in DNA repair following damage by endogenous or exogenous processes. It has become clear over the past decade that inhibition of PARP in the context of defects in other DNA repair mechanisms provide a tumor specific way to kill cancer cells. We describe the rationale for this approach and the design and discovery of niraparib, a potent PARP-1/2 inhibitor with good cell based activity, selectivity for cancer over normal cells, and oral bioavailability. Niraparib was characterized in a number of preclinical models before moving to phase I clinical trials, where it showed excellent human pharmacokinetics suitable for once a day oral dosing, achieved its pharmacodynamic target for PARP inhibition, and had promising activity in cancer patients. It is currently being tested in phase 3 clinical trials as maintenance therapy in ovarian cancer and as a treatment for breast cancer.

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RATIONALE DNA Repair Response. DNA repair processes are critical for accurate cell replication and maintenance of genomic stability, as cells are continually faced by an onslaught of DNA damage. This occurs both through normal cellular functions, such as metabolic processes resulting in the generation of reactive oxygen radicals or through replication errors, but also as a result of exogenous damage such as UV light, ambient and therapeutic radiation, and through exposure to chemicals, including those in diets. To cope with these injuries, cells have evolved sophisticated defense mechanisms, with multiple overlapping DNA damage repair responses, focused on resolution of both DNA single-strand breaks (SSB) as well as DNA double-strand breaks (DSB) (Figure 1). There are at least five major DNA damage repair mechanisms operational in mammalian cells,1 which include base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR), which are the primary mechanisms to resolve SSB. Homologous recombination (HR) and nonhomologous end joining (NHEJ) are the two pathways responsible for repairing DSB. HR is a high fidelity repair system which relies on a homologous sequence of chromosomes adjacent to the site of breakage as a template to ensure accurate restoration of the DNA sequence. NHEJ is an error prone pathway with lower fidelity: it dissects the DNA damage and joins unrelated DNA strands, thereby causing altered nucleotide sequences and gene rearrangements. DSB are deleterious lesions to cells and are difficult to repair. If these lesions remain uncorrected they result in mutagenesis, or in the case of gross DNA damage, in cell death. All five pathways are complex processes involving an orchestrated interplay of many distinct proteins, and loss-of© XXXX American Chemical Society

function in one or more enzyme in these detection and repair processes results in an accumulation of mutations and DNA defects and predisposition to cancer. Given that DNA damage is the cornerstone of chemo- and radiotherapy widely used for the treatment of cancer and that DNA repair pathways have the ability to overcome the cytotoxic lesions induced by these treatments, there has been more than two decades of interest in targeting DNA repair pathways for clinical benefit.1 However, it is only within the past decade, with the development2 of PARP inhibitors such as niraparib and related compounds described herein, that significant treatment advances have been made. These advances have been enabled by an improved understanding of how critical alternative DNA repair pathways become in the context of defects in DNA DSB repair, allowing them to be exploited for therapeutic gain.3−5 PARP Family. Poly(ADP-ribose) polymerase (PARP)-1 is the founding and most abundant member of poly(ADPribosyl)ating proteins, also known as the ADP-ribosyltransferase diphtheria toxin-like proteins (ARTD), a family of some 17 proteins.6 The primary function of the PARPs is to posttranslationally modify target proteins with ADP-ribose units using NAD+ as substrate. The best characterized PARPs, PARP1, -2, -5A, and -5B, all possess the ability to catalyze the transfer of multiple ADP-ribose units, resulting in the formation of long, branched poly(ADP-ribose) (PAR) chains. In contrast, the remaining PARPs transfer single ADP-ribose units onto target proteins.7,8 Received: November 25, 2014

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DOI: 10.1021/jm5018237 J. Med. Chem. XXXX, XXX, XXX−XXX

Drug Annotation

Journal of Medicinal Chemistry

Figure 1. DNA repair pathways.

Figure 2. Schematic describing role of PARP-1/2 in base excision repair pathways.

inhibitors have also shown applicability in models of reperfusion injury, inflammation, cardiovascular disease, and neurodegenerative disorders. BRCA1/2 Connection and Synthetic Lethality. As noted above, tumor cells frequently possess defects in DNA repair mechanisms and these promote growth and survival advantages; however, unlike normal cells, these defects provide an opportunity for therapeutic exploitation. In 2005, a major breakthrough occurred when two seminal papers appeared in Nature from independent groups demonstrating that targeting more than one DNA repair pathway in tumor cells could induce “synthetic lethality”,12−14 achieving profound cytotoxicity within the tumor cells while sparing the normal cells. Two genes are described as being synthetic lethal if mutation/loss/ inhibition of either alone is compatible with cell viability, but loss of function of both concurrently results in cell death.15 Specifically, the publications described the selective killing of BRCA1 or BRCA2 deficient tumor cells by PARP inhibitors. In contrast, normal cells with an intact BRCA pathway are viable when treated with a PARP inhibitor, and minimal cytotoxicity is seen. BRCA1 and BRCA2 are known tumor suppressors and are key components involved in the repair of DNA doublestrand breaks by HR, and mutations in these genes predispose individuals to hereditary breast and ovarian cancer. It has also been shown that BRCA1/2 mutations have been found in prostate, pancreatic, and other types of cancer.16 Germline BRCA1/2 mutations are inherited in the heterozygous state, with one wild-type allele, and this second allele is lost to confer complete loss of BRCA function. This accelerates genomic instability, acquisition of mutations, and tumor development. In contrast, normal cells retain the wild-type copy, thus maintaining HR repair function. It is the differential HR capacity between the tumor and the surrounding tissue that allows a unique opportunity for targeting PARP activity to achieve cell killing.

PARP-1 and the closely related PARP-2 are nuclear proteins and possess DNA binding domains with zinc fingers.9 The basal catalytic activity of PARP-1 is low, but upon DNA damage to cells the DNA binding domains within PARP-1 serve to localize and rapidly bind at the sites of damage (Figure 2). Upon binding at the lesion, conformational rearrangement occurs throughout the protein, relaying information from the DNA binding domain to the catalytic domain, thereby increasing the catalytic activity up to 500-fold. This activation results in the addition of PAR chains on several proteins associated with chromatin, including: histones, p53, topoisomerases, and PARP itself.10 The addition of these negatively charged PAR chains relaxes chromatin and results in the fast recruitment of DNA repair factors such as XRCC1, which access the DNA breaks and repair them through BER. Additionally, PARP is involved in several other nuclear processes including acting as a facilitator of HR. It has also been implicated in NHEJ, and PARP-1 also regulates transcription by modulating chromatin structure, acting as a coregulator of transcription factors and interacting with chromatin insulators. The knockout of PARP-1 significantly impairs repair of DNA damage following exposure to radiation or cytotoxic insult,11 with the residual PARP dependent repair activity being due to PARP-2, which contributes about 10% to nuclear PARP activity.9 Similarly, PARP-1 and -2 inhibition with small molecules has been demonstrated to sensitize tumor cells to cytotoxic agents and ionizing radiation. On the basis of these findings, beginning in the 1990s multiple companies and academic groups developed PARP inhibitors as chemo- and radiopotentiators and progressed molecules into early stage clinical trials.2 However, despite these agents being efficacious in initial clinical trials, they proved to be highly toxic in combination with chemo- and radiotherapy, and they were not advanced into late stage clinical testing. It should be noted, although it is outside the scope of this manuscript, that PARP plays a role in multiple pathological conditions,2,7 and PARP B


Drug Annotation

Journal of Medicinal Chemistry Farmer and Ashworth,13 in collaboration with scientists at KuDOS Pharmaceuticals, described the hypersensitivity of BRCA1 and BRCA2 deficient cells to both silencing of PARP-1 by RNA interference and also inhibition with small molecule PARP-1/2 inhibitors. Both genetic knockdown and pharmacological inhibition of PARP activity profoundly sensitized these HR deficient cells, resulting in a reduction of clonogenic survival, chromosomal instability, cell cycle arrest, and subsequent apoptosis. In clonogenic assays, BRCA1 and BRCA2 deficient cells were extremely sensitive to PARP inhibitors, with some compounds showing IC50s in the nanomolar range. In contrast, wild-type cells were 2 orders of magnitude less sensitive to PARP inhibition, with IC50s around the 2 micromolar range. The effects of one compound, KU0058684 (1, Figure 3), were also studied in vivo, and this

administration of 1 failed to prevent tumor formation. In follow up studies in a genetically engineered mouse model for BRCA1 associated breast cancer, treatment of tumor bearing mice with the PARP inhibitor olaparib17 resulted in inhibition of tumor growth without signs of toxicity.18 In related work, Bryant and Helleday described the acute sensitivity of BRCA2 deficient cell lines to PARP inhibitors AG14361 (2) and NU1025 (3).12 They demonstrated the HR defective cell lines irs1 and irs1SF are sensitive to PARP inhibition, and correction of the HR defects in these cell lines by reintroduction of XRCC2 or XRCC3 reversed that sensitivity. They also showed that the BRCA2 deficient cell line V-C8 was profoundly sensitive to PARP inhibitors 2 and 3 compared to the isogenic cell line V-C8+B2 with functional BRCA2. To demonstrate the therapeutic potential of PARP inhibition for the treatment of BRCA2 deficient tumors the VC8 cells, and the isogenic V-C8+B2 cells, were implanted into mice, and the growth rates following treatment with 2 monitored. The PARP inhibitor had no effect on the outgrowth of the BRCA2 proficient cells compared to vehicle treatment. In contrast, three out of five mice bearing the BRCA deficient V-C8 xenograft responded with complete remission. Cell death arises from combining both conditions: PARP inhibition resulting in blockage of BER and defects in DSB repair through loss of BRCA1/2. Neither alone is itself sufficient to affect cell viability. This is illustrated in Figure 4, whereby DNA damage in the presence of a PARP inhibitor results in persistent SSB. During replication, these SSBs cause stalled replication forks and subsequently develop into DSB. In normal patients, and in normal tissue of BRCA1/2 mutant patients, these DSB can be repaired uneventfully through HR repair. But in BRCA1 and BRCA2 deficient tumor cells, where

Figure 3. Tool compounds.

PARP inhibitor was demonstrated to prevent formation of HRdeficient tumors. When BRCA2 deficient embryonic stem (ES) cells were injected into athymic mice and allowed to form teratocarcinomas, well tolerated doses of 1 severely inhibited tumor formation from these BRCA2 deficient cells. These effects were selective for HR deficient tumors, as injection of the corresponding wild type ES cells with concomitant

Figure 4. Homologous recombination defective cells are hypersensitive to PARP inhibition. C



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Drug Annotation

COMPOUND DESIGN AND DISCOVERY There has been an interest in the discovery and development of PARP inhibitors for a number of years.7 Efforts from many groups have resulted in a good understanding of key pharmacophore elements for the binding of small molecules to PARP-1/-2 enzymes. This allowed us to take a design approach to hit finding, incorporating prior knowledge with novel approaches, in order to find starting points for lead optimization. Early inhibitors23,24 were based on the knowledge that nicotinamide (4) is a weak PARP inhibitor, with one of the key elements in improving potency to constrain the amide bond (which can rotate in nicotinamide). This is supported by X-ray structures,25 in which three key hydrogen bonding interactions are seen between the inhibitors with backbone CO and NH of glycine, and the hydroxyl group of serine (Figure 5).

both alleles have been lost, these lesions are not repaired by the homologous recombination repair pathway. Instead these HR deficient cells rely on the error prone NHEJ repair pathway, resulting in gross genomic instability, chromosomal mutations, cell cycle arrest, and eventually apoptosis. Of note, more recent evidence19,20 suggests that additional mechanisms, including PARP-1 trapping on the SSB during BER, as well as a direct role of PARP-1 in reactivating stalled replication forks, might be responsible for the synthetic lethality of PARP inhibitors in HR-deficient cells. These reports triggered great excitement and led to drug discovery efforts across the industry, including the initiation of our program at the IRBM (Merck Research Laboratories, Rome), which resulted in the identification of niraparib. Defects in Homologous Recombination. Although the initial data focused on tumors with inactivation mutations in BRCA1/2, there is a growing body of evidence supporting the expansion of this strategy beyond hereditary cancers into sporadic (i.e., nonfamiliar) tumors bearing defects in other HR related genes.3−5 It is widely accepted that HR deficiency is the main determinant for PARP inhibitor sensitivity in BRCA1/2 mutant tumors and, given that the HR repair machinery involves multiple interacting genes,7,13 disruption of any of the HR genes should have a deleterious effect on HR and confer hypersensitivity to PARP inhibition. For instance, it is estimated that in high grade serous ovarian cancer, 15% of women have germline mutations in BRCA1/2, yet large scale genomic evidence suggest up to half of patients may have acquired defects in HR and thereby be sensitive to PARP inhibition. HR deficiency may arise through genetic or epigenetic mechanisms, sometimes called “BRCAness”, which is used to describe the phenotype of HR deficient sporadic cancers that share phenotypes with BRCA1/2 associated hereditary cancers.21 For example, genes involved in HR that have been identified to contribute to hereditary epithelial ovarian cancer include PALB2, Rad51C, Rad51D, BRIP1, BARD1, Chk2, Mre11A, MSH6, NBN, and Rad50,3−5,21 and it has been shown that approximately half of high-grade serous ovarian cancers have defects in HR.22 Epigenetic silencing of BRCA1 (11−35% of epithelial cancers) and FANCF (5−20%) genes through promoter methylation has been shown to confer sensitivity to PARP inhibition, as has overexpression of the EMSY gene, which is known to suppress BRCA2 transcription. Similarly, several other genes including ATM, CHK2, and Fancomi anemia genes, which are essential for the repair of stalled replication forks, have been reported to be disrupted in sporadic tumors and can sensitize to PARP inhibition. Studies have also demonstrated that loss of the tumor suppressor PTEN in endometrial cancers causes sensitivity to PARP inhibition. Given the complexity of the HR DNA damage response, rather than genotyping at the single gene level, it may be more instructive to use functional assays that can identify tumors with defects in the HR repair pathway. BRCA1/2 loss through epigenetic silencing can be identified through immunohistochemistry and the formation of RAD51 nuclear foci could possibly be used to identify sporadic tumors with HR defects. These foci form at the site of DNA DSB in HR repair competent cells, but not in HR defective tumors, hence detection of RAD51 by immunofluorescence may serve as a measurement of HR integrity.

Figure 5. Nicotinamide and a schematic of the H-bond pattern of PARP-1 inhibitors.

The restraint in amide rotation is made (Figure 6) either by constraining the amide into a ring such as rucaparib26 (5, AG-

Figure 6. Structures of PARP inhibitors in mid to late stage clinical development.

14699, PF-01367338) and olaparib17 (6, KU 0059436, AZD2281), or by placing a hydrogen bond accepting group such that the amide NH anti to the carbonyl forms a ring via an intramolecular H-bond as in veliparib27 (7, ABT-888). We utilized both approaches; however, it was the latter that led to the discovery of niraparib (8, MK-4827) and is discussed below. At the outset,28 we designed a number of [6,5] fused aromatic azabicycles, in which a nitrogen atom in the fivemembered ring was placed to make the key intramolecular Hbond to the anti NH of the amide (Figure 7). This proved a fruitful exercise, with several examples showing moderate to good inhibition of PARP-1 activity in an in vitro enzyme assay. The most promising of these early analogues was the 2Hindazole 10, showing IC50 = 24 nM in the PARP-1 assay and some, albeit weak, activity (EC50 = 3 700 nM) in a cell based PARylation assay. Compound 10 also had promising rat pharmacokinetics (F 41%, CLp 30 mL/min/kg, terminal halflife 5.1 h). We explored structure−activity relationships on D


Drug Annotation


high in vivo clearance in rats (14 CLp = 450; 15 CLp = 220 mL/min/kg), in excess of liver blood flow. Compounds 14 and 15 showed a similar rat plasma free fraction, were stable in rat plasma, and did not partition into blood cells, leading us to hypothesize extrahepatic metabolism as a route of clearance. Compound 14 was radiolabeled, dosed intravenously (iv) to rats, and urine and bile examined; the majority of the dosed radioactivity was excreted as the carboxylic acid 16 and its acylglucuronide. These results pointed to oxidation as a major clearance route, and we believed this to be nonhepatic. To test this hypothesis, microsomes from lung, kidney, intestine, and heart were screened: compound 14 showed turnover in lung and kidney, in addition to liver and microsomes. Cytochrome P450 phenotyping showed that metabolism was very largely via CYPs 1A1 and 1A2 (which are expressed in the lung), so we placed turnover via recombinant CYP1A1 into our in vitro screening cascade in order to drive down clearance. Because compounds 14 and 15 showed appropriate levels of PARP inhibition in vitro and in cells, our strategy focused on maintaining the major features of these molecules (the 2phenyl-2H-indazole-7-carboxamide with a basic nitrogen appended to the para position of the phenyl group) and to attempt to reduce benzylic oxidation. Among the approaches we took (Table 1), blocking the benzylic methylene (17) and constraining the amine into a ring (18) proved promising, yielding compounds with similar potency, reduced in vitro clearance in both rat liver microsomes and CYP1A1, and correspondingly reduced plasma clearance in rat. As an alternative to fusing a ring as in 18, we also appended saturated nitrogen heterocycles to the para position of the phenyl group (Table 2). With the basic nitrogen in the benzylic position (19), we still observed high turnover by CYP1A1, and the 4-substituted piperidine 20 showed reduced PARP-1

Figure 7. Heterobicylic carboxamides as PARP inhibitors.

compound 10 as a scaffold, with the most interesting finding being that incorporating a basic group off the para position of the phenyl group (dimethylaminomethyl derivative 14, Figure 8) gave an improvement in enzyme inhibition (PARP-1 IC50 =

Figure 8. Functionalizing the core PARP inhibitor.

3.7 nM). More importantly, 14 showed a significant improvement in the cell based PARP inhibition assay (PARylation EC50 = 110 nM), presumably in large part due to improved physicochemical properties allowing better cell penetration. Compound 14 was also active against a HeLa cell line in which BRCA1 had been silenced by shRNA (CC50 [concentration required to reduce cell viability by 50%] = 520 nM), with more than 10-fold selectivity over the nonsilenced control. The monomethyl derivative (15, PARP-1 IC 50 = 3.8 nM, PARylation EC50 = 68 nM) had a similar profile to 14. Where these two molecules differed was in their in vitro clearance profiles: in rat liver microsomes, 14 had rather high turnover (Clint 177 μL/min/mg protein), 15 considerably lower (Clint 28 μL/min/mg protein), but both showed very Table 1. Blocking Benzylic Oxidation

a

Inhibition of PARP-1 enzymatic activity. bInhibition of hydrogen peroxide induced PARylation in HeLa cells. cRat liver microsome intrinsic clearance: units are μL per min per mg of protein. dRecombinant CYP1A1 intrinsic clearance. eRat in vivo total clearance following iv dosing. E


Drug Annotation

Journal of Medicinal Chemistry Table 2. Appending a Basic Heterocycle

a

Inhibition of PARP-1 enzymatic activity. bInhibition of hydrogen peroxide induced PARylation in HeLa cells. cRat liver microsome intrinsic clearance: units are μL per min per mg of protein. dRecombinant CYP1A1 intrinsic clearance. eRat in vivo total clearance following iv dosing.

Figure 9. Representative HPLC radiochromatograms of 8 incubated in the presence of rat, dog, and human hepatocytes.

(niraparib). As well as its moderate clearance in rat, 8 showed good bioavailability (65%) and a high volume of distribution (Vdss = 6.9 L/kg), leading to a reasonably long terminal half-life (t1/2 = 3.4 h). During the course of its preclinical development, we needed to address the issue of species specific pharmacokinetics. Although rat pharmacokinetics was acceptable, 8 showed high clearance in dog (CLp = 31 mL/min/kg). Despite this, it had good oral bioavailability (F = 57%), a high volume of distribution (Vdss = 12.3 L/kg), and a good terminal half-life (t1/2 = 5.7 h). 8 was stable in blood from rat, dog, and human, but experiments in hepatocytes showed high turnover in dogs to the product of amide hydrolysis, acid 23 (Figure 9). This was

inhibition and activity in BRCA1 silenced HeLa cells (CC50 410 nM). However, the 3-substituted piperidine (21) proved very promising, with good enzyme and cell potency, reduced liver microsome and CYP1A1 in vitro clearance, and moderate in vivo plasma clearance in rat. Resolution of 21 gave compounds 22 and 8, both showing excellent inhibition of PARP-1. The Renantiomer 22 had somewhat lower in vitro metabolic clearance than the S-enantiomer 8 in rat liver microsomes, but compound 8 was more potent in cell based assays (PARylation EC50, 22 = 30 nM, 8 = 4.0 nM; BRCA1-HeLa CC50, 22 = 470, 8 = 34 nM). Given this improved potency and similar in vitro turnover in human liver microsomes (HLM Clint, 22 = 4, 8 = 3 μL/min/mgP) we focused on compound 8 F


Drug Annotation

Journal of Medicinal Chemistry confirmed with in vivo experiments in dogs, in which 52% of the radioactivity of [14C]-8 dosed iv to bile duct cannulated dogs was recovered as 23, with less than 5% recovered as parent. As expected, given the lack of the key H-bond donor, 23 is not a potent PARP-1 inhibitor (PARP-1 IC50 > 500 nM). Unlike dogs, [14C]-8 dosed iv to bile duct cannulated rats gave 45% of recovered radioactivity as parent drug along with numerous metabolites, none of which accounted for more than 5% of the dose. Because this metabolism appeared to be a dogspecific phenomenon, we were not concerned that this hydrolysis would have a major impact on human pharmacokinetics. With the different routes of clearance in preclinical species, the quantitative prediction of human pharmacokinetics was difficult. Nonetheless, given good physical properties, preclinical bioavailability, and low metabolic turnover in human in vitro systems, we had reasonable confidence that human PK would be good. This, indeed, turned out to be the case as discussed below. An important lesson that this illustrates is the value of mechanistic PK studies. The understanding of extrahepatic oxidative metabolism in rats helped find stable compounds, and an understanding of the dog specific clearance mechanism allowed us to go forward despite high dog in vivo clearance.

Table 4. Inhibition of Proliferation of BRCA1 and BRCA2 Deficient Cancer Cells

Table 3. Inhibition of Intracellular PARylation PARylation IC50 (nM)

PARylation IC90 (nM)

HeLa A2780 CAPAN-1

wild type wild type BRCA2 deficient

4±2 4+1 3.5 ± 1

46 ± 6 52 ± 5 50 ± 9

wild-type HeLa HeLa BRCA1 minus wild-type A549 A549 BRCA-2 minus UWB1.289 UWB1.289+BRCA1 SUM149PT SUM1315MO2 BT-20 DoTc2-4510

no yes (shRNA) no yes (shRNA) BRCA1 mutant no (BRCA1 restored) BRCA1 mutant BRCA1 mutant no BRCA2 mutant

CC50 (nM) 852 34 1760 11 56 975 24 20 2,200 23

± ± ± ± ± ± ± ± ± ±

262 17 670 5 20 40 7 6 180 6

are derived from a tumor of a patient with papillary serous adenocarcinoma histology and that carry an inactivating mutation in the BRCA1 gene.29 The CC50 for the UWB1.289 line was 56 nM, with 18-fold selectivity over the UWB1.289+BRCA1 cell line, a stable cell line derived from UWB1.289 cells in which expression of wild-type BRCA1 has been restored. In agreement with data published for other PARP inhibitors,13 mechanism of action studies in cell culture demonstrated that niraparib exerted its cytotoxic activity on BRCA-defective cells by inducing cell cycle arrest at the G2/M phase followed by apoptosis and then mitotic catastrophe. This last event is presumably due to inhibition of PARP, leading to the persistence of DNA double-strand break lesions that cannot be repaired by HR and the consequent use of alternative errorprone pathways such as single-strand annealing and/or nonhomologous end joining. Monotherapy Activity in Preclinical in Vivo Models. Xenograft studies were performed on the hydrochloride salt of niraparib. In a single-dose pharmacokinetic study in CD-1 mice, the plasma concentration was approximately 100 nM 24 h after oral administration of 50 mg/kg. Free fraction in mouse and human plasma are 26% and 16%, respectively. Niraparib inhibits the proliferation of BRCA1 mutant MDA-MB-436 cells with CC50 = 18 nM. These human mammary adenocarcinoma cells form tumors when implanted subcutaneously in the flank of immunocompromised mice and were used as a model system to test niraparib in vivo activity on BRCA1 mutant cells. Preliminary dose-dependent experiments demonstrated that efficacy was achieved at a daily oral dose of 50 mg/kg. As shown in Figure 10, at 80 mg/kg, a significant inhibition of tumor growth was observed after only 1 or 2 weeks of treatment. Continuous daily dosing for 3 or 4 weeks was more efficacious and induced tumor shrinkage, with complete and sustained regression observed after 4-week treatment. Niraparib was well-tolerated. as indicated by the less than 10% reduction in body weight in all treatment groups. Niraparib was also shown to be active in a CAPAN-1 pancreatic cancer cell xenograft model. CAPAN-1 cells are BRCA2 deficient cells that are sensitive to niraparib (CC50 = 90 nM). Niraparib was dosed using a similar schedule as for the BRCA1 mutant xenograft model (80 mg/kg for 2, 3, or 4 weeks) and showed around 60% tumor growth inhibition

PRECLINICAL CHARACTERIZATION Niraparib is a potent and selective PARP-1 and PARP-2 inhibitor with in vitro IC50 = 3.8 and 2.1 nM, respectively, and displays at least a 100-fold window over the other PARP-family members PARP-3, v-PARP, and Tankyrase-1 (TANK-1).28 Monotherapy Activity in Cell Based Assays. The ability of niraparib to inhibit intracellular PARP-1/-2 activity was evaluated in an assay that measures the amount of poly(ADPribose) (PAR) chains formed in cells as a result of DNA damage induced by exposure to hydrogen peroxide (H2O2). In these conditions, niraparib inhibited intracellular PARylation in three different cell lines, with IC50 and IC90 of about 4 and 50 nM, respectively (Table 3).

BRCA status

BRCA defect

Proliferation was measured after 12 days (for SUM1315MO2) or 5−7 days (for all other cell lines) after addition of niraparib to the medium, using the CellTiter-Blue assay (Promega) according to manufacturer’s instruction. Values represent the mean CC50 ± SD derived from a minimum of four experiments

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cell line

cell line

Values represent the mean ± SD derived from a minimum of three experiments.

The selectivity of niraparib for BRCA1 or BRCA2 deficient cells was demonstrated in several cell lines. Niraparib selectively inhibited proliferation of cancer cell lines which had been stably silenced for BRCA1 or BRCA2 compared to their wild-type counterparts. The CC50 for HeLa BRCA1-deficient cells was 34 nM, with a 25-fold selectivity window over the BRCA1wt matched pair HeLa cells. Niraparib inhibited the proliferation of BRCA2 silenced A549 human lung cancer cells lines, with CC50 = 11 nM and more than 100-fold selectivity window over the BRCA2wt matched pair A549 cells (Table 4). It also strongly inhibited the growth of cancer cell lines carrying inactivating mutations of BRCA1 (SUM149PT and SUM1315MO2) or BRCA2 (DoTc2-4510) genes with CC50s of approximately 20 nM. The selectivity of niraparib for BRCA mutant cells was confirmed using UWB1.289 cell lines, which G


Drug Annotation


Figure 10. Efficacy in BRCA1 mutant mouse xenograft assay following 1, 2, 3, or 4 weeks once daily oral dosing.

(Figure 11). Again, it was well-tolerated, with less than 10% reduction in body weight in all treatment groups.

Figure 12. Pharmacodynamic activity of niraparib in MDA-MB-436 xenografts. % PARP activity is shown at 4, 8, and 24 h post po dosing.

chemotherapy. In some cases, the specific role of PARP has yet not been fully elucidated,31 but PARP-1 is known to be required to release trapped topoisomerase I/irinotecan complexes from DNA and Temozolomide induced DNA damage is repaired by the BER pathway which requires PARP to recruit repair proteins.32,33 It is noteworthy that, because of the myelosuppression induced by PARP inhibitors, it is of paramount importance to their clinical development as chemosensitizers to establish proper dose and schedule in combination, in addition to monotherapy. Niraparib strongly enhanced the therapeutic effect of radiation administered using a clinically relevant dose fractionation protocol in breast and lung cancer models.34 The mechanism of radiosensitization is p53-independent and seems to depend on the conversion of radiation induced sublethal SSBs into lethal DSBs as a consequence of BER inhibition.35 Niraparib has also been shown to sensitize colorectal cancer cell lines in vitro and in vivo to irinotecan. Interestingly, we demonstrated that in subcutaneous xenograft models oral doses of niraparib 50% lower than those efficacious in monotherapy administered for not more than 5 days can still strongly potentiates irinotecan activity in vivo.36 Short-term treatment (5 days) has also been shown to potentiate Temozolomide activity in preclinical models, but in this case no significant efficacy was observed at niraparib doses lower than those used in monotherapy. The reasons underlying this difference between irinotecan and Temozolomide remain to be investigated and might be related to the different mechanism of action of the two drugs.

Figure 11. Efficacy in BRCA2 mutant mouse xenograft assay following 2, 3, or 4 weeks once daily oral dosing.

A PARP pharmacodynamic (PD) assay was developed to monitor PARP activity in tumor extracts and in peripheral blood mononuclear cells (PBMCs). This assay measures PARP activity by detecting the incorporation of 3H-NAD in PAR chains synthesized in response to nicked DNA. Tumor or PBMC homogenates are normalized for PARP-1 levels, and the PARylation reaction is initiated by addition of a nicked DNA strand. Using this assay, we showed that maximum efficacious doses of niraparib strongly (>90%) and continuously inhibited PARP activity in MDA-MB-436 and CAPAN-1 BRCA mutant tumors over 24 h. Data are shown for the MDA-MB-436 model in Figure 12 (data in the CAPAN-1 model are similar). This level of intratumoral inhibition correlated with 50% or greater inhibition observed peripherally in PBMCs at 24 h postdose. These results suggested that PARP inhibition in a peripheral tissue like PBMCs can be explored as a pharmacodynamic biomarker in the clinic to determine whether niraparib has sufficient activity (i.e., inhibits PARP activity in PBMC by >50%) at the maximum tolerated dose (MTD) to warrant further development. Combination Therapy. PARP-1/2 inhibitors have been demonstrated to be effective in preclinical models in combination with platinum, alkylating and methylation agents, radiation therapy, and topoisomerase I inhibitors.30 In contrast to the rationale for monotherapy, in which PARP inhibitors causes DNA damage in HR deficient cancers, PARP is required for repair of DNA damage induced by standard cytotoxic

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OTHER PARP INHIBITORS IN DEVELOPMENT There has been, and continues to be,37 considerable interest in the discovery and development of PARP inhibitors. For the sake of brevity, we limit ourselves to the discussion of PARP inhibitors in mid- or late-stage clinical development. Structures are shown in Figure 6. Rucaparib (5) was characterized26 as a potent PARP inhibitor in cell based assays of radio- and chemosensitization and is a very potent (PARP-1 IC50 = 2 nM) enzyme inhibitor. It was in development by Pfizer but was outlicensed to Clovis in June, 2011. Rucaparib is currently being evaluated in a phase 3 ovarian cancer trial and is in earlier phase clinical trials for other cancer indications. Olaparib (6) was discovered17 by KuDOS. In 2005, AstraZeneca bought KuDOS H


Drug Annotation


1a) had cancers of any histological subtype. However, enrichment for germline BRCA1 mutation (gBRCA1mut) and gBRCA2mut carriers was conducted prospectively in phase 1a to establish evidence that niraparib could have anticancer activity in tumors defective in HR function. Patients were considered to have a gBRCA1mut or gBRCA2mut if they had previous confirmation of a pathological BRCA mutation as determined by Myriad Genetics BRCA mutation testing (BRAC Analysis). Dose expansion (phase 1b) was conducted in patients with sporadic platinum-resistant high-grade serous ovarian cancer and castration-resistant prostate cancer, as it was believed at the time these patients may harbor defects in DNA repair. The starting dose of 30 mg/day once daily orally in phase 1a was selected based on preclinical safety studies. A modified 3 + 3 design48 was pursued for dose escalation at approximately 40% dose increments. Tumor response was assessed by response evaluation criteria in solid tumors (RECIST). Bone scans and prostate-specific antigen (PSA) levels were obtained for patients with castration-resistant prostate cancer. Serial serum CA125 levels were obtained for high-grade serous ovarian cancer patients. Pharmacokinetic parameters were determined at all dose levels. Pharmacodynamic measurements consisted of PARP activity in PBMCs, changes in circulating tumor cells, and molecular characterization of tumor tissue if available. Ten dose levels were investigated between 30 and 400 mg/ day in both BRCA1/2 mutant and BRCA1/2 wild-type patients, with the latter dose considered dose limiting due the occurrence of reversible grade 4 thrombocytopenia. A dose reduction to 300 mg/day was evaluated in an additional 10 patients and determined to be the recommended phase 1b dose due to the absence of previously defined dose-limiting toxicities. In addition to these 10 patients, a further 40 were treated in phase 1b at this dose of 300 mg/day. Consistent with known toxicities of other PARP inhibitors, dose proportional hematological toxicities were observed. All hematological toxicities were reversible and manageable with dose interruptions or reductions. The most common nonhematological treatment related adverse events were nausea, fatigue, constipation, anorexia, vomiting, and insomnia. However, these were considered mild to moderate in severity. Pharmacokinetic analysis indicated exposure to niraparib was dose proportional between the 30 and 400 mg/day doses, with moderate variability between patients at the same dose. It was rapidly absorbed, with peak plasma concentrations occurring 3−4 h after dosing and had a mean terminal half-life of 36.4 h. It is currently believed that it is important to maintain continuous inhibition of PARP-1 enzyme for monotherapy regimens. Importantly, the plasma trough concentrations were >2 μM at the 300 mg/day dose, with all doses of 40 mg/day or above achieving plasma trough concentrations of 288 nM or greater which, based on xenograft studies, may be expected to be efficacious. Peripheral blood mononuclear cells (PBMCs) were collected at predose on days 1, 5, and 21, and at 4 h postdose on days 5 and 21. Pharmacodynamic evaluation showed PARP inhibition in PBMCs exceeded 50% at doses of 80 mg/day or greater.47 Induction of gH2AX foci was observed in post-treatment tumor tissue and in circulating tumor cells in patients, confirming the presence of DNA double-strand breaks consistent with PARP inhibition. Antitumor activity was documented in patients with and without germline BRCA mutations, with particularly compelling efficacy observed in ovarian cancer patients (Figure 14). In

while olaparib was in phase I and continues its development. It is a potent PARP-1/2 inhibitor (PARP-1 IC50 = 5 nM) with good activity in cell based assays and bioavailable in rats and dogs. Olaparib is dosed at 300 mg twice daily (bid) and is currently in a number of phase 2 or 3 clinical trials alone or in combination for the treatment of breast, ovarian, pancreatic, and prostate cancer. Veliparib27 (7) is a potent PARP-1/2 inhibitor (PARP-1 Ki = 5 nM) with nanomolar cell based activity and good bioavailability that was discovered and is in development by AbbVie (previously Abbott). It is dosed bid orally and is in multiple combination studies in a variety or tumor types, including breast, ovarian, lung, and pancreatic. BMN 673 (9)38 is a subnanomolar PARP-1/2 inhibitor (PARP1 IC50 = 0.6 nM) with good activity (EC50 = 2.5 nM) in the cell based PARylation assay and good oral bioavailability that is in development by BioMarin. Similarly to niraparib, 9 is dosed once daily, unlike rucaparib, olaparib, and veliparib, which require bid dosing. 9 has started a phase 3 study, dosed at 1 mg qd in breast cancer with germline BRCA mutations, along with other earlier stage cancer trials. While there was growing excitement about PARP inhibitors in the early years of this century, the field received two setbacks at the beginning of its second decade. The first39 came from clinical trials with iniparib (25, Figure 13), a molecule licensed

Figure 13. Iniparib.

by Sanofi from BiPar and reported to be a prodrug of a covalent PARP inhibitor (via reduction of the nitro group to the corresponding nitroso analogue). Despite a promising phase 2 trial40 in triple-negative breast cancer in combination with gemcitabine and carboplatin, it was disclosed in 2011 that there was no positive effect in a phase 3 trial.41 It was later shown that iniparib is not, in fact, a PARP inhibitor.42 Second, despite promising early trials, AstraZeneca announced43 in 2011 that olaparib would not be progressed to phase 3 trials. Reasons given were that it was believed unlikely that it would improve overall survival and issues in developing a formulation. However, a later analysis of the data44 by BRCA status was performed, and it was observed that ovarian cancer patients harboring a BRCA mutation achieved significant clinical benefit from the treatment of olaparib in the maintenance setting. AstraZeneca restarted45 its clinical development program after a nearly two-year hiatus. Meanwhile, in 2011, Pfizer out-licensed rucaparib to Clovis and in 2012 Merck out-licensed niraparib to Tesaro.

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CLINICAL EXPERIENCE Phase 1. Niraparib entered clinical studies in the fall of 2008. The form being developed is the para-toluenesulfonate salt.46 The first human study was a nonrandomized, open-label, dose-comparison, single-group assignment, safety and efficacy phase 1 trial at centers in the US and UK (NCT00749502).47 The trial consisted of a phase 1a portion in patients (n = 60) with advanced solid tumors and a phase 1b portion in patients (n = 40) with high-grade serous ovarian cancer or castrationresistant prostate cancer. Patients enrolled into the doseescalation and dose-confirmation phases of the study (phase I


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Figure 14. Antitumor activity in ovarian cancer *BRCA1/2 mutation carriers. †Reduction in overall sum of measurable disease but new lesion seen (overall: PD). Refractory patient (BRCA mutated) not included.

20 radiologically assessable gBRCAmutovarian cancer patients, eight (40%) achieved a confirmed RECIST and CA125 GCIG partial response at doses ranging from 80 to 400 mg/day with a median duration of response of 387 days. (CA125 is a serum marker adopted by the Gynecological Cancer Intergroup (GCIG) that is often used a biomarker of response in ovarian cancer.) Three of these responding patients were platinum resistant and five were platinum sensitive. Stable disease of 120 days or greater was reported in two additional gBRCAmut ovarian cancer patients. Four gBRCAmut breast cancer patients were evaluable in this study, and two (50%) achieved partial responses lasting approximately 3 months at starting doses of 210 and 150 mg/day. One nonsmall cell lung cancer (NSCLC) patient with a documented gBRCA2mut had stable disease for 175 days at 110 mg/day. In 22 germline BRCA wild type highgrade serous ovarian cancer patients, five (23%) achieved a durable partial response by RECIST or CA125 GCIG at starting doses of 60 mg/day or greater. Two of these responding patients were platinum resistant, and three were platinum sensitive. Stable disease of 120 days or greater was reported in three additional gBRCAwt high-grade serous ovarian cancer patients. An additional NSCLC patient experienced sustained RECIST stable disease for 316 days on 40 mg/day. Nine of 21 (43%) castration-resistant prostate cancer patients had stable disease for a median duration of 254 days. Collectively, niraparib demonstrated clinical validation of its mechanism of action and pharmacological properties amenable to once daily administration and resulted in significant antitumor activity in gBRCAmut and sporadic cancers within a large therapeutically effective dosing window (60−300 mg/ day). In February 2011, an open-label, nonrandomized, phase I trial (NCT01294735) was initiated in the US to evaluate niraparib given with Temozolomide in patients with advanced cancer. The trial was completed in May 2012 following recruitment of 19 patients. Patients received niraparib (30, 40, and 70 mg/day orally, days 1−8 of a 28 day cycle) plus Temozolomide (150 mg/m2 orally, days 4−8 of the 28 day cycle). The maximum tolerated dose was found to be niraparib (40 mg/day) plus Temozolomide (150 mg/m2/day). Overall, the highest incidence of adverse events observed was myelosuppression, including thrombocytopenia (63%) and

leukopenia (42%, 8/19), as well as nausea (42%) and fatigue (37%). One glioblastoma patient exhibited a partial response after six treatment cycles of niraparib (40 mg), and stable disease was reported in a malignant melanoma and a serous ovarian cancer patient after 4 cycles of niraparib, 40 and 30 mg/ day, respectively. No pharmacodynamic markers of target engagement were evaluated. This study highlights one of the challenges in developing PARP inhibitors in combination with traditional chemotherapies, particularly when the chemotherapeutic agents are administered at full doses. Doses and schedules of a single agent PARP inhibitor determined as safe in phase I monotherapy clinical trials are likely to be toxic when given in combination with anticancer cytotoxic chemotherapy, while the dosing regimen determined as safe in combination with a cytotoxic is unlikely to be sufficient as a single agent.49 Higher doses are probably required for single-agent activity because PARP activity needs to be nearly completely suppressed to ensure that the relatively modest levels of endogenous damage persist and are rendered cytotoxic. Conversely, for chemo-and radiopotentiation, where high levels of damage are induced in a short period, PARP does not need to be totally suppressed for such levels of damage to be cytotoxic. Starting in the summer of 2010, three additional phase 1 studies were opened. In July 2010, an open-label, nonrandomized, dose-escalation, phase 1b trial (NCT01110603) was initiated in the US to evaluate niraparib in combination with approved doses of common standard of care agents carboplatin, or carboplatin/paclitaxel, or carboplatin/liposomal doxorubicin used for the treatment of breast and ovarian cancers and establish the recommended phase 2 dose in adults with advanced solid cancers. In November 2010, an open-label, n o n r a n d o m iz e d , d o s e - e s c a l a t i o n , p h a s e 1 b t r i a l (NCT01227941) was initiated in the US to evaluate niraparib in combination with pegylated liposomal doxorubicin in patients with advanced solid tumors. In November 2010, an open-label, nonrandomized, phase 1 trial (NCT01226901) was initiated in Japan in patients with advanced solid tumors. None of these three phase 1 studies were completed; niraparib was out-licensed by Merck to Tesaro, and the clinical development program became focused on bringing forward the benefits of a J


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other DNA repair mechanisms was toxic to tumor cells (synthetic lethal) that spurred enormous interest in the field. The chemical series that led to niraparib was designed based on knowledge of the pharmacophore necessary for PARP inhibition. A number of challenges were addressed during its discovery, including at the outset obtaining good cell based activity. The preclinical pharmacokinetic profiles at first were suboptimal, but good mechanistic studies of clearance provided solutions, in rats showing that extrahepatic oxidation played a major role and that a hydrolysis seen in dogs was species specific. Niraparib was shown to have cytotoxic activity in a number of BRCA1 and BRCA2 negative or mutant cells lines with good selectivity over normal cells. Preclinical xenograft experiments showed proof of principle that niraparib would inhibit relevant tumor cell growth in vivo, and characterization of the extent of PARP inhibition in tumor and in PBMCs allowed development of a peripheral biomarker that could be used in the clinic. Niraparib has excellent human pharmacokinetics with a long half-life allowing once daily oral dosing, and doses of 80 mg/day or more show PARP inhibition expected to provide clinical benefit. In a phase 1 trial niraparib has shown very promising activity in BRCA mutant patients and in sporadic cancers, and these activities are being further investigated in two phase 3 trials in breast and in ovarian cancer. In addition to patients with germline BRCA mutations, there is increasing evidence that other deficiencies in homologous recombination may impart sensitivity to PARP inhibitors such as niraparib. A significant activity in its future development will include methods of identifying patients other than those that carry a germline BRCA mutation that would most probably gain clinical benefit from niraparib treatment. After great enthusiasm 10 years ago, a hiccup along the way around the beginning of the current decade but recent studies showing great promise, it appears that PARP inhibitors are back on track.51 The outcomes of ongoing late stage trials of niraparib and other PARP inhibitors are awaited with excitement, and it is hoped that this will provide significant benefit to patients suffering from cancer.

PARP inhibitor to patients with breast and ovarian cancer as quickly as possible. Currently, there is one ongoing phase 1 niraparib combination study. In the fall of 2013, a phase 1 trial was initiated to assess whether Temozolomide could sensitize tumor cells to niraparib in patients with pretreated, incurable Ewing’s sarcoma based on efficacy in preclinical models. A key element in this clinical investigation will be to identify the optimal efficacious dosing regimen of both agents and pharmacodynamic markers validating target engagement and mechanism(s) of efficacy. Phase 3. On the basis of phase 1 results with niraparib and evidence of clinical efficacy with other PARP inhibitors,44,50 two phase 3 studies of niraparib were initiated in 2013. The first study initiated was NOVA, a phase 3 randomized double-blind trial of maintenance with niraparib versus placebo in patients with platinum sensitive ovarian cancer who have either gBRCAmut or a tumor with high-grade serous histology (NCT01847274). The patients must have received at least two platinum based regimens, had a response to their last regimen, have no measurable disease >2 cm, and normal CA125 (or >90% decrease) following their last treatment. The study is designed to assess the primary end point of whether maintenance with niraparib will extend progression free survival (PFS) in this population. There are two independent patient cohorts, one with deleterious gBRCAmut and the other with high-grade serous histology but without such germline BRCA mutations. Each cohort will contain 180 patients, with 120 being treated with niraparib at 300 mg/day continuously daily and 60 receiving placebo. It is anticipated that gBRCAmut patients will experience an increased PFS versus placebo and some patients in the non-gBRCAmut population will experience an increased PFS due to other deficiencies in DNA damage repair. Analysis of biomarkers from blood and archival tumor tissue predicting increased PFS in this cohort will be valuable in understanding sensitivity to niraparib beyond germline BRCA mutations such as deficiencies in homologous recombination. The second phase 3 study was initiated in late 2013 and referred to as BRAVO. This study is a randomized, open-label, multicenter, controlled trial to compare niraparib versus physician’s choice, in patients with germline BRCA mutations who have advanced HER2 negative breast cancer. Eligible patients are randomized 2:1 for receiving niraparib orally at a dose of 300 mg once daily on a continuous dosing regimen or physician’s choice among one of the following four single agents: eribulin, vinorelbine, gemcitabine, or capecitabine. The primary end point is PFS. The hypothesized median PFS improvement is 6 versus 3 months (hazard ratio = 0.5) for niraparib versus physician’s choice. The study has 99.6% power for the primary PFS analysis at a 1-sided α of 0.025. Assuming an increase in overall survival from 9 to 13 months (hazard ratio = 0.69), the study has 80% power for the overall survival comparison at a 1-sided α of 0.025. The evaluation of predictive biomarkers related to efficacy of niraparib will include tumor biomarkers, assessment of circulating plasma DNA, humoral immune response monitoring, and PBMCs. Additional phase 3 studies in ovarian cancer and small cell lung cancer have been announced, but details are not available publically yet.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +41 413182426. E-mail: [email protected]. Present Addresses §

Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Unit 1956, PO Box 1956, Houston, Texas 77230-1429, United States. ∥ MSD Research GmbH, Ringstrasse 27, Kriens 6010, Switzerland. Notes

The authors declare the following competing financial interest(s): PJ owns shares in Tesaro, KW is an employee of Tesaro, Inc., MR is an employee of MSD and holds restricted stock in Merck.

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ACKNOWLEDGMENTS We would like to thank our many colleagues at the IRBM, Merck Research Laboratories and at Tesaro who contributed to discovering and advancing niraparib through discovery and clinical development.

CONCLUSIONS Although there has been interest in PARP inhibitors for a variety of therapeutic applications for some time, it was the discovery that PARP inhibition in the context of defects in K


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ABBREVIATIONS USED BER, base excision repair; CLp, plasma clearance; DSB, (DNA) double-strand break; GCIG, gynecological cancer intergroup; HR, homologous recombination; IRBM, Istituto di Ricerche di Biologia Molecolare; MMR, mismatch repair; NER, nucleotide excision repair; NHEJ, nonhomologous end joining; PAR, poly(ADP-ribose); PARP, poly(ADP-ribose) polymerase; PBMC, peripheral blood mononuclear cell; PFS, progression free survival; PSA, prostate specific antigen; RECIST, response evaluation criteria in solid tumors; shRNA, small hairpin RNA; SSB, (DNA) single-strand break; UK, United Kingdom (of Great Britain and Northern Ireland); US, United States (of America); Vdss, volume of distribution at steady state

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NOTE ADDED IN PROOF The FDA in December 2014 approved olaparib (Lynparza) as monotherapy in patients with heavily pretreated ovarian cancer that is associated with defective BRCA genes.

M


Niraparib: A Poly(ADP-ribose) Polymerase (PARP) Inhibitor for the

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