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Drug Development Unit, Division of Cancer Therapeutics and Division of Clinical Studies, The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research, Downs Road, Sutton, Surrey SM2 5PT, UK (J. Mateo, M. Ong, D. S. P. Tan, M. A. Gonzalez, J. S. de Bono).

Correspondence to: J. S. de Bono johann.de-bono@ icr.ac.uk

Appraising iniparib, the PARP inhibitor that never was—what must we learn?Joaquin Mateo, Michael Ong, David S. P. Tan, Michael A. Gonzalez and Johann S. de Bono

Abstract | Several drugs targeting poly(ADP-ribose) polymerase (PARP) enzymes are under development. Responses have been observed in patients with germline mutations in BRCA1 and BRCA2, with further data supporting antitumour activity of PARP inhibitors in sporadic ovarian cancer. Strategies to identify other predictive biomarkers remain under investigation. Iniparib was purported to be a PARP inhibitor that showed promising results in randomized phase II trials in patients with triple-negative breast cancer. Negative results from a phase III study in this disease setting, however, tempered enthusiasm for this agent. Recently, data from in vitro experiments suggest that iniparib is not only structurally distinct from other described PARP inhibitors, but is also a poor inhibitor of PARP activity. In this context, the negative iniparib phase III data might have erroneously promulgated the notion that PARP inhibition is not an effective therapeutic strategy. Here, we scrutinize the development of iniparib from preclinical studies to registration trials, and identify and discuss the pitfalls in the development of anticancer drugs to prevent future late-stage trial failures.

Mateo, J. et al. Nat. Rev. Clin. Oncol. 10, 688–696 (2013); published online 15 October 2013; doi:10.1038/nrclinonc.2013.177

IntroductionOver the past 5 years, around 4,800 early phase clini­cal trials have been initiated in cancer medicine. During this period, more than 2,300 phase III studies were initi­ated. Sadly, the rate of failure in oncology for novel com­pounds undergoing clinical evaluation continues to be high compared with other medical specialties, such as cardio vascular diseases, and might even be rising with the advent of targeted therapies.1–3 Costs of drug develop­ment also continue to increase, and may now be more than US$3 billion per drug approved; moreover, the market narrows the room for new approvals,4 impacting the price of compounds when approved and jeopard­izing the sustainability of health services.5,6 Failures in late­phase registration clinical trials are especially disappoint ing, as they represent an enormous expendi­ture of money and time from pharmaceutical companies and academic institutions, and signify potential suffering of huge numbers of patients with advanced­stage cancer and their families.

Why are promising results observed in preclini­cal research often not translated into clinical success? Several causes contribute to failures including: limited know ledge of cancer biology (despite ongoing and encouraging advances), selection of pharmacological compounds with suboptimal pharmacological proper­ties, poorly predictive preclinical models, inappropriate trial designs, or decision­making based on nonrelevant end points.7–11

Iniparib (BiPar Sciences and Sanofi) is a compound that was initially developed as a poly(ADP–ribose)

polymerase (PARP) inhibitor, a class of drugs that impairs single­stranded DNA break repair. In general, PARP inhibitors have been useful in one of two strat­egies: first, ‘synthetic lethality’, capitalizing on the sensi­tivity of cells with defective homologous­recombination (HR)­mediated DNA repair, and second, sensitization of cells to DNA­damaging therapies. Indeed, several PARP inhibitors have demonstrated promising preclinical and clinical antitumour activity with wide therapeutic indices in the setting of tumours with BRCA1 or BRCA2 germ­line inactivating mutations.12–15 Moreover, numerous preclinical models have also demonstrated that PARP inhibition can sensitize cells to the DNA­damaging effects of ionizing radiation, alkylating agents, and t opoisomerase I targeting.16–19

Recent studies have raised concerns that iniparib is not a bona fide PARP inhibitor.20,21 The implications of these new data must be carefully considered, because over 2,500 patients have been treated in clinical trials of iniparib that were designed with PARP inhibition as a therapeutic goal (Table 1). These new data should caution against generalizing the negative results of i niparib trials to the ongoing development of other PARP inhibitors. In this Review, we retrace the development of iniparib from preclinical studies through to phase I–III studies and analyse lessons we must learn to optimize the d evelopment of other t argeted drugs.

Preclinical studiesIniparib was developed as a prodrug of the more reac­tive, but unstable, 4­iodo­3­nitrosobenzamide (INOBA). INOBA inactivates PARP via two mechanisms: first, zinc­ejection following oxidation of the first zinc­finger

Competing interestsThe authors declare no competing interests.

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domain of the PARP protein resulting in loss of DNA­stimulated PARP activity without loss of DNA binding capacity; second, induction of PARP­degrading amino­peptidases that generate a characteristic polypeptide degradation product.22–25 Importantly, these purported mechanisms of action for iniparib differed signifi­cantly from ‘classic’ PARP inhibitors, which specifi­cally target the NAD+ binding site of PARP1 or PARP2, mimicking the NAD+ substrate by resembling the NAD+ moiety and competitively blocking PARP activity.26

Interestingly, the antitumour activity of INOBA also seemed to depend on the levels of glutathione and other reducing compounds. Depletion of glutathione in vitro enhanced antitumour activity and the lack of reducing flavoproteins in malignant cells, that convert INO2BA into nontoxic amines rather than to INOBA, was pro­posed as a mechanism of selective tumoricidal action of INO2BA in cancer cell cultures, but not in fibroblast cultures.27 Importantly, with or without glutathione depletion, in vitro experiments required substantial concen trations of INO2BA for inhibition of PARP a ctivity and antitumour activity.

There are limited published preclinical studies of inipa rib either as a single agent or in combi nation with chemotherapy; antiproliferative effects have been reported in cancer cell lines including triple­ negative breast cancer (TNBC; oestrogen [ER]­receptor­negative, progesterone [PR]­receptor­negative, and HER2­negative) in which iniparib caused cell­cycle arrest in the G2/M phase.27,28 However, whereas synthetic lethal­ity in BRCA­mutant cell lines was confirmed for other NAD+­competitive PARP inhibitors before clinical evalu­ation,12,26 potent activity of iniparib related to selective inhibition of PARP in BRCA­deficient cultured cells has yet to be reported. Moreover, very few results were avail­able within the public domain on the capacity of inipa­rib to sensitize cells to DNA­damaging chemotherapy, including p latinum agents,29,30 at the time the clinical trials started.

In vitro studies, however, have suggested that i niparib does not exhibit the properties of a classic PARP inhibi­tor. The effects of two different structural classes of NAD+­competitive PARP inhibitors (benzimidazole and pyridazinone derivatives) were compared with the effects of iniparib and its C­nitroso metabolite (INOBA).26 The effects of these compounds were tested on BRCA1­deficient (MDA­MB­436, exon 20 mutation), BRCA2­deficient (DLD1–/–) and BRCA1/2­proficient (MDA­MB­231 and DLD1+/+) breast cancer cell lines. All NAD+­competitive PARP inhibitors showed high selectivity for PARP, inhibited PARP enzymatic activ­ity at nanomolar concentrations, inhibited autoribosy­lation of PARP­1, potentiated alkylating chemotherapy (temozolomide), and showed selective activity in BRCA­deficient tumour cell lines and xenograft models. By con­trast, iniparib and its metabolite were not able to inhibit PARP enzymatic activity, diminish poly(ADP–ribose) formation, potentiate temozolomide, or show activity in either BRCA­deficient or BRCA­proficient cell lines or xenograft models.26 The doses of iniparib required to

achieve a cytotoxic effect were very high (>40 μmol/l). Furthermore, depletion of glutathione, a potential mediator of resistance, did not appreciably alter these observations. Instead of selective potent activity, inipa­rib was found to nonspecifically react and form adducts with proteins containing cysteine residues, including the PARP­1 zinc finger domain.

In a separate study, three different BRCA­proficient TNBC cell lines were tested against iniparib and three different NAD+­competitive PARP inhibitors: AG­014699 (rucaparib, Pfizer­Clovis), AZD­2281 (olaparib, Astra­Zeneca) and ABT­888 (veliparib, Abott Laboratories),21 showing lower potency of PARP inhibition and anti­tumour activity for iniparib. BRCA1 knockdown sensi­tized cells to iniparib, but it did not result in an increase of γ­H2AX formation as occurred with rucaparib, olapa­rib, and veliparib. An in vitro study of 12 breast cancer cell lines demonstrated higher IC50 concentrations for iniparib (13–70 μM) when compared with olaparib (IC50 range 3.7–31 μM).31

Another study exposed HR­deficient cells (BRCA2­deficient PEO1 human ovarian cancer cells and ATM­deficient GM16666 fibroblasts) and HR­proficient cells (BRCA2­revertant PEO4 ovarian cancer cells and ATM­restored GM16667 fibroblasts) to veliparib, olapa­rib, and iniparib.32 The HR­deficient cells were selectively sensitive to veliparib and olaparib; however, iniparib did not selectively target HR­deficient cells. Furthermore, iniparib—unlike the other tested agents—did not syner gize with either topoisomerase I poisons, cispla­tin, gemcitabine, or paclitaxel in various cell lines, and failed to inhibit poly(ADP–ribose) formation even at c oncentrations of 100 μmol/l.

Overall, these preclinical data suggest that the poten­tial cytotoxic effects of iniparib are not mediated by PARP inhibition, but by mechanisms that are yet to be elucidated that might be related to stimulation of intra­cellular production of reactive oxygen species.33 These findings became apparent only after the pursuit of a large and expensive drug development programme, and clearly question whether the preclinical evidence was strong enough to justify the initiation of clinical trials.

Key points

■ Iniparib is not a bona fide inhibitor of poly(ADP-ribose) polymerase (PARP), so the clinical results in this context should not be extrapolated to other PARP inhibitors in development

■ Preclinical data on iniparib did not sufficiently elucidate the mechanism of action of this agent before clinical trials were initiated

■ Phase I trials should provide proof of mechanism and, ideally, proof of concept, in expansion cohorts to test biological hypotheses; early clinical trials of iniparib lacked proof of mechanism

■ Selection of a patient population, and implementation and validation of predictive biomarkers, are critical to optimize drug development

■ Randomized phase II trials have a significant rate of false positivity, so promising results should be interpreted prudently until other confirmatory studies are reported

■ Preclinical and clinical studies with negative results and efforts evaluating reproducibility of previously published data should be publically available to minimize the risk of publication bias

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Clinical studiesPhase I trialsSingle­agent iniparib was first evaluated in patients with advanced solid cancer in a phase I study with a ‘3 + 3’ dose­escalation design (NCT00298675) (Table 1).34 Results were reported for 24 patients treated at eight dose levels (0.5–8.0 mg/kg intravenous [IV] adminis­tration per day, twice weekly). No dose­limiting toxici­ties were observed and the most common adverse events were gastro intestinal disorders (39%). Best response of stable disease for ≥2 months was documented for six out of the 24 patients. Pharmacokinetic analysis showed rapid conversion of iniparib (half­life [T1/2] 4 min) into its active metabolites, but a later study reported that most of the drug is rapidly transformed into inactive com­pounds.35 At a dose of 1.4 mg/kg, the maximum achieved concen tration (Cmax) was above the threshold for efficacy

in preclinical models. Pharmacodynamic data showed inhibition of PARP in peripheral blood mononuclear cells (PBMCs) by >50% after a single dose of 2.8 mg/kg, and greater PARP inhibition after multiple doses.34

Studies of other biomarkers of PARP inhibition, such as induction of γ­H2AX foci, were not reported and pharmaco dynamic analysis in tumour tissue was not pursued. Unfortunately, most of the information about the assays used for the pharmaco dynamic studies and their validity were not detailed at the time the results were communicated. What was defined as the ‘biologi­cally relevant dose’ was estimated by pharmaco kinetic and pharmacodynamic studies; moreover, a maximum toler ated dose (MTD) was never defined and specific expansion cohorts to prove target modulation in popu­lations with known HR­mediated DNA repair system a berrations were not conducted.

Table 1 | Clinical studies of iniparib as a single agent or combined with other drugs

Trial initiation Trial identifier n Phase Tumour type Treatment arms

Phase I trials

March 200634 NCT00298675 58 I ASC Iniparib; iniparib + irinotecan

January 200736,37 NCT00422682 84 I ASC Iniparib + topotecanIniparib + temozolamideIniparib + gemcitabineIniparib + carbo/paclitaxel

March 200848 NCT00687765 120* I/II Gliomas Iniparib + temozolamide

July 2010 NCT01161836 7* I ASC Iniparib (ECG-effect study)

September 2010 NCT01213381 18* I ASC Iniparib + gemcitabine/carboplatin

November 2011 NCT01455532 160* I/IB ASC IniparibIniparib + gemcitabine + carboplatinIniparib + paclitaxelIniparib + PEG-doxorubicin

September 2012 NCT01551680 30* I Brain M1 Iniparib + radiotherapy

Phase II trials

October 200741 NCT00540358 123 II TNBC Iniparib + gemcitabine + carboplatinvs gemcitabine + carboplatin

December 200843 NCT00813956 80 II TNBC (neoadjuvant) Iniparib + gemcitabine + carboplatin

May 200849 NCT00687687 22 II Uterine carcinosarcoma Iniparib + carboplatin + paclitaxel

June 2008 NCT00677079 12* II BRCA1/2 ovarian Iniparib

December 200945 NCT01033123 41 II Ovarian (platinum-sensitive) Iniparib + gemcitabine + carboplatin

December 200946 NCT01033292 43 II Ovarian (platinum-resistant) Iniparib + gemcitabine + carboplatin

February 2010 NCT01045304 163* II TNBC Iniparib (weekly vs twice a week) + gemcitabine + carboplatin

May 201047 NCT01086254 116 II NSCLC Iniparib + gemcitabine + cisplatinvs gemcitabine + cisplatin

July 201083 NCT01173497 37 II TNBC-brain M1 Iniparib + irinotecan

September 201042 NCT01204125 141 II TNBC (neoadjuvant) Iniparib + paclitaxel

Phase III trials

July 200944 NCT00938652 519 III TNBC Iniparib + gemcitabine + carboplatinvs gemcitabine + carboplatin

March 201051 NCT01082549 780* III SCC lung Iniparib + gemcitabine + carboplatinvs gemcitabine + carboplatin

Other

May 2012 NCT01593228 – III ASC Iniparib extension programme

*Planned accrual as per ClinicalTrial.gov website; final results not reported. Abbreviations: ASC, advanced solid cancers; ECG, electrocardiogram; NSCLC, non-small-cell lung cancer; SCC, squamous cell carcinoma; TNBC, triple-negative breast cancer; vs, versus.

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A phase Ib study combined iniparib with four differ ent chemotherapy regimens: topotecan (n = 14), gemcita bine (n = 22), temozolomide (n = 17), and carbo platin plus paclitaxel (n = 13) (NCT00422682).36 The doses of inipa­rib explored ranged from 1.1–11.2 mg/kg (IV, days 1 and 4 of every week). Surprisingly, no dose­limiting toxicities were reported, and seven of 66 patients (10%) achieved radiological partial or complete responses as their best response to treatment. Further safety data from an expan­sion cohort who received carbo platin (AUC6, day 1) plus paclitaxel (200 mg/m2, day 1) with iniparib (5.6 mg/kg, IV, days 1, 4, 8, and 11) again showed a low incidence of grade 3 or 4 adverse effects neutropenia (13.3%) and anaemia (6.7%).37 These data contrast with PARP inhibitor trials in which single­agent dose­escalation was limited by haematological toxi city, which was further potentiated by combination with chemotherapy.14,38,39

Phase II trials in TNBCAn open­label, randomized phase II study in 123 patients with TNBC evaluated gemcitabine (1,000 mg/m2, days 1 and 8) plus carboplatin (AUC 2, days 1 and 8)40 with or without iniparib (initially 4.0 mg/kg, days 1, 4, 8, and 11; later amended to 5.6 mg/kg on the same schedule) once every 3 weeks (NCT00540358).41 The majority of patients (58.5%) had no prior systemic treatment for metastatic disease. The efficacy analyses evaluated the intention­to­treat population but, unlike the later phase III trial, radiological response assessments were performed by the investigators and were not subjected to an external blinded review. Patients with or without BRCA mutations were eligible, but the number of patients with BRCA mutations and their outcome have not been reported.

The clinical benefit rate (CBR), which is the rate of com­plete or partial radiological response together with stable disease >6 months, and objective response rate (ORR) were 34% and 32% in the chemotherapy alone arm, and 56% and 52% in the iniparib arm, respectively. The trial was designed to detect an improvement in CBR, assum­ing a rate of 0.45 in the control group and expecting a CBR >0.60 in the experimental arm (with a power of 80% and accepting a two­sided α error of 0.05). Consequently, the study was considered positive (P = 0.001 for CBR; P = 0.002 for ORR). Secondary survival end points were also positive, but subject to very broad 95% confidence intervals; median progression­free survival (PFS) was 3.6 (2.6–5.2) months in the chemotherapy alone group versus 5.9 (4.5–7.2) months in the iniparib group (P = 0.01). Overall survival was 7.7 (6.5–13.3) months compared with 12.3 (9.8–21.5) months (P = 0.01) for the iniparib arm. Interestingly, no difference in the rate of adverse events, including haemato logical events, was observed between the two arms, again raising concerns regarding the lack of class­type t oxicities associated with PARP inhibitors.

A neoadjuvant trial of weekly paclitaxel (80 mg/m2, day 1) alone or with iniparib once weekly (11.2 mg/kg, IV, day 1) or iniparib twice weekly (5.6 mg/kg, IV) was also pursued in patients with TNBC (NCT01204125).42 Overall, 141 patients were recruited; the trial did not detect differences in the primary end point, rate of

pathological complete response (pCR) of the primary breast tumour. In another trial, 80 patients received 4–6 cycles of neoadjuvant gemcita bine (1,000 mg/m2, IV), carboplatin (AUC2, IV, days 1 and 8) and iniparib (5.6 mg/kg, IV, days 1, 4, 8, and 11) once every 3 weeks (NCT00813956). A pCR rate of 47% (90% CI 27–69%) was reported for the 19 carriers of BRCA mutation treated; for the BRCA wild­type p articipants, the pCR rate was 33% (90% CI 23–44%).43

Phase III trial in TNBCIniparib quickly entered into phase III trials, labelled as a PARP inhibitor following the promising phase II data.41 A randomized open­label phase III study enrolled patients with TNBC who had undergone up to two prior lines of treatment.44 Patients were randomly assigned (1:1) to gemcitabine–carboplatin alone (gemcitabine 1,000 mg/m2, IV) and carboplatin (AUC 2; , IV, days 1 and 8) or the same regimen plus iniparib (5.6 mg/kg, IV, days 1, 4, 8, and 11) every 3 weeks. The trial was planned with two co­primary end points: overall survival and PFS. The sample size was calculated with the aim of detecting, with a power of 90%, a HR of 0.65 for PFS and 0.66 for overall survival between the two arms. The type 1 error accepted (0.05, two­sided) was divided for the two co­primary end points (0.04 for overall survival and 0.01 for PFS). The trial was to be considered posi­tive if either one of the two primary end points was met. Between July 2009 and March 2010, 519 patients were enrolled and randomly assigned, which constituted over­recruitment of patients as the statistical design demanded 420 patients to test the hypothesis. Patient demographics suggested a similar population to the prior phase II trial. In total, 152 of 258 patients (59%) on the chemotherapy­ only arm crossed over to receive chemotherapy and iniparib following disease progression. ORR was not signifi cantly different (30% versus 34%), nor was the study positive for either of the co­primary end points.44

Clinical trials in other tumour typesTwo single­arm trials in both platinum­sensitive and platinum­ resistant recurrent ovarian carcinoma have tested iniparib in combination with carboplatin and gem­citabine (NCT01033123 and NCT01033292).45,46 A signifi­cant number of BRCA­mutation carriers were enrolled, but no relationship between BRCA status and objective response was observed.14,38

Other trials have explored the antitumour activity of ini­parib in combination with several chemotherapy regimens in lung cancer, gliomas, and uterine carcino sarcoma.47–49 Overall, none of these trials has provi ded either proof of concept of chemosensitization with inipa rib or identi­fied predictive biomarkers of response. A phase II trial in patients with lung cancers and all histologies but largely non­squamous did not report improvement of overall sur­vival or PFS.47 In parallel, a large randomized phase III study in patients with newly diagnosed squamous­cell lung cancer was pursued (NCT01082549).50 Final results are yet to be presented, but a negative outcome has been announced in a press note (Table 1).51

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Appraising iniparib: critical questionsWas sufficient preclinical data acquired?When the early clinical trials began, limited published preclinical data supported the clinical evaluation of inipa rib in patients with cancer. Iniparib was ‘labelled’ as a potent and selective PARP inhibitor, and clinical trials were designed to recruit patients with presumed DNA repair deficiency. Later attempts to evaluate inipa­rib by independent investigators failed to find supporting evidence of potent and selective PARP inhibition. This inability to validate the mechanism of action, the lack of in vitro data supporting a selective effect on HR­deficient cell lines, and the lack of robust evidence of synergy in combination with chemotherapy raises signifi cant con­cerns that the preclinical evidence was insufficient to initiate clinical studies.

Reproducibility of research results is an often under­estimated concern, at least in part because of academic and/or industry pressures to accelerate drug develop­ment programmes, maximize fiscal return, and achieve academic recognition. When selecting a compound to enter clinical evaluation, careful scrutiny is needed of the available preclinical data, the validity of the assays used, and the robustness of the results to justify the high amount of personal and monetary resources to be employed. Two independent studies evaluating the reproducibility of results published by either academic researchers or pharma ceutical industry investigators in high­impact publications have recently exposed the problem: only 11–25% of published results could be replicated by two groups of industry investigators, even when using identical reagents (usually provided by the original laboratories).52,53 The issue is also relevant when developing drug combinations, as the concept of ‘synergy’ is often overestimated and clinical trials too often fail to confirm the expectations derived from preclinical results.54 Investigators, industry, and editors should also be encouraged to report and accept for publication ‘nega­tive’ preclinical studies to limit such publication bias, and to await a robust confirmation and appraisal of preclinical data before clinical trials are commenced.

Were proof of mechanism and concept pursued?Given the extremely short reported T1/2 of iniparib (4–11 min),34 a major concern is whether systemic expo­sure to iniparib or its active metabolites are significant enough for clinical activity. Although Cmax plasma levels of iniparib exceeded concentrations necessary for in vitro activity, few data are available regarding plasma expo­sure (such as AUC) necessary for antitumour activity. Although early phase trials reported a T1/2 of 2–4 h for the active metabolites, Verweij et al.35 presented contra dicting data that T1/2 of metabolites was <2 h, with substantial inactivation of the parent compound by gluta thione. Furthermore, pharmacokinetic exposures were not c orrelated with target modulation in tumour tissue, as no pharmaco dynamic data from paired tumour samples were reported. This issue is particularly important because no radiological antitumour activity was observed in the original single­agent iniparib dose­findin g trial.34

The low incidence of haematological adverse events and lack of single­agent antitumour activity in early phase trials could prompt concerns of a possible alterna­tive mechanism of action.14,38 In this situation, proof­of­mechanism or proof­of­concept studies are particularly critical to demonstrate robust target modulation via validated pharmacodynamic assays in samples of rel­evance. As mentioned, the effect of iniparib on target PARP modulation was reported only in PBMCs, but not in paired tumour tissue biopsies. Although normal tissue pharmacodynamic studies (in PBMCs, skin, or hair folli­cles) might suggest biological target modulation and assist in identifying the optimal time points to perform tumour biopsies, such analyses are limited in predict­ing effects in tumour tissue owing to limitations in drug delivery and tumour heterogeneity.55,56 Ultimately, in the absence of observed radiological responses and/or clini­cal toxicity, analysis of paired tumour biopsies are neces­sary to understand the relationship between drug dose and potency and duration of target pharmaco dynamic modulation.57,58 Ideally, evaluation of PARP inhibition by assessment of diminished formation of poly(ADP–ribose) polymerase polymers, inhibition of PARP­1 activity, and induction of γ­H2AX in tumour samples would have provided s ignificant support of the proof of the mechanism for iniparib.

The ‘biologically active dose’ and recommended phase II dose for iniparib (5.6 mg/kg, IV, twice weekly) was—critically—not defined on the basis of evidence of objective single­agent radiological response, dose­limiting toxicities, or robust pharmacodynamic data. Such data should be crucial to make informed go/no­go decisions in drug development.59 Defining antitumour activity and toxicity of drug doses well above the lowest biologically active dose is important, because poor drug penetration is commonly a mechanism of treatment resistance.60 It is advisable to evaluate the antitumour activity of the MTD versus the minimum biologically active dose of novel agents while acquiring detailed pharmaco dynamic data. Ideally, this should be done in specific expansion cohorts of phase I trials while focusing on the appropriate population for the biological context.

Indeed, in the field of PARP inhibition, some evi­dence suggests a dose­activity relationship above the lowest biologically active dose. In the phase I trial of olaparib, the pharmacodynamic studies defined 100 mg twice daily as a biologically effective dose whereas the MTD of the drug, based on the tolerability profile, was 400 mg.14 Phase II trials comparing 100 mg versus 400 mg twice daily in sequentially enrolled cohorts showed clear superior ity for 400 mg twice daily dose in both patients with breast cancer and ovarian cancer.61,62

What was the target population?The advent of targeted therapies has necessitated a shift in histologically driven selection of target populations to molecularly selected groups of patients. Characterization of the appropriate population should happen in concert with the preclinical studies characterizing the properties of a new compound and establishing practical methods

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to identify those patients with a validated biomarker. As a purported PARP inhibitor, iniparib was developed in populations thought to be defective in HR DNA repair, as well as populations thought to benefit from potential chemosensitization.

Several trials of iniparib were conducted in women with TNBC on the basis of favourable preclinical data, an unmet medical need, and the suggestion that the basal­like group of breast cancers is associated with functional defects in HR­mediated DNA repair.63–65 Moreover, pre­clinical data showed significant upregulation of PARP in TNBC in comparison with other subtypes of breast cancer.66 However, defining TNBC as a target population is challenging as it is characterized by the absence of three biomarkers (ER, PR and HER2) rather than by a positive selection biomarker indicative of HR­mediated DNA­repair deficiency. Moreover, although the TNBC popula­tion has been shown to be enriched (perhaps up to 71%) in those with basal­like breast cancers,67 the explora tory Affimetrix gene­expression profiling of the inipa rib phase III study revealed considerable molecular hetero­geneity in the trial population.44 Even if iniparib were a potent PARP inhibitor, the estimated frequency of BRCA1 and BRCA2 germline mutations in unselected TNBC is approximately 10–20%.68,69 Furthermore, even accepting that there is accumulation of PARP­1 in several tumour types, including TNBC,66 and that PARP­1 might act as a BRCA2­expression downregulator,70 no clini­cal data supports PARP­1 upregulation as a predictive marker of sensitivity to PARP inhibitors.

At present, no validated strategy is available to identi fy patients with TNBC who have DNA­repair deficiencies to participate in trials of PARP inhibitors other than testing for germline BRCA1/2 mutations, which might have been used to demonstrate proof of concept for single­ agent iniparib. An alternative approach is to design trials attempting clinical validation of other candidate biomarkers, such as a panel of immuno histochemical markers, namely ER, HER2, CK5, CK6, and EGFR, which have been used to retrospectively identify basal­like tumours with high specificity and sensitivity71 or functional assays for HR competency, which are currently being developed.72

The optimal target population for PARP inhibitors has been explored in a number of trials, for example, Gelmon et al.13 recruited patients with ovarian cancer and TNBC to receive olaparib, and prospectively evaluated the results on the basis of the presence of BRCA1/2 muta­tions. Among non­BRCA­mutated patients, the cohort of patients with ‘sporadic’ TNBC was rapidly closed after no signs of anticancer activity were detected in the first 15 patients. However, consistent with earlier studies,73 a cohort of predominantly platinum­ sensitive non­BRCA-mutated patients with ovarian cancer responded to olaparib treatment. One potential reason contributing to the lower number of responses in the BRCA-mutated breast cancer cohort and the complete lack of benefit for the unselected TNBC group might be the high number of prior treatment regimens received by these patients (the median prior lines of chemotherapies was

three, and ranged from one to seven), which included patients with platinum­resistant or refractory disease; however, detailed information on prior treatments was not a vailable for this study.

Overall, these findings strongly reinforce the import­ance of pursuing proof of concept in carefully selected target populations when designing clinical studies. In hindsight, the iniparib trials might have taken both attention and resources away from the patients proven to benefit most from PARP inhibitors, the BRCA m utation carriers.

Transition from phase I to phase III Frameworks such as the Pharmacological Audit Trail (PhAT)58,74 can subject a new compound that is transition ing from preclinical to clinical trials to critical performance criteria. These frameworks ask a series of questions that require demonstration of proof of concept and testing of mechanistic hypotheses (Figure 1) to better select drugs that merit further evaluation. In this trans­ition to phase III trials, we must continually reinforce and match knowledge gained from preclinical and clini­cal studies so that each stage of development benefits from all available information.

When iniparib was entering phase II trials, preclinical synergistic cytotoxicity data was reported for iniparib when combined with gemcitabine and carboplatin in TNBC cell lines;75 however, activity and toxicity of the gemcitabine–carboplatin combination was not studied in earlier phase I trials. A dose­finding study of iniparib in combination with carboplatin and gemcitabione was initiated in Japan at the request of the Japanese regulatory authorities, after the phase II/III trials in patients with TNBC were recruiting (NCT01213381).76 These find­ings bring into question how the backbone of carboplatin (AUC2, days 1 and 8) in combination with gemcitabine (1,000 mg/m2, days 1 and 8) every 3 weeks was chosen for the subsequent phase II and phase III trials, consider­ing prior trials that used gemcitabine–carboplatin with targeted agents in breast cancer had used a more­intense dosing of carboplatin.77,78 Since the CBR of 34% (primary end point of the iniparib randomized phase II trial) in the control group was lower than expected (45%), one must question whether this relates to underdosing of carboplatin.

Allowing crossover in randomized trials is another factor that merits discussion in the development of inipa rib. Crossover was permitted in the phase II study, which can be justified and frequently practiced for ‘ethical reasons’. In this case, 30 of 59 (51%) patients randomly assigned to the gemcitabine–carboplatin arm received gemcitabine–carboplatin–iniparib at disease progres­sion, but 83% of those patients discontinued the triple treatment after two cycles or fewer, so the crossover did not translate into any significant signal of benefit. Despite these findings, the phase III trial also allowed patients in the gemcitabine–carboplatin arm to receive gemcitabine–carboplatin–iniparib at disease progression. Considering how crossover undeniably complicates the interpretation of overall survival results, because patients in both arms

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receive the experimental agent, the phase III design should have considered the lack of benefit patients would receive who were crossed over in the previous study. One addi­tional consideration is that if gemcitabine–ca rboplatin–iniparib is truly inferior to gemcitabine–carboplatin alone, a crossover design might benefit the experi mental arm because those who crossover to iniparib remain on ineffec tive treatment after disease p rogression when receiving gemcitabine–carboplatin.79

End point selection is also a critical factor in the design of registration studies. The phase III trial of gemcitabine–carboplatin–iniparib in patients with TBNC was designed with co­primary end points of overall survival and PFS, but it is important to mention that PFS has not been demonstrated to be a surrogate marker of overall sur­vival in metastatic breast cancer, as was shown for bevaci­zumab.80–82 Despite the benefits of using PFS, especially if presented together with improvement in quality­of­life parameters, concerns should be raised about the contin­ued selection of PFS as a primary end point in registration trials, particularly when p ermitting the potential impact of crossover on overall survival.

ConclusionsA successful drug development programme demands a plausible biological hypothesis supported by robust and reproducible preclinical data, early trials providing proof of desired target modulation, analysis and/or validation of putative predictive/patient enrichment bio markers, transparent rules to make ‘no­go’ decisions early in develop ment, and, finally, a multidisciplinary trans­lational strategy that produces continuous r eiterative f eedback between preclinical and clinical research.

In hindsight, the development of iniparib proceeded without sufficient preclinical and pharmacodynamic data to warrant larger trials. This included a failure to acquire proof of target modulation and antitumour activ­ity in the BRCA-mutated population on the basis of a pre­dictive biomarker. Ultimately, the phase III trial in TNBC failed to meet its co­primary end points, promulgating the notion that PARP is not a good therapeutic target, despite significant antitumour activity demonstrated by other PARP inhibitors.38,60,61 Ironically, negative results from the phase III trials in TNBC were communicated to the media only 22 days after the phase II study was

Population identi�cation

Targeted drug candidate

Validated predictive assayfor molecular aberration

Pharmacokinetics

Pharmacodynamics

Biochemical pathwaymodulation

Achievement ofbiological effect

Hypothesis testing usingintermediate end points

of clinical response

Reassessment of molecularalterations at disease

progression

Inhibition of resistantbiological pathways

4-iodo-3-nitrobenzamide(Iniparib)

Generic PhAT Questions of PhAT

Yes, adequate concentrationsin plasma achieved

Unknown—no tumour biopsy datashowing PARP inhibition

PARP zinc-ejection (inactivation)Induction of PARP proteases

Sensitization of chemotherapy

BRCA1/2 germline mutation valid‘BRCAness’ not validated

No marker for chemosensitization

PBMC PARP inhibition seenbut insuf�ciently robust data

No tumour biopsy data available

No

No

Are adequate concentrations of drugand metabolites in plasma achieved?

Are adequate concentrations of drugand metabolites in tumour achieved?

What are the proposed mechanism(s)of the novel drug candidate?

What are the validated predictiveassays for the target of the drug?

What are the pharmacodynamic effectsin surrogate normal cells?

What are the pharmacodynamiceffects in tumour cells?

Is there a proposed population to betargeted by the novel drug?

Is there modulation of thetargeted biochemical pathway?

Is there evidence of wanted biologicaleffect or clinical response?

Is there modulation of circulating tumourcells, tumour markers, or other markers?

Is there evidence of mechanism(s)of resistance to drug?

Is there a mechanism of overcomingmechanisms of resistance?

BRCA1/2 germline mutation‘BRCAness’ in TNBC and ovarian cancer

population sensitive to DNA-damaging agents

Glutathione inactivation has notbeen pursued

Single-agent activity very limitedCombination data heterogeneous

In plasma perhapsNo tumour biopsy data

Figure 1 | Confronting the Pharmacological Audit Trail (PhAT). Adapted from Yap et al.58 (left) with the published data from the iniparib preclinical and clinical studies (right). Permission obtained from Nature Publishing Group © Yap, T. A. et al. Nat. Rev. Cancer 10, 514–523 (2010).

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published in a high­impact journal. Overall, the story of iniparib is a compendium of results released in abstract form or never published, with few results in peer­review publications, making it difficult to examine by i ndependent investigators.

Overall, these outcomes should challenge us to systematic ally scrutinize each step during drug discov­ery and development, as well as understand the context of phase II trials and the significant risk of false­positive results from randomized phase II studies. This case should at least guide the scientific community to a con­sensus about the characteristics a PARP­1 inhibitor must exhibit in preclinical and early clinical studies.

Clinical development of iniparib has now been inter­rupted; considering the enormous time and money invested on it, the story of iniparib clearly merits the attention of academics and industry and the reflection

that without a clear understanding of the mechanism of action of a new compound, analytically validated biomarkers to guide dose and schedule selection, and predictive biomarkers to define the optimal target popu­lation, drug development efforts remain at high risk of failure.

Review criteria

We searched the MEDLINE and PubMed databases for original articles focusing on iniparib published between 2005 and 2013, as well as abstract databases from ASCO, ESMO and AACR between 2005 and 2013. The search terms we used were “iniparib”, “BSI-201” or “4-iodo-3-nitrobenzamide” and “PARP”. All papers and abstracts identified were English-language full-text papers or abstracts. We also searched the reference lists of identified articles for further papers.

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AcknowledgementsThe Drug Development Unit of the Royal Marsden NHS Foundation Trust and The Institute of Cancer Research is supported in part by a programme grant from Cancer Research UK. Support was also provided by the Experimental Cancer Medicine Centre (to The Institute of Cancer Research) and the National Institute for Health Research Biomedical Research Centre (jointly to the Royal Marsden NHS Foundation Trust and The Institute of Cancer Research).

Author contributionsAll authors researched data for the article and made a substantial contribution to discussions of the content and contributed to writing the manuscript. J. Mateo, M. Ong, and J. S. de Bono reviewed and edited the manuscript before submission.

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