L Cappuzzo

E B Horn

M Chatterjee

D C Turner

E Felip

H Lena

F Cappuzzo

L Horn

E B Garon

R Hui

H.-T Arkenau

M A Gubens

M. D Hellmann

D Dong

C Li

K Mayawala

T Freshwater

M Ahamadi

J Stone

G M Lubiniecki

J Zhang

E Im

D P De Alwis

A G Kondic

Dr Øystein Fløtten
email: oystein.flotten@helse-bergen.no

Systematic evaluation of pembrolizumab dosing in patients with advanced non-small cell lung cancer

Keywords: exposure-response, immunotherapy, non-small cell lung cancer, pembrolizumab, PD-L1, tumor size modeling

Systematic evaluation of pembrolizumab dosing in patients with advanced non-small cell lung cancer

INTRODUCTION

Pembrolizumab is a potent, highly selective, humanized IgG4 monoclonal antibody against the immune checkpoint programmed death 1 (PD-1) that has a binding affinity for the PD-1 ligands PD-L1 and PD-L2 in the low nanomolar concentrations (PD-L1 half-maximal inhibitory concentration [IC50], ~0.1 to 0.3 nM and PD-L2 IC50, ~0.5 to 0.9 nM). Consistent with other monoclonal antibodies, pembrolizumab has a low clearance (0.2 L/day), limited central (3.7 L) and peripheral (4.4 L) volume of distribution, and low to moderate variability (22% to 41%) [START_REF] Dirks | Population pharmacokinetics of therapeutic monoclonal antibodies[END_REF][START_REF] Gangadhar | Population pharmacokinetic (popPK) model of pembrolizumab (pembo; MK-3475) in patients (pts) treated inKEYNOTE-001 and KEYNOTE-002[END_REF][START_REF] Patnaik | Phase I study of pembrolizumab (MK-3475; anti-PD-1 monoclonal antibody) in patients with advanced solid tumors[END_REF]. The half-life is 14-22 days, and serum exposure appears linear over the range of 0.1 to 10 mg/kg at steady-state dosing. Pembrolizumab has demonstrated robust antitumor activity and manageable toxicity across multiple dosages in several advanced malignancies. Pembrolizumab 2 mg/kg every 3 weeks (Q3W) was approved through the US Food and Drug Administration accelerated approval program for previously treated, PD-L1-positive advanced non-small cell lung cancer (NSCLC). A pooled analysis of the first 495 patients with previously treated or treatment-naive advanced NSCLC enrolled in the multicohort phase Ib KEYNOTE-001 study (ClinicalTrials.gov identifier, NCT01295827) demonstrated acceptable toxicity and durable antitumor activity for pembrolizumab, the magnitude of which was dependent on tumor PD-L1 expression [START_REF] Garon | Pembrolizumab for the treatment of non-small-cell lung cancer[END_REF].

We present an integrated analysis including efficacy and safety data from the final NSCLC expansion cohort of KEYNOTE-001. This final cohort, which included patients with previously treated NSCLC who received pembrolizumab 2 mg/kg Q3W only, was excluded from the initial publication by Garon et al [START_REF] Garon | Pembrolizumab for the treatment of non-small-cell lung cancer[END_REF] because it was not part of the planned training or validation sets for PD-L1 expression. We also describe a comprehensive exposure-response model based on tumor size data that was developed using all available NSCLC data from KEYNOTE-001 and employed for pembrolizumab dose selection in advanced NSCLC. This approach has become increasingly common for evaluating growth dynamics and treatment response in oncology [START_REF] Wang | Elucidation of relationship between tumor size and survival in non-small-cell lung cancer patients can aid early decision making in clinical drug development[END_REF][START_REF] Claret | Exploratory modeling and simulation to support development of motesanib in Asian patients with non-small cell lung cancer based on MONET1 study results[END_REF][START_REF] Ribba | A review of mixed-effects models of tumor growth and effects of anticancer drug treatment used in population analysis[END_REF] and is well suited for integrating and normalizing data from different time points and treatment durations.

METHODS

Study design

KEYNOTE-001 is a multicenter, open-label, phase Ib trial that included multiple advanced NSCLC expansion cohorts. Eligibility criteria for the first 495 patients enrolled were reported previously [START_REF] Garon | Pembrolizumab for the treatment of non-small-cell lung cancer[END_REF]. Key eligibility criteria for the final cohort included age ≥18 years, locally advanced or metastatic NSCLC, disease progression after platinum-based chemotherapy and an appropriate tyrosine kinase inhibitor for a sensitizing EGFR mutation or ALK translocation if present, Eastern Cooperative Oncology Group (ECOG) performance status 0-1, PD-L1 positivity, adequate organ function, no history of pneumonitis, and no systemic immunosuppressive therapy or active autoimmune disease.

All patients provided written informed consent. The study was conducted in accordance with the protocol, good clinical practice standards, and the Declaration of Helsinki. All protocols and amendments were approved by the appropriate institutional review board or ethics committee at each participating institution.

Treatment and assessments

In the initial KEYNOTE-001 NSCLC cohorts, 489 of 495 patients received pembrolizumab 10 mg/kg Q3W or 10 mg/kg every 2 weeks (Q2W); the final six patients received pembrolizumab 2 mg/kg Q3W before the protocol was amended [START_REF] Garon | Pembrolizumab for the treatment of non-small-cell lung cancer[END_REF]. Based on data from a randomized comparison in melanoma showing no difference between pembrolizumab 2 and 10 mg/kg Q3W [START_REF] Robert | Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dosecomparison cohort of a phase 1 trial[END_REF], a final NSCLC cohort was added in which patients received pembrolizumab 2 mg/kg Q3W.

Patients received pembrolizumab until disease progression assessed per immune-related response criteria [START_REF] Wolchok | Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria[END_REF] by investigator review, intolerable toxicity, or investigator or patient decision. Dose delay, prolonged dosing interval, or discontinuation were used to manage toxicity; dose reduction was not allowed. Tumor lesions were measured using computed tomography or magnetic resonance imaging at baseline and every 9 weeks thereafter. Tumor size was recorded as the sum of the longest diameters (SLD) assessed per RECIST v1.1 [START_REF] Eisenhauer | New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)[END_REF] by independent central review. Adverse events (AEs) were collected throughout the study and for 30 days after treatment discontinuation (90 days for serious AEs) and graded according to National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0. AEs of special interest based on immune etiology ("immune-mediated AEs") were identified from a prespecified list of terms (Table S1) and reported regardless of attribution to treatment by the investigator. PD-L1 expression was assessed in contemporaneous biopsy samples using immunohistochemistry and the 22C3 anti-human PD-1 antibody (Merck) [START_REF] Garon | Pembrolizumab for the treatment of non-small-cell lung cancer[END_REF]. For enrollment, expression was prospectively assessed using a prototype assay, with positivity defined as membranous staining on ≥1% of cells within tumor nests or staining in stroma. The PD-L1 tumor proportion score (TPS), defined as the percentage of tumor cells with membranous PD-L1 staining, was retrospectively assessed using a clinical trial assay, with positivity defined as TPS ≥1%. Based on the findings by Garon et al [START_REF] Garon | Pembrolizumab for the treatment of non-small-cell lung cancer[END_REF], PD-L1 positivity was further categorized as TPS 1% to 49% or ≥50%.

Blood samples (3.5 mL) for peak and trough pharmacokinetic assessment were collected regularly throughout treatment (Supplementary Materials). Regardless of treatment schedule, samples were collected at baseline and week 6. Pembrolizumab serum concentration was assessed using an electrochemiluminescence-based immunoassay with a 10-ng/mL limit of quantitation.

Exposure-Efficacy Analysis

A tiered evaluation approach was employed as part of a comprehensive evaluation, starting with more traditional comparisons of observed efficacy data (exploratory regression analyses) and followed by nonlinear mixed effects (NLME) modeling. Data analysis conducted in a stratified manner for the early analyses was pooled for the NLME model of change from baseline in tumor size because the model's statistical framework was better suited for integrating data.

All patients who had pharmacokinetic data and measurable disease per RECIST v1.1 by central review at baseline were included in the exposure-efficacy regression and NLME modeling analyses (n = 496: n = 6 treatment-naive and 47 previously treated patients received 2 Q3W, n = 45 treatment-naive and 216 previously treated received 10 Q3W, and n = 39 treatment-naive and 143 previously treated received 10 Q2W). Exposure was defined as the area under the concentration-time curve at steady state over 6 weeks (AUCss-6weeks), derived from an independent population pharmacokinetic model (manuscript submitted for publication). AUCss-6weeks was chosen as the exposure metric because it provided an integer number of dosing intervals across Q2W and Q3W regimens; steady state was selected for convenience and because pembrolizumab exhibits linear pharmacokinetics. A common steady-state exposure metric was used to avoid potential confounding between early study drop-out unrelated to dose/pharmacokinetic variability and cumulative exposure (ie, patients who progressed early and discontinued treatment ultimately had lower total exposure to pembrolizumab than those treated for a longer duration). Such a correlation could artificially manifest as a positive exposureresponse relationship if a time-dependent exposure metric was chosen. Moreover, because pembrolizumab exhibits linear and time-independent pharmacokinetic behavior, AUCss-6weeks was expected to be a reasonable proxy exposure for all patients (eg, those who had lower AUCss-6weeks are expected to have proportionally lower AUC0-anytime earlier during treatment).

Efficacy was defined as change from baseline in the SLD of target lesions (ie, tumor size).

Change in tumor size was considered an appropriate efficacy measure given the demonstrated relationship between changes in tumor size and overall survival in NSCLC [START_REF] Wang | Elucidation of relationship between tumor size and survival in non-small-cell lung cancer patients can aid early decision making in clinical drug development[END_REF][START_REF] Blumenthal | Overall response rate, progression-free survival, and overall survival with targeted and standard therapies in advanced non-smallcell lung cancer: US Food and Drug Administration trial-level and patient-level analyses[END_REF][START_REF] Jain | Change in tumor size by RECIST correlates linearly with overall survival in phase I oncology studies[END_REF]. Before NLME modeling, an exploratory regression analysis was performed to evaluate observed change in SLD versus pembrolizumab exposure at a single post-baseline time point. Particular emphasis was placed on weeks 18 and 27 because at the time of analysis, these were the latest common imaging time points reached by the majority of patients remaining on study who were treated at 2 mg/kg and 10 mg/kg.

Tumor size NLME model structure

All tumor size data were used simultaneously to fit the NLME model. At 2 mg/kg, the majority of data were up to 18 weeks of follow-up, although six patients were observed for >1 year; together with the 10-mg/kg dose groups, these data were used to inform long-term model behavior.

The tumor size model is illustrated in Figure S1 and is described mathematically as where "Baseline" is the actual measured tumor size (SLD) at initial screening, kgrowth is the firstorder tumor growth rate, kdeath is the rate constant that captures the kinetics of net removal in the responding portion of the tumor, and "delay" is the delay between baseline and the first dose.

Both kgrowth and kdeath were constrained to be positive during estimation, with individual parameters log normally distributed.

A fraction (f) of total tumor diameter was assumed to be accessible to treatment, with the remaining portion (1-f) undergoing exponential growth. This model parameterization is similar to previous models in the literature and was sufficiently flexible to capture different patterns of tumor growth observed for NSCLC, as well as for many other solid tumors in pembrolizumabtreated patients. modeled exposure-tumor size response were determined using uncertainty simulations based on the final model-estimated parameters. Briefly, 1000 draws were made from the parameter distributions, and for each set of population parameters, a trial with 1000 patients resampled with replacement from the observed dataset at each dose was simulated, accounting for interindividual and residual variability. SLD output from model simulations was categorized as response (SLD reduction from baseline ≥30%), stable disease (change in SLD from baseline between -30% and +20%), and progressive disease (≥20% increase in SLD from baseline); these categories were analogous to standard RECIST v1.1 categories [START_REF] Eisenhauer | New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)[END_REF] except that nontarget and new lesions were not considered when categorizing response. The median and 90% confidence interval (CI) of the proportion of patients in each category were tabulated across the 1000 uncertainty replicate simulations and plotted across the range of doses studied to graphically demonstrate the relationship between tumor size and exposure.

Exposure-Safety Relationship

Patients enrolled in all NSCLC cohorts of KEYNOTE-001 who had pharmacokinetic data were included in an analysis of the relationship between pembrolizumab AUCss-6weeks and the incidence of immune-mediated AEs. Logistic regression was used to analyze the frequency of immune-mediated AEs.

RESULTS

Efficacy and Safety of Pembrolizumab 2 mg/kg Q3W: Clinical Observations

Between April 3, 2014, and July 14, 2014, 55 patients with previously treated NSCLC received pembrolizumab 2 mg/kg Q3W. Patient characteristics were as expected for a previously treated advanced NSCLC population (Table S7). As of the January 23, 2015, data cutoff date, all patients had a minimum follow-up duration of 27 weeks; 15 (27%) patients remained on pembrolizumab. The most common reason for discontinuation was disease progression (n = 20; 36%).

The overall response (ORR) and disease control (DCR) rates per RECIST v1.1 by central review were 15% and 50%, respectively, in patients with measurable disease at baseline (n = 52) (Table 1). ORR was 30% in patients with PD-L1 TPS ≥50% (n = 23), 0% in patients with TPS 1% to 49% (n = 23), and 25% in patients with TPS <1% (n = 4). Decreases from baseline in tumor size were observed for 67% of patients with known PD-L1 expression treated at 2 mg/kg (Figure 1).

Among patients treated at 10 mg/kg in randomized cohorts with similar inclusion criteria as the 2-mg/kg cohort, including the amount of prior therapy and requirement for PD-L1 positivity per the prototype assay at baseline, decreases from baseline were observed in 66% of patients treated at Q3W and 63% treated at Q2W (Figure 1); ORR and DCR were similar to those of patients treated with pembrolizumab 2 mg/kg (Table 1).

Treatment-related AEs were reported for 26 (47%) patients treated with pembrolizumab 2 mg/kg Q3W. Five (9%) patients reported grade 3-5 treatment-related AEs (n = 2 grade 3 colitis, n = 1 grade 5 cardiorespiratory arrest, n = 1 grade 4 pneumonitis, and n = 1 grade 3 pneumonitis). The treatment-related death occurred in a 75-year-old man who was hospitalized on day 30 for possible pneumonia; 3 days later, he died from cardiopulmonary arrest considered by the investigator to be possibly related to pembrolizumab. Three (5%) patients discontinued treatment because of drug-related AEs (n = 1 each grade 5 cardiorespiratory arrest, grade 4 pneumonitis, and grade 3 pneumonitis). Immune-mediated AEs occurred in 9 (15%) patients: colitis (n = 2 grade 3, n = 1 grade 1), hypothyroidism (n = 2 grade 2, n = 1 grade 1), pneumonitis (n = 1 grade 3, n = 1 grade 4), and exfoliative dermatitis (n = 1 grade 1). Considering all 550 patients with NSCLC enrolled in KEYNOTE-001, the AE profile observed at 2 mg/kg Q3W was mostly similar to that observed in patients treated at higher dosages (Table 2).

Exploratory Regression and Model-Based Analyses of the Exposure-Efficacy Relationship

Observed tumor size data showed a wide range of longitudinal response patterns across the previously treated population. At week 18, 170 previously treated patients had both tumor size and exposure data. Exploratory graphical analysis of observed tumor size and exposure data from these patients showed a flat relationship between exposure and change from baseline in tumor size at 18 weeks, with overlapping CIs observed between subsets defined by binned AUCss-6weeks (Figure 2). The linear regression slope estimates were not significantly different from zero, with P values greater than the prespecified significance level (>0.05), regardless of whether the data were pooled or stratified by PD-L1 expression (Figure S5).

In agreement with the exploratory graphical and linear regression analyses of the data observed at week 18, individual pembrolizumab exposures (across all patients) also showed no statistically significant influence on the model-estimated tumor shrinkage rate in an NLME analysis of the exposure-response relationship (P = 0.54 based on -2 log-likelihood reduction and χ 2 test). The 95% CIs of the exposure response parameter were found to overlap with zero (point estimate, 0.196; range, -0.0784 to 0.47), consistent with no significant difference from a flat exposureresponse relationship. PD-L1 expression (Figure 3 and Table S5) and EGFR mutation (Table S5) status were the only factors found that explained a significant portion of interindividual variability in longitudinal tumor size patterns, with the impact of these factors found to be independent of dose. (Full details of structural model selection and analysis of covariate effects are found in the Supplementary Materials.)

Exposure-Response Simulations

Model-simulated median response rates at week 27 for patients with PD-L1 TPS ≥50% were 39% (90% CI 31% to 46%) at 2 mg/kg Q3W, 40% (90% CI 34% to 45%) at 10 mg/kg Q3W and 44% (90% CI 37% to 49%) at 10 mg/kg Q2W (Figure 3A). The CIs for patients with PD-L1 TPS 1% to 49% also showed overlap (Figure 3B), and the relationship between kdeath and exposure for PD-L1 TPS <1% was similarly flat (data not shown).

Exposure-Safety Relationship

A total of 544 patients were evaluable for the relationship between exposure and safety. Logistic regression analysis identified treatment duration as a significant factor for occurrence of immune-mediated AEs. After inclusion of treatment duration in the model, no significant relationship between pembrolizumab exposure assessed as AUCss-6weeks and immune-mediated AEs was found (P = 0.57) (Figure S6). Similarly, pembrolizumab exposure was not significantly correlated with the hazard for the occurrence of immune-mediated AEs in the time-to-event analysis (P = 1.0). Apart from treatment duration, no other investigated covariate was a significant predictor of the probability of experiencing an immune-mediated AE. Based on simulations from the final logistic regression model, even when forcing a relationship with pembrolizumab exposure, the predicted immune-mediated AE incidence at 9 months was similar for 2 mg/kg Q3W (26%), 10 mg/kg Q3W (27%), and 10 mg/kg Q2W (28%).

DISCUSSION

Based on the observed clinical data and comprehensive clinical pharmacology modeling and simulation, the approved 2-mg/kg Q3W dose of pembrolizumab provides clinically significant antitumor activity in NSCLC, with an efficacy and safety profile comparable to those observed with doses of 10 mg/kg Q3W or 10 mg/kg Q2W. Given that no dose-exposure-response dependency for efficacy or safety was identified between the 2-mg/kg and 10-mg/kg doses, the benefit-risk profile at the higher dose levels is not expected to be better than at 2 mg/kg Q3W.

The analysis supporting these conclusions represents the first comprehensive population pharmacokinetic/pharmacodynamics study of a therapy targeting PD-1/PD-L1 signaling. In this analysis, a NLME modeling framework was used to describe the relationship between systemic pembrolizumab exposure and antitumor efficacy in patients with NSCLC. The implemented model described the treatment effect on tumor size and captured kinetics of the underlying intratumoral heterogeneity of response, including the tumor shrinkage rate, underlying tumor growth for unresponsive tumor cells, and the extent to which tumors respond in an individual patient. Inclusion of f allowed for an empirical description of profiles in patients whose tumor size increases or stabilizes to a smaller size after an initial decrease and is coherent with the general understanding of intratumoral response heterogeneity. One could speculate that the remaining 1-f tumor fraction may represent tumor cells with lower PD-L1 expression or poorly perfused areas of tumor inaccessible to pembrolizumab, but these hypotheses have yet to be explored.

To build the final model, a covariate search was conducted to identify factors important to NSCLC tumor growth/shrinkage patterns under pembrolizumab treatment that could be accounted for as part of the final exposure-response evaluation. In this way, the potential for imbalances and gaps in the available data to influence the exposure-response assessment was lessened, enhancing the robustness of the results. A relatively stringent significance level was used in covariate testing (P < 0.01 in the forward step, P < 0.001 in the backward step) to mitigate the likelihood of false positives given that multiple hypothesis testing was applied during the search. Using these criteria, EGFR mutation and PD-L1 expression status were selected as predictors of the fraction of tumor affected by treatment and the tumor kill rate, respectively. The impact of PD-L1 and EGFR as predictors of tumor size kinetics is not surprising given their established role in NSCLC cancer cell growth and the known mechanism of pembrolizumab. Aside from target lesion SLD, other factors can influence RECIST-based response assessments, including shrinkage in nontarget tumors (eg, pathologic lymph nodes) and appearance of malignant lesions indicative of disease progression. Because the model only accounted for target lesion SLD, such nuances of RECIST were not accounted for in the simulations. Therefore, caution is urged in interpreting the results and making direct comparisons with RECIST v1.1 response categories.

The efficacy profile of the 2-mg/kg Q3W dose is further supported by early translational and biomarker pharmacokinetic/pharmacodynamic results, whereby potential clinical efficacy was predicted by integrating available preclinical pharmacokinetics, PD-1 receptor occupancy and antitumor data from a syngeneic mouse model, early clinical pharmacokinetic data, and human disease properties [START_REF] Lindauer | Translational pharmacokinetic/pharmacodynamic model of tumor growth inhibition by the new anti-PD1 monoclonal antibody MK-3475. 23rd Meeting of the Population Approach Group in Europe[END_REF]. Data from the KEYNOTE-010 study of pembrolizumab 2 and 10 mg/kg Q3W versus docetaxel for previously treated NSCLC support the similar efficacy and safety of pembrolizumab 2 and 10 mg/kg Q3W [START_REF] Herbst | Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial[END_REF].

The demonstrated lack of a dose-exposure-response relationship for pembrolizumab raises the question of how to best determine the appropriate dose for immunotherapy. Recently, there has been considerable interest in optimizing dose selection for immunotherapies and other anticancer therapies [START_REF] Sachs | Optimal dosing for targeted therapies in oncology: Drug development cases leading by example[END_REF][START_REF] Postel-Vinay | Towards new methods for the determination of dose limiting toxicities and the assessment of the recommended dose for further studies of molecularly targeted agents-Dose-Limiting Toxicity and Toxicity Assessment Recommendation Group for Early Trials of Targeted therapies, an European Organisation for Research and Treatment of Cancer-led study[END_REF]. Currently, most oncology dose-finding studies are designed to determine the maximum tolerated dose (MTD) based on the rate of prespecified dose-limiting toxicities that occur within a prespecified period of time, usually the first treatment cycle. However, this method may be outmoded for targeted therapies and immunotherapies, for which the biologically effective dose (BED) may be much lower than the MTD [START_REF] Sachs | Optimal dosing for targeted therapies in oncology: Drug development cases leading by example[END_REF]. Using the MTD rather than the BED could expose patients to a higher dose than that necessary to achieve clinical effect and may increase toxicity, which could lower overall clinical effectiveness. Therefore, dose determination in oncology should use a multifactorial approach that includes not only clinical data from the first treatment cycle but extended clinical data, preclinical models, pharmacokinetics, pharmacodynamics, and integrated modeling and simulation [START_REF] Sachs | Optimal dosing for targeted therapies in oncology: Drug development cases leading by example[END_REF][START_REF] Postel-Vinay | Towards new methods for the determination of dose limiting toxicities and the assessment of the recommended dose for further studies of molecularly targeted agents-Dose-Limiting Toxicity and Toxicity Assessment Recommendation Group for Early Trials of Targeted therapies, an European Organisation for Research and Treatment of Cancer-led study[END_REF]. Ideally, this multifactorial process would lead to a randomized dose-ranging study appropriately powered to identify the BED.

In summary, the approach reported here provides an integrated framework for exposure-efficacy analysis that accounts for imbalances in data and effects of explanatory covariates more thoroughly than those that rely exclusively on categorical end points (eg, RECIST). The final model adequately captured the array of diverse tumor size profiles observed in the pembrolizumab-treated NSCLC population and provided strong evidence for a lack of exposure dependency on response for the clinical dose range of 2 mg/kg Q3W to 10 mg/kg Q3W/Q2W. Normalization of trial data through simulations also revealed considerable overlap of response at these dosages. Because the model-estimated exposure-slope point estimate was slightly positive, the median simulated response and upper uncertainty bounds supported the possibility that there is a modest trend of increasing response rate and decreasing stable disease rate as dose or frequency is increased. However, the significant overlap in confidence intervals of the simulated response categories across a wide spectrum of exposures suggests antitumor response is likely saturated in this dose range. Therefore, it is likely that between-patient differences in pembrolizumab pharmacokinetics do not result in clinically relevant differences in efficacy over the dose range of 2 mg/kg Q3W to 10 mg/kg Q2W. Overall, the clinical data and model-based statistical testing and trial simulations of the magnitude of the exposure-response relationship support 2 mg/kg Q3W as an appropriate dose for pembrolizumab in patients with NSCLC. M. Gubens reports the following disclosures outside the submitted work: personal fees from Boehringer Ingelheim, Bristol-Myers Squibb, Calithera, Clovis, and Pfizer for serving as an advisory board member; grants from Abbvie, Celgene, Genentech/Roche, Merck, Novartis, and OncoMed for serving as the principal investigator of clinical trials; and nonfinancial support from Celgene and Genentech/Roche for serving as an advisory board member.

H. Hellmann reports funding to the institution from Merck for conduct of this study and, outside the submitted work, grant support from Bristol-Myers Squibb and personal fees from AstraZeneca, Genentech, Inovio, and Neon.

L. Horn reports personal fees from Merck for serving as an advisory board member and, outside the submitted work, personal fees from Genentech for serving as an advisory board member; personal fees from Biodesix for serving as a speaker; and serving as an uncompensated consultant for Bayer, Bristol-Myers Squibb, and Xcovery. Error bars represent the 90% confidence intervals around the estimates. Response was defined as a ≥30% decrease from baseline in SLD, stable disease was defined as a <30% decrease but <20% increase from baseline in SLD, and progression was defined as a ≥20% increase from baseline in SLD). CI, confidence interval; SLD, sum of the longest diameters.

Figure 1 .

 1 Figure 1. Best percentage change from baseline in sum of the longest diameters of target lesions

Figure 2 .

 2 Figure 2. Observed percentage change from baseline in tumor size at 18 weeks by

Figure 3 .

 3 Figure 3. Median simulated response rates by pembrolizumab dose spanning the observed range

  Melanie Leiby, PhD, of the ApotheCom Merck oncology team, Yardley, PA, USA; M. Leiby is currently a full-time employee of Merck & Co., Inc., Kenilworth, NJ, USA. This assistance was funded by Merck & Co., Inc., Kenilworth, NJ, USA. Funding for this research was provided by Merck & Co., Inc., Kenilworth, NJ USA.There is no grant number associated with this sponsorship. Garon reports funding to the institution from Merck for conduct of the study.

	Funding: Disclosures
	E.B.

at University of California, Santa Barbara on May 4, 2016 http://annonc.oxfordjournals.org/ Downloaded from

Acknowledgments

We thank the patients and their families and caregivers for participating in this study; Rik de Greef and Jeroen Elassaiss-Schaap (Merck & Co., Inc., Kenilworth, NJ, USA) for technical support; and Roger Dansey (Merck & Co., Inc., Kenilworth, NJ, USA) for critical review of the manuscript. Medical writing and editorial assistance was provided by Sarah Adai, MS, and

To account for the effect of drug exposure, AUCss-6weeks was incorporated into the structural model parameterization on the tumor kill rate by assuming a log-linear relationship: Results from the independent population pharmacokinetics model provided post hoc clearance (CL) estimates, with plasma exposure within the dosing interval at steady state calculated as dose/CL. Here, TVkdeath denotes the typical value of kdeath in the population; "AUCtypical,ss-6weeks" (7079 mg/L × day) is used to normalize exposure values. The estimated value of θ determines the extent of the pembrolizumab exposure-response in NSCLC. Only observed tumor sizes were used for the modeling, with no imputations for missing data.

The patient-specific factors of PD-L1 expression level, smoking history, ECOG performance status, demographics (age, sex, and weight), baseline tumor size, prior treatment, and EGFR mutation status were tested for inclusion in the model using the stepwise covariate modeling function of PsN [START_REF] Lindbom | PsN-Toolkit--a collection of computer intensive statistical methods for non-linear mixed effect modeling using NONMEM[END_REF] (forward inclusion at P < 0.01 and backward exclusion at P < 0.001). The Supplementary Methods, including Figures S1-S4 and Tables S2-S6, provide further details on the handling of covariates.

Trial simulations

Response rate simulations were conducted to normalize for potential data imbalances with respect to covariates and dose/exposure. The expected dose-response relationships based on the R. Hui reports the following disclosures outside the submitted work: personal fees from Merck Sharp & Dohme for serving as an advisory board member and speaker; personal fees from AstraZeneca for serving as an advisory board member and consultant; personal fees from Pfizer for serving as an advisory board member; and personal fees from Boehringer Ingelheim and Roche for serving as a speaker.

H. Lena


Desmond E Schipper

Djamila Ikhlef

Samila Khalal

Jean-Yves Saillard

Kenton H Whitmire

A New Main Group Element-rich nido-Octahedral Cluster System: Synthesis and Characterization of [Et 4 N][Fe 2 (CO) 6 (μ 3 -As){μ 3 -EFe(CO) 4 } 2 ]

A series of clusters of the form [Et4N][Fe2(CO)6(μ3-As)}(μ3-EFe(CO)4)] where E is either P or As were synthesized from [Et4N]2[HAs{Fe(CO)4}3] and ECl3. AsCl3 gives the Asonly compound; PCl3 produces compounds having two As atoms with one P atom, or one As atom and two P atoms and can exist as two possible isomers, one of which is chiral. The As2P and AsP2 clusters co-crystallize and their structure as determined by single-crystal X-ray A c c e p t e d m a n u s c r i p t diffraction is given along with the structure of the As-only cluster. Analytical data as well as DFT calculations support the formation and geometries of the new molecules.

Introduction

Transition metal organometallic clusters have proven to have a very rich structural chemistry, which can be rationalized within the framework of the so-called Wade-Mingos electron counting rules. 1 This delocalized bonding formalism, originally developed for boron hydride clusters, 2 was, with the help of the isolobal analogy, 3 extended to transition metal clusters as well as to main group clusters and hybrid systems made of both main group elements and transition metals. In the latter case, the validity of the isolobal analogy is sometimes borderline because of the mismatch of the main group element and transition metal orbitals, leading to deviations from the Wade-Mingos rules. [4][5][6] This specific feature, together with the potential for skeletal isomerism arising from the metal/main group chemical heterogeneity, renders the structural chemistry of mixed main group/transition metal clusters particularly diverse and original from the point of view of bonding.

In our pursuit of single-source precursors to metal pnictide phases, we have explored routes to combine phosphorus and arsenic with iron in single source precursors with the goal of producing Fex(As1-yPy) (x = 1 -3; y = 0 -1) and related heterometallic compounds for their important electronic, magnetic, and magneto-caloric effects. [7][8][9][10][11][12][13] We have been particularly interested in [Et4N]2[HAs{Fe(CO)4}3] ([Et4N]2[I]) as a starting material because it can be prepared cleanly in very high yields without using volatile arsine or organoarsenic compounds. 14 Starting with As or P. These compounds possess a previously unobserved nido-octahedral E3M2 core.

Results and Discussion

Synthesis and Characterization

Immediately The structures of both the As3 and AsxPy compounds (Figure 1) were determined by single crystal X-ray diffraction. Interestingly, in the case of the mixed P-As system there was a nonstatistical distribution of P and As over the flanking sites. As shown in Figure 2, six different compounds are possible, of which two (As-As-P and P-P-As) exist as enantiomers. The X-ray refinement showed those crystals to be composed of a mixture of several of the compositional isomers of [III] -wherein the flanking "E" sites were a mixture of arsenic and phosphorus, with 50% and 66% P at the two Fe(CO)4-bound positions.

That the two sites refined to different values was especially surprising given the centrosymmetric nature of the space group. The central, naked main group atom refined to essentially only As and was fixed completely as that element. The overall composition indicated by the refinement was As1.83P1.17.

The related M3E2 nido-octahedral compounds have been known for some time. [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] and the compounds reported here may be viewed as an isolobal replacement of Fe(CO)3 -by P or As.

[Et4N][II] and [Et4N][III] are electron precise, and the core electron count of each is consistent with that predicted by Wade-Mingos's rules for a nido-octahedron with seven skeletal electron pairs distributed across five vertices. 1 Nido-octahedra in an M2E3 configuration in which E, taken generally as any nonmetal, are unknown outside a handful of structures with a carbon at the bridging "E" vertex. [START_REF] Aumann | Naturforschung Teil B Anorg[END_REF][32][33][34][35][36][37][38][39] In those structures, the idealized square-pyramidal geometry is highly distorted and the bridge-carbon bears an alkyl or phosphine group. Anions [II] -and [III] -are related to the metallated polyphosphine structural class of which there are numerous examples, 30,40,41 but this is the first time this unit has been observed as part of an octahedral cluster. Moreover, with its mixed P/As ligand composition, [III] -is related to a small collection of structures in which the pnictogens are found at mixed positions as ligands in metallated systems. [42][43][44][45][46][47] With an As/P ratio of 1.83 to 1.17 indicated by the crystal data and only two compositional isomers possible, the implied percentages of 83% of the As2P isomer and 17% of the AsP2 isomer could be estimated. The composition of the crystals as a mixture of the As2P and AsP2 isomers is further supported by the analytical data; the As2P:AsP2 ratio of Theoretical calculations (Table 1) support a ~327-389 ppm location for the flanking phosphorus atoms and a ~185-287 ppm location for the bridging phosphorus atoms. While the expected phosphorus-phosphorus couplings were not directly observed, such couplings may be too broad to be observed. Among other examples of mixed P/As metallated ligand systems, there are only a few documented instances of P-P coupling. 42,49 [Et4N][III] is not susceptible to protonation. Treatment with the super acid HSO3CF3 was insufficient to protonate the bridging pnictogen atom. Efforts to alkylate and metallate the bridge pnictogen with ethyl bromide and Mn(CO)5Br were also unsuccessful, probably because of steric crowding at this position.

Theoretical Studies

Full geometry optimizations of the various compounds shown in Figure 2 have been carried out by DFT calculations at the PBE0/Def2TZVP level (see Computational Details).

Relevant computed NMR data are given in Table 1 and energetic data are provided in Table 2.

In the case of the As2P and AsP2 systems, the more stable isomers were found to be isomers B and A, respectively, i.e., those for which the naked main-group atom is arsenic, in full agreement with the X-ray data for which this position is found to be of 100% As occupation (see above).

The free energy preference for these isomers is not very large (4.4 and 2.7 kcal/mol, respectively) but it appears to be enough for avoiding the isolation of the other isomers. In the case of the less stable isomers (as well as in the P3 species) the naked phosphorus atom is negatively charged (NPA charge range: -0.02/-0.05), suggesting it should be subject to electrophilic attack. This is not the case when phosphorus is bonded to an Fe(CO)4 group nor for naked arsenic (range: +0.06/+0.14). This is in line with the fact that naked phosphorus atoms are rare in main-group transition-metal organometallic cluster chemistry. 53 The same authors noted that RPCl2 and Co2(CO)8 gave Co3(CO)9PR along with CoCl2. 54 Various observations leading to spirocyclic or spiked tetrahedral structures were already postulated in the earlier report. These same pathways could explain the route to [Et4N][III] as well. This discussion is more speculative and, since multiple products are formed, a variety of these pathways may be operating simultaneously. In some ways, having both As and P present in We cannot rule out the possibility that the isolation of [Et4N][III] may be a matter of the stability of the products; the less stable species generated in the PCl3 addition process may simply decompose leaving the observed mixture of products.

Conclusions

The

ASSOCIATED CONTENT

Computational details: Density functional theory geometry optimizations of the five cationic clusters shown in Figure 2 were performed using the Gaussian 09 program, [START_REF] Gaussian | Gaussian[END_REF] with the PBE1PBE (PBE0) 56-57 hybrid functional and the general Def2TZVP (triple- polarized) basis set from the EMSL Basis Set Exchange Library. 58 Frequency calculations of normal modes of vibration were carried out in order to ascertain that the optimized structure are true minima. The natural A c c e p t e d m a n u s c r i p t population analysis (NPA) was performed with the NBO 5.0 program. 59 Calculations of the 31 P NMR chemical shifts were performed according to the Gauge invariant atomic orbital (GIAO) method. 60 No. CHE-1411495. GENCI-CINES and GENCI-IDRISS French national computer centers are acknowledged for computational resources (grant 2015-087367).

Synopsis

[

  [HAs{Fe(CO)4}{Fe2(CO)7H}], [Et4N][{Fe3(CO)10}As{Fe(CO)4}], [Et4N][{H2Fe3(CO)9}-A c c e p t e d m a n u s c r i p t As{Fe(CO)4}], [Et4N][As{HFe2(CO)7}{FeMn(CO)8}], [As{Fe2(CO)8}{FeMn(CO)8}], [{H2Fe3(CO)9}As{Mn(CO)5}] and [Et4N][{Fe3(CO)10}As{Mn(CO)4Br}] by using various oxidants in the absence or presence of Mn(CO)5Br. 15 As an additional strategy we have examined the addition of ECl3 to [Et4N]2[I] as a means of introducing additional main-group elements into the cluster framework. Herein we report the synthesis, structures and theoretical descriptions of a series of compounds [Et4N][Fe2(CO)6(μ3-As){μ3-EFe(CO)4}2] where E is either

  upon reaction of [Et4N]2[I] with ECl3 (E = P, As) in the presence of triethylamine, a blanketing mist forms in the flask above the reaction solution with rapid bubbling. The color transitions from a deep brick-red to a yellow-brown over the course of the addition and maintains this color for approximately 20 minutes. Then the mist disappears and the solution color transitions to a deep orange-red. The IR of the solution in the first 20 minutes suggests a complex mixture of products. For E = As, the spectrum at 30 min primarily shows the CO stretching bands associated with the known [Et4N][Fe3(CO)9{μ3-AsFe(CO)4}2], the major product of the reaction (Scheme 1). Extraction of the crude product with diethyl ether yielded small amounts of [Et4N][Fe2(CO)6(μ3-As){μ3-AsFe(CO)4}2], [Et4N][II], along with [Et4N][Fe3(CO)9(μ-CO){μ3-AsFe(CO)4}].15 In contrast, for E = P, the IR spectrum after 30 min largely resembled that of [Et4N][II] and was proven to contain a mixture of P and As in the flanking positions (vide infra). These was the only isolated product and is designated collectively as [Et4N][III]. That spectrum showed little change over the following 24 hours as well.

A c c e p t e d m a n u s c r i p t

  Scheme 1. Reaction products from treatment of [Et4N]2[I] with AsCl3.

A c c e p t e d m a n u s c r i p tFigure 1 .

 1 Figure 1. Structure of the anion [Fe2(CO)6(μ3-As){μ3-EFe(CO)4}2] -, [III] -, as determined by

A c c e p t e d m a n u s c r i p t

Figure 2 .

 2 Figure 2. The various [Fe2(CO)6(μ3-E)}{μ3-EFe(CO)4}2] -compounds possible with an

A c c e p t e d m a n u s c r i p t

31 P

 31 NMR data obtained for the crude reaction mixture show the two additional peaks at 147.5 and 153.90 ppm as compared to the solution NMR of the recrystallized sample. While the previously mentioned signals at 307 and 323 ppm respectively can be attributed to the As-As-P and P-As-P signals, the 147.5 and 153.9 signals could be attributed to phosphorus at the bridging position. It may also be that one among the 147.5 and 153.9 signals can be attributed to the phosphorus atom in [Et4N]2[Fe3(CO)9{μ3-PFe(CO)4}{μ3-AsFe(CO)4}], identified in the ESI/MS of the crude product. For comparison, white phosphorus (P4) shows one signal at ~-525 ppm.48

A c c e

  p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t

  Mechanistic Considerations:The mechanism by which these new clusters are formed is clearly complicated and must involve recombination of a variety of E, E{Fe(CO)4} and/ or E{Fe(CO)4}2 fragments, which

  the bridging position would have made the mechanistic discussion easier, as that would imply a completely random combination of E{Fe(CO)x}y fragments. Steric crowding may prevent formation of [Fe2(CO)6{E{Fe(CO)4}3] -explaining the lack of an Fe(CO)4 group on the central main group atom. Loss of an Fe(CO)4 group would occur more readily from As than P, which is consistent with the lack of negative charge found at the As atom in the DFT calculations. The appearance of the thick mist above the reaction solution upon introduction of PCl3 could result from HCl formation to produce an intermediate such as [{(CO)4Fe}3AsPCl2] 2-with an As-P bond. Reduction of the PCl2 by iron carbonyl fragments suggests an intriguing possibility -the A c c e p t e d m a n u s c r i p t intermediacy of a multiply bonded species such as (CO)4Fe-EE'-Fe(CO)4. Both E = E' = P and E = As, E' = P could exist. Addition of an [As{Fe(CO)x}2] -fragment across the multiple bond could explain the preference for P at the end positions and the presence of only As in the central location. The appearance of [Et4N]2[Fe3(CO)9{μ3-AsFe(CO)4}2] in the reaction solution (1b, Supporting Information) implies an oxidative process is taking place, but its structural similarity to the E3M2 compound suggested that it could be an intermediate in the formation of [III] -. To test this hypothesis, [Et4N]2[Fe3(CO)9{μ3-AsFe(CO)4}2] was treated with PCl3 (1c, Supporting Information). This led to the formation of small amounts of the AsP2 compound, but also the P3 variant, unseen in the original reaction of [Et4N]2[I] with PCl3 after 48 hours of reaction time (1b, Supporting Information). This would suggest vertex substitution is possible, but the low yield indicates it is not the prevailing mechanism for the formation of the AsP2 and As2P variants.

  tendency of [Et4N]2[HAs{Fe(CO)4], [Et4N]2[I], to react with PCl3 to form the mixed P/As [Et4N][III] is similar to the manner in which [Et4N]2[I] forms [Et4N]2[Fe3(CO)9{μ3-AsFe(CO)4}2] as the majority product upon treatment with one equivalent of a one-electron oxidant. Both products share square-pyramidal cores, with the former bearing an M2E3 core and the latter an M3E2 core. This would suggest PCl3 interacts in an oxidative manner withA c c e p t e d m a n u s c r i p t [Et4N]2[I], ultimately supplying neutral phosphorus atoms to the recombining fragments to form the core. These P atoms are found at the flanking positions of the core. If P atoms do take the bridge position during the M2E3 core formation, the clusters in which they are incorporated are unstable and may be subject to electrophilic attack. In the case of [Et4N]2[I] reacting with AsCl3, [Et4N][{Fe3(CO)10}As{Fe(CO)4}] is the majority product with a small amount of [Et4N][II] formed. The appearance of the latter suggests that the reactivity of [Et4N]2[I] towards AsCl3 mirrors at least in part its reactivity with PCl3 although another pathway, perhaps the same observed for the formation of [Et4N][{Fe3(CO)10}As{Fe(CO)4}] from the reaction of [Et4N]2[I] with hydride abstraction agent [CPh3][BF4] may be involved.15 With these clusters, the structural diversity exhibited by the metal carbonyls expands further. The cluster series reported herein also points to the possibility of other M2E3 clusters. A reexamination of some of the known M3E2 clusters might yield insights into other instances in which PCl3 and a suitable starting material might yield an M2E3-type cluster with metals in addition to iron and main-group elements other than phosphorus and arsenic.EXPERIMENTALGeneral Considerations: All reactions were performed under a dry, oxygen-free argon atmosphere according to standard Schlenk techniques. The solvents dichloromethane, tetrahydrofuran, and ethyl ether were dried with a Pure Process Technology solvent purification system and degassed prior to use. [Et4N]2[HAs{Fe(CO)4}3] was prepared according to literature methods.14 PCl3 was obtained from Sigma Aldrich, distilled, and stored under argon.Triethylamine was obtained from Sigma Aldrich, dried over KOH, and distilled under argon.

A c c e p t e d m a n u s c r i p t

  AgPF6 was obtained from Strem Chemicals and used without further purification. IR measurements were obtained using a Perkin Elmer Spectrum Two FTIR spectrometer.1 H and 31 P NMR data were recorded on a 500 MHz Bruker spectrometer (202 MHz for 31 P). H3PO4 (85%) was used as the reference standard for the[START_REF] Aumann | Naturforschung Teil B Anorg[END_REF] P NMR spectra. ESI mass spectra were obtained on a Bruker Daltonics MicroTOF analyzer equipped with an Agilent 1200 HPLC system. 1a) Synthesis of [Et4N][Fe2(CO)6(µ3-As){µ3-EFe(CO)4}2], E = P/As [Et4N][III]: 1.4 g of (Et4N)2{H-As(Fe(CO)4)3} (1.6 mmol) was dissolved in 20 mL of DCM or THF at room temperature. To this solution 0.5 mL of triethylamine was added (3.6 mmol) with stirring. After 5 minutes, a solution of 0.14 mL of PCl3 (1.6 mmol) in 20 mL DCM or THF was added dropwise (2 mL/min) and the mixture stirred overnight. The following day, the solution was reduced to dryness in vacuo and 60 mL dry, degassed Et2O was added. This was allowed to stir for 3 h whereafter the solution was filtered and stored in a refrigerator at -10 °C for 3 weeks. Rhombohedral red crystals of [Et4N][III] grew along the walls of the flask above and below the solution level. Yield 225 mg, 15% (Based on As). IR, νCO (DCM) 2064 (vw), 2032 (vs), 2019, (s), 1983 (w), 1960 (w), and 1935 (m) cm -1 .

A c c e p t e d m a n u s c r i p t of 0 .

 0 14 mL of AsCl3 (1.6 mmol) in 20 mL DCM or THF was added dropwise (2 mL/min) and the mixture stirred overnight. The following day, the solution was reduced to dryness in vacuo and 60 mL dry, degassed Et2O was added. This was allowed to stir for 3 h whereafter the solution was filtered and stored in a refrigerator at -10 °C for 3 weeks. Brownish rhombs of product were obtained as a mixture with crystals of [Et4N][Fe3(CO)9(μ-CO){μ3-AsFe(CO)4}]. Yield Trace, not isolated. νCO (DCM) 2062 (vw), 2030 (vs), 2014, (s), 2001 (m), 1980 (w), 1960 (w), and 1936 (w) cm -1 .ESI-MS (m/z, (relative %)) 840.4 (4) [Fe2(CO)6As3{Fe(CO)4}2] -) calcd for Fe4As3C14O14 840.29.. 1c) Preparation and Reaction of [Et4N]2[Fe3(CO)9{μ3-AsFe(CO)4}2] with PCl3 and Et3N: Preparation of [Et4N]2[Fe3(CO)9{μ3-AsFe(CO)4}2]: 1.4 g of [Et4N]2[HAs{Fe(CO)4}3] (1.6mmol) was dissolved in 20 mL of THF. To this was added dropwise a solution of 0.4 g AgPF6 (1.6 mmol) in 20 mL of THF. This was allowed to stir for 1 h and then 0.5 mL of triethylamine was added (3.6 mmol). The resulting solution was allowed to stir for 20 min. FT-IR of the solution after 20 min of stirring showed only the presence of [Fe3(CO)9{μ3-AsFe(CO)4}2] 2-. To this solution was then added a solution of 0.14 mL PCl3 (1.6 mmol) in 20 mL of THF via cannula. This was allowed to stir for 48 h at room temperature, filtered, and taken to dryness in vacuo.

  : A series of clusters of the form [Et4N][Fe2(CO)6(μ3-As){μ3-EFe(CO)4}2] where E is either P or As were synthesized from [Et4N]2[HAs{Fe(CO)4}3] and ECl3. AsCl3 gives the Asonly compound; PCl3 produces compounds having two As atoms with one P atom, or two P atoms and one As atom and can exist as two possible isomers, one of which is chiral. The As2P and AsP2 clusters co-crystallize and their structure as determined by single-crystal X-ray diffraction is given along with the structure of the As-only cluster. The new cluster series has a unique square-pyramidal M2E3 core, structurally analogous to clusters with the well-known M3E2 core configuration. Analytical data as well as DFT calculations support the formation and geometries of the new molecules.

A c c e p t e d m a n u s c r i p t

Table 1 .

 1 DFT-computed[START_REF] Aumann | Naturforschung Teil B Anorg[END_REF] P NMR chemical shifts (ppm) of the P-containing compounds of Figure2. The atom numbering corresponds to that of Figure1.

	P(1)	P(3)	P(2)

Table 2 .

 2 DFT-computed HOMO-LUMO gaps and relative energies between the various compounds of Figure 2. E 0 = ZPE-corrected energy, G = free energy, H = enthalpy.

	m a n u s c r i p t
	As2P (isomer A) 5.3 4.4 5.3 3.99 A c c e p t e d As2P (isomer B) E 0 (kcal/mol) 0.0 G (kcal/mol) 0.0 H (kcal/mol) 0.0 EHOMO-LUMO (eV) 4.03	AsP2 (isomer A) 0.0 0.0 0.0 4.17	AsP2 (isomer B) 3.5 2.7 3.7 4.06

Supporting Information

Electronic Supplementary Information (ESI) available: Experimental procedures, ESI-MS data,


Richard-Ferroudji Audrey

Nicolas

Menon Ragunath

Rinaudo Jean-Daniel

« Co-designing Research on Social Transformations to Sustainability » Title: The DIALAQ project on sustainable groundwater management: a transdisciplinary and transcultural approachtoparticipatory foresight

In the past decades, groundwater over-exploitation has increased the vulnerability of users, social inequalities and environmental degradation. In this context, the DIALAQ project aimed to experiment and disseminate a participatory approach intended to strengthen stakeholders" capacity to implement more sustainable agricultural and groundwater management. DIALAQ"s network encompasses 8 regions in 4 countries (India, Morocco, France and the United States) including groups of farmers, administration"s representatives, NGOs, elected representatives and researchers from several disciplines. A seed funding enabled cooperation between academics and non-academic partners that led to the consolidation of the network and enabled the design of the project. Firstly, a focused review of literature on participatory foresight exercises in the field of groundwater management is presented. Secondly, the challenges and pathways taken in designing the research is described.This process resulted in a common methodological and ethical framework presented in conclusion.

Highlights

Groundwater governance is a major issue in many regions of the world today Participatory foresight approaches can promote changes towards more sustainable environment management

The grassroots based research network involved partners ready to explore pathways for adaptation Co-design resulted in a common methodological and ethical framework adaptable to the diversity of local situations

Introduction

In the past decades, groundwater over-exploitation has increased the vulnerability of users, social inequalities and environmental degradation. The DIALAQ 1 research project was designed to address these challenges. This project aims to experiment and disseminate participatory approaches intended to strengthen stakeholders" capacity to envision, plan and implement more sustainable and integrated agricultural and groundwater management strategies. DIALAQ is based on the hypothesis that scenarios are intermediary objects that can help, through participatory processes, to [START_REF] Molle | The groundswell of pumps: Multilevel impacts of a silent revolution: paper prepared for the ICID-Asia Meeting[END_REF] bridge the gaps between the knowledge needed, produced and put in practice by scientists and stakeholders and (2) foster transformation to sustainability. The aim of DIALAQ was to implement foresight approaches in four countries (India, Morocco, France and the United States).Within this international project, we assumed that the diversity of cases and a transversal approach at an international level may help create knowledge on the use and abuse of groundwater, its impacts and the possible transformative changes in governance and practices. The project involved building communication between different cases where groundwater resources were overused (or at significant risk of overuse) to learn from this diversity. This kind of project usually faces two challenges: [START_REF] Molle | The groundswell of pumps: Multilevel impacts of a silent revolution: paper prepared for the ICID-Asia Meeting[END_REF] to engage academic and non-academic partners, and (2) to implement comparable approaches in different countrieswith contrasting situations in terms of legal frameworks, policies, types of agriculture and practices of future studies.Indeed, the main challenge related to transcultural approaches of envisioning possible futures.This paper focuses on the co-design phase of a joint research project. Firstly, we present a focused review of the literature on participatory foresight exercises in the field of groundwater management. Secondly, we describe the challenges (especially taking into account the diversity of actors within our network) and the choice made in project design.The co-design of the research involved consolidating the research network, selecting sites for project implementation and co-designing the objectives and activities.Cooperation between academic and non-academic partners led to the choice of a common methodological and ethical framework that we present in the concluding section.

Participatory foresight for sustainable groundwater management

The challenge of groundwater governance

Groundwater is an invisible resource of critical concern. In the past 40 years, the"pump revolution" has played a key role in the development of agriculture, providing economic development to rural communities [START_REF] Molle | The groundswell of pumps: Multilevel impacts of a silent revolution: paper prepared for the ICID-Asia Meeting[END_REF][START_REF] Kuper | When groundwater takes-over: linking 40 years of agricultural and groundwater dynamics in large-scale irrigation scheme in Morocco[END_REF][START_REF] Giordano | Global groundwater? Issues and solutions[END_REF]. Yet, groundwater resources are increasingly overexploited [START_REF] Molle | The groundswell of pumps: Multilevel impacts of a silent revolution: paper prepared for the ICID-Asia Meeting[END_REF] and depleted [START_REF]World Bank: Deep Wells and Prudence : Towards Pragmatic Action for Addressing Groundwater Overexploitation in India[END_REF]. This is accentuated by climate change because in many areas irrigation requirements are projected to increase while rainfall may decrease [START_REF]Groundwater and global change: Trends, opportunities and challenges[END_REF][START_REF] Shah | Climate change and groundwater: India's opportunities for mitigation and adaptation[END_REF]. In many regions of the world, groundwater overuse has already led to a collapse of local economies, and caused or worsened social and ecological crises [START_REF]World Bank: Deep Wells and Prudence : Towards Pragmatic Action for Addressing Groundwater Overexploitation in India[END_REF][START_REF] Moench | Groundwater and poverty: exploring the connections[END_REF]. As groundwater tables decline, access to groundwater is increasingly skewed towards wealthy users, generating accentuated inequalities [START_REF] Srinivasan | Examining the emerging role of groundwater in water inequity in India[END_REF]. Moving from this situation to a more sustainable one calls for transformative adaptations of technical, socio-economic and institutional types. Governance of groundwater is of increasing concern globally [START_REF] Shah | Taming the anarchy groundwater governance in South Asia[END_REF][START_REF] Mukherji | Groundwater socio-ecology and governance: a review of institutions and policies in selected countries[END_REF]. However, within the fields of research dedicated to groundwater issues, interdisciplinary projects are rare [START_REF]Groundwater governance: Learning from local experiences -Irrigation and drainage[END_REF] and there has been limited social research in the broad arena of groundwater management [START_REF] Mitchell | Directions for social research to underpin improved groundwater management[END_REF] and limited communication between social research "traditions" [START_REF] Faysse | Convergent readings of groundwater governance? Engaging exchanges between different research perspectives[END_REF]. Notable works have been undertaken in economics, building on Ostrom [START_REF] Ostrom | Governing the Commons: The Evolution of Institutions for Collective Action[END_REF]. These studies mainly assessed the individual, fragmented and diffuse use of groundwater and groundwater users" strategies that are often considered to focus on short-term profits [START_REF] Bekkar | On the difficulty of managing an invisible resource: Farmers' strategies and perceptions of groundwater use, field evidence from Morocco[END_REF]. Other studies discussed possible modes of regulation of groundwater [START_REF] Shah | Taming the anarchy groundwater governance in South Asia[END_REF][START_REF] Diwakara | Determinants of trust and cooperation: Case study of self-managed tubewell organizations in North Gujarat, India[END_REF] and the equity related issues [START_REF] Hoogesteger | Intensive groundwater use and (in)equity: Processes and governance challenges[END_REF]. Socio-economic and anthropological approaches also described the vulnerability of farmers [START_REF] Janakarajan | Are wells a potential threat to farmers' well-being? Case of deteriorating groundwater irrigation in Tamil Nadu[END_REF], the connections between the intensive use of groundwater and poverty [START_REF] Moench | Groundwater and poverty: Exploring the connections[END_REF], farmers" knowledge and social status provided by groundwater access [START_REF] Aubriot | Tank and well irrigation crisis: Spatial, environmental and social issues. Cases in Puducherry and Villupuram districts[END_REF], groundwater related policy implementation [START_REF] Mukherji | Political ecology of groundwater: The contrasting case of water-abundant West Bengal and water-scarce Gujarat, India[END_REF] and farmer"s resistance to these policies [START_REF] Birkenholtz | Groundwater governmentality: Hegemony and technologies of resistance in Rajasthan's (India) groundwater governance[END_REF]. These studies unveiledthe inequalitiesrelated to the access and use of this invisible resource [START_REF] Srinivasan | Examining the emerging role of groundwater in water inequity in India[END_REF][START_REF] Aubriot | Tank and well irrigation crisis: Spatial, environmental and social issues. Cases in Puducherry and Villupuram districts[END_REF]. They showed theconflicts and contradictions between multiple interests, values and attachments. Actors of agricultural territories are ambivalent toward groundwater because it may become a resource for economic development and "liberation" but it may also lead to possible "trap" that towards more inequity and more poverty [START_REF] Kuper | Liberation or Anarchy? The Janus Nature of Groundwater Use on North Africa's New Irrigation Frontiers[END_REF].

Dialogic democracy and future studies to improve groundwater management

There is recognition today that groundwater over-exploitation urgently needs to be curtailed but there is little consensus on how best this can be achieved [START_REF]Integrated Groundwater Management: Concepts, Approaches and Challenges[END_REF]. In many cases, the most promising solutions may lie outside the groundwater sector and within a broader approach to resource systems [START_REF] Giordano | Global groundwater? Issues and solutions[END_REF][START_REF]Integrated Groundwater Management: Concepts, Approaches and Challenges[END_REF]. The participation of local actors in the development of adaptive management to climate change is considered by some authors as a cornerstone to its success [START_REF] Faysse | Convergent readings of groundwater governance? Engaging exchanges between different research perspectives[END_REF]. Designing and implementingparticipatory processes are often proposed with reference to the dialogic democracy model [START_REF] Callon | Acting in an Uncertain World[END_REF] and to the framework of future studies [START_REF]Futures methodologies[END_REF]. Within dialogic democracy, collective decision-making emerges through a deliberative process which favours the collective exploration of identities and problems [START_REF] Callon | Acting in an Uncertain World[END_REF].Only such collaborative research is found to enable the exploration of multidimensional uncertainties [START_REF] Callon | Acting in an Uncertain World[END_REF]. This can be referred to as post-normal science [START_REF] Funtowicz | Science for the post-normal age[END_REF]. The different purposes and effects of participatory dialogic settings for water issues have long been debated [START_REF] Korff | Implementing participatory water management: recent advances in theory, practice, and evaluation[END_REF]. Experiences that have taken placeto date show that it is possible to implement participation with a variety of approaches ranging from modelling to methods based on arts and creative activities [START_REF] Richard-Ferroudji | Assembling different forms of knowledge for participative water management -Insights from the Concert'eau game[END_REF]. More specifically, transdisciplinarity hasbeen implemented in the groundwater field [START_REF] Scholz | Transdisciplinarity in groundwater managementtowards mutual learning of science and society[END_REF]. Moreover, the crossover between future studies and environmental research has proven effective in understanding long-term environmental dynamics, and offers frameworks to make explicit the choices available to address wicked environmental problems [START_REF]Futures methodologies[END_REF]. 3 Designing the research: challenges and pathways

Consolidating the network: interconnection of pioneers

DIALAQ enrolled academics and non-academics from four continents. It relied on farmers"organizations and on-going partnerships between researchers and stakeholders to consolidate the project network. Connection between countries was initially made through the academic partnersbringing together a wide range of skills in the social sciences (anthropology, economics, geography, sociology) and bio-physical sciences (agronomy, ecology, engineering, hydro-geology). The project was then introduced to stakeholders (NGOs, public administrations, municipalities and farmers organisations) already engaged in previous projects and with whom trust hadalready been established. Such choice was made, first, because enrolling non-academic partners is a challenge. They may consider that adaptation to groundwater overuse isnot an urgent issue as compared to more serious problems such as labour availability or volatility of markets for farmers.

The selectedsites for DIALAQwereagricultural regions where groundwater resources were under pressure and where stakeholders expressed concern about the level of groundwater exploitation. These case studieswere diverse in terms of scale, types of water resources, degree of groundwater overuse, types of crops, institutions and legal context. The common pattern of the selected siteswas the existence of collective action among actors, which could be strengthened, and the potential for transformation of practices and governance.For example, in Ain Timguenay (Morocco), the ongoing increase of land planted with irrigated orchardshas led to increased farmers" concern about groundwater depletion. Their willingness to consider and discuss possible groundwater management strategies is exceptional in Morocco (and in North Africa more generally). In this area, within a previous project (www.groundwater-arena.net), discussions were initiatedbetween academics and non-academics about ways to support a sustainable use of the aquifer for sustainable agriculture, involvinga farmers" cooperative, the catchment management agency and the regional office of the department of agriculture. DIALAQ aimed to continuethis multi-stakeholder process by exploring strategies for changes (e.g. changes in irrigation techniques, changes in crop patterns, etc.).Willing partners accepted being pioneers in groundwater adaptation. They were ready to explore and ready to participate in a multi-stakeholder dialogue with researchers and partners from other countries.In India, for example,a farmer association in Bahoor became involved in the project because they were interested in the scenario building exercise to adjust their strategies. They also expressed their motivation in the inter-case exchanges. Some years ago, with their own budget, they had already travelled across India to learn from other farmers" experiences.

Benchmarking: A co-design workshop

In 2014-2015, the inter-case and transdisciplinary dialogue process began. Preparatory meetings and field trips occurred at the country level. Yet, the key step in the co-design of the project was a 4-day workshop, organized in Morocco in February 2015. Partners from the four countries participated, including academics and non-academics (at least two participants per country). This workshop included a one-day field trip. On this day, discussions were held with staff from the Department of Agriculture in Sefrou, Morocco, about the main actions taken by the department to foster agricultural development. Then, the group visited a large-scale farm (Figure 1) and a smallscale one (less than 5ha) that belonged to a member of an agricultural cooperative. Through discussions with farmers and managers, participants explored different models of production and identified common patterns and differences with other case studies. Finally, a meeting with the Mayor of the Ain Timguenay rural municipality, village representatives and a leader of the cooperative gave another opportunity to discuss the challenges with regards to the sustainable use of groundwater. This field trip was essential to strengthen the network and share knowledge among countries and disciplines.Relevant similarities and differences between case studieswere discussed. For example, the development of intensive farming relying on groundwater and supported by public policies in Morocco was comparable to the situation in Nebraska (USA). By contrast, the average farm size in the study case in India is smaller than the one in the Moroccan case, and Indian agricultural policies are much less developed.Indeed sharing observations during the field work served as a benchmark for the co-design of the joint-research proposal. The last two days of the workshop focused on the joint design of the project using several participatory methods (elicitation, group discussions, etc.). The discussions were framed by the needs for developing a joint proposal. The objectives of DIALAQ, the content of the work packages and the organization of the activities were discussed. Participants questioned the relevance and feasibility ofimplementing the same kind of exercise in 8 regions in 4 continents with such differences in terms of groundwater crisis, farmers" situation, scientific data that was available and needed. Cultural differences were considered in the use of foresight exercise both in terms of power structures and the way local actors dealt with uncertainty. The co-design led to collectively acknowledge the relevance of the international comparison and to a common framework anticipating differences in the implementation phase.

Conclusion: A common methodological and ethical framework

The co-design resulted in(1) a common framework to describe the participatory process to be implemented in each case study in ways that account for the diversity of local situations in terms of culture, institutions, issues, etc.; and (2) the organization of the cross-case interactions for sharing experiences insights, and scaling up.Three stages for the participatory process were defined for implementation at the case study level with groups of farmers and otheractors (NGOs, elected representatives, etc.), at first in paralleland later jointly: (1)group workshops at the different research sites to collectively assess the current dynamics and sustainability of agriculture and groundwater uses and to discuss existing scientific knowledge;

(2) at each site, collective framing and discussion of scenarios of evolution and conceptualization of pathways towards sustainability, and (3)a final workshop involving all groups to share learning that occurred within groups, and discuss the transformative pathways. Within this general framework, case study teams would use different tools to trigger dialogue and collective exploration of pathways. The methodological choices made in each case study would be compared, especially in terms of how participants were involved and the way participatory processes were implemented, to take stock of these experiences acknowledging cultural differences. A project task was dedicated to fostering cross-case interaction involving both academics and actors from each case study to share learning. The expected output wasa road map document on "designing alternatives pathways to reduce vulnerability of farming on overexploited aquifers" translated in the main language used in each case study. Across cases, it was also planned to use video in order to share information and to provide a space for stakeholders to voice their viewpoints.

Above all, the co-design led to discuss and clarify ethical issues. Groundwater access is often seen as a source of welfare for communities. Yet, it is also a source of ill fare. There are strong inequities in terms of farmers" capacities to use groundwater, and their access to it, but also in terms of actors" capacities to adapt to groundwater depletion and to have a voicein the water debate. Our project aimed to improve the sustainability and resilience of groundwater-dependent and agriculture-based socio-ecological systems. The team acknowledged its collective concern with distributional equity and awareness of local power structures and inequalities. The differences among farmers were consideredin planning to work with different socio-economical groups (including small holders and large-scale farmers), giving special attention to under-represented groups, e.g., in India, marginalized castes, women and small holders. Finally, the transcultural approach that was developed mainly aimed atachieving procedural equity while increasing knowledge and providing opportunities for actors to voice their concerns and proposals. We considered experiential knowledge of stakeholders while demystifying scientific knowledge. DIALAQ is a solutionoriented research. As social and natural scientists, we acknowledge being actors in solutionbuilding. But DIALAQ will not promote any specific pre-defined solution. We aim at supporting stakeholders in building their own strategies for changes towards sustainability and build their capacity to trigger changes so that initiatives towards sustainability continue after completion of the research project.

Figure 1 :

 1 Figure 1: Visit of a large scale (more than 200ha) farm, recently installed in the Sefrou region (Morocco), representative of a "modern and capital-intensive" production of apples and plums, Source: A. Richard, 2015

  31. * Faysse N, Rinaudo JD, Bento S, Richard-Ferroudji A, Errahj M, Varanda M, Imache A, Dionnet M, Rollin D, Garin P, et al.: Participatory analysis for adaptation to climate change in Mediterranean agricultural systems: Possible choices in process design. Regional Environmental Change 2014, 14:57-70. This paper examines the methodological choices made by three research teams in the design and implementation of participatory foresight analyses to explore agricultural and water management options for adaptation to climate change. 32. Rinaudo JD, Montginoul M, Varanda M, Bento S: Envisionning innovative groundwater regulation policies through scenario workshops in France and portugal. Irrigation and Drainage 2012, 61:65-74. 33. *Figureau AG., Montginoul M. and Rinaudo JD: Policy instruments for decentralized management of agricultural groundwater abstraction: a participatory approach.Ecological Economics 2015 119: 147-157. This paper proposes and analyses three policy instruments which can be used to enhancefarmers" compliance with individual water allocations in a decentralized management context. Three regulation strategies are proposed for the case of groundwater allocations for irrigation: the first relies on economic instruments; the second is based on tools designed to promote pro-social behaviors; and the third combines assumptions from the first two approaches. 34.van Klink I, Richard-Ferroudji A, Venkatasubramanian G, Aubriot O, Prabhakar I, G. M: From climate changes to changes in agriculture, land and water at the village level: a foresight approach in India. Sciences Eaux & Territoires 2016 (Forthcoming).

DIALogic exploration of futures and pathways for sustainable farming on overexploited AQuifers

Aknowledgements:

The authors gratefully acknowledge all the participants who were involved in the DIALAQ project (see http://www.ifpindia.org/content/dialaq). The co-design was supported by seed grants from the ISSC under the Transformations to Sustainability Programme. The Programme is funded by the Swedish International Development Cooperation Agency (Sida) and serves as a contribution to Future Earth. Supplementary support for seed grants is provided by the Swedish Secretariat for Environmental Earth System Sciences (SSEESS), the Netherlands Organisation for Scientific Research (NWO), the Economic and Social Research Council (ESRC) UK through the Newton Fund and the National Research Foundation of South Africa. The opinions, findings and conclusions or recommendations expressed in thismaterial are those of the author(s) and do not necessarily reflect the views of the ISSC or Sida.