Background: We performed a phase II trial of pembrolizumab in patients with NSCLC or melanoma with untreated brain metastases to determine the activity of PD-1 blockade in the CNS. Interim results were previously published, and we now report an updated analysis of the full NSCLC cohort. Methods: This was an open-label, single-institution, phase 2 study. Eligible patients were ≥ 18 years of age with advanced NSCLC with ≥1 brain metastasis 5-20mm not previously treated or progressing after prior radiation, no neurologic symptoms or corticosteroid requirement, and performance status <2. Patients were treated with pembrolizumab 10 mg/kg IV every 2 weeks. Cohort 1 was for patients with PD-L1 ≥1% and cohort 2 PD-L1 <1% or unevaluable. The primary endpoint was the proportion of patients achieving a brain metastasis response. All treated patients were analyzed for response and safety endpoints. This study is closed to accrual and is registered with Clinicaltrials.gov, number NCT02085070. Here we report the updated results of the NSCLC cohort. Findings: Between March 31, 2014 and May 21, 2018, 42 patients were treated. Median followup was 8.3 months (IQR 4.5 to 26.2 months). Eleven of 37 patients in cohort 1 had a brain metastasis response (29.7% [95% CI, 15•9-47•0%]). There were no responses in cohort 2. Grade 3-4 AEs related to treatment included 2 patients with pneumonitis, and 1 each with constitutional symptoms, colitis, adrenal insufficiency, hyperglycemia, and hypokalemia. Treatment-related serious adverse events occurred in 6 (14%) patients and included pneumonitis acute kidney injury, colitis, hypokalemia, and adrenal insufficiency. There were no treatment-related deaths. Interpretation: Pembrolizumab has activity in brain metastases from NSCLC with PD-L1 expression ≥1% and is safe in select patients with untreated brain metastases. Further investigation of immunotherapy in patients with CNS disease from NSCLC is warranted.

The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been maximized.

Purpose Pembrolizumab is active in melanoma, but activity in patients with untreated brain metastasis is less established. We present long-term follow-up of pembrolizumab-treated patients with new or progressing brain metastases treated on a phase II clinical trial ( ClinicalTrials.gov identifier: NCT02085070). Patients and Methods We enrolled 23 patients with melanoma with one or more asymptomatic, untreated 5- to 20-mm brain metastasis not requiring corticosteroids; 70% of patients had prior systemic therapy. Pembrolizumab was administered for up to 24 months. Brain metastasis response, the primary end point, was assessed by modified Response Evaluation Criteria in Solid Tumors (RECIST). Pretreatment tumors were analyzed for T-cell infiltrate and programmed death ligand 1. Results Six patients (26%) had a brain metastasis response. Eight patients (35%) did not reach a protocol evaluation scan and were unevaluable for brain metastasis response as a result of progression or need for radiation. Brain metastasis and systemic responses were concordant, with all ongoing at 24 months. The median progression-free and overall survival times were 2 and 17 months, respectively. Eleven patients (48%) were alive at 24 months. This included three unevaluable patients. One of these three patients had hemorrhaged, and two had symptoms from perilesional edema requiring radiosurgery, but all three patients remained on commercial pembrolizumab more than 24 months later. None of the 24-month survivors received subsequent BRAF inhibitors. Neurologic adverse events occurred in 65% of patients; all adverse events but one were grade 1 or 2. Three patients had seizures, which were treated with anticonvulsants. Most responders had higher pretreatment tumor CD8 cell density and programmed death ligand 1 expression, whereas all nonresponders did not. Conclusion Pembrolizumab is active in melanoma brain metastases with acceptable toxicity and durable responses. Multidisciplinary care is required to optimally manage patients with brain metastases, including consideration of radiation to large or symptomatic lesions, which were excluded in this trial. Two-year survival was similar to patients without brain metastasis treated with anti–programmed cell death 1 agents. Concordant brain and extracerebral responses support use of pembrolizumab to treat small, asymptomatic brain metastases.

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The multidrug resistance transporter P-glycoprotein (P-gp) effluxes a wide range of substrates and can be affected by a wide range of inhibitors or modulators. Many studies have presented classifications for these binding interactions, within either the context of equilibrium binding or the Michaelis-Menten enzyme analysis of the ATPase activity of P-gp. Our approach is to study P-gp transport and its inhibition using a physiologically relevant confluent monolayer of hMDR1-MDCKII cells. We measure the elementary rate constants for P-gp efflux of substrates and study inhibition using pairwise combinations with a different unlabeled substrate acting as the inhibitor. Our current kinetic model for P-gp has only a single binding site, because a previous study proved that the mass-action kinetics of efflux of a single substrate were not sensitive to whether there are one or more substrate-binding and efflux sites. In this study, using this one-site model, we found that, with "high" concentrations of either a substrate or an inhibitor, the elementary rate constants fitted independently for each of the substrates alone quantitatively predicted the efflux curves, simply applying the assumption that binding at the "one site" was competitive. On the other hand, at "low" concentrations of both the substrate and inhibitor, we found no inhibition of the substrate efflux, despite the fact that both the substrate and inhibitor were being well-effluxed. This was not an effect of excess "empty" P-gp molecules, because the competitive efflux model takes site occupancy into account. Rather, it is quantitative evidence that the substrate and inhibitor are being effluxed by multiple pathways within P-gp. Remarkably, increasing the substrate concentration above the "low" concentration, caused the inhibition to become competitive; i.e., the inhibitor became effective. These data and their analysis show that the binding of these substrates must be cooperative, either positive or negative.

The fitted steady-state Vmax values from the analysis correlated to within a factor of 2-3 with the values predicted from the elementary parameters. However, the fitted Km value could be generated by a wide range of underlying "molecular" Km values. This is because of the convolution of the drug passive permeability kinetics into the fitted Km. This implies that Km values measured in simpler systems, e.g., microsomes or proteoliposomes, even if accurate, would not predict the Km values for the confluent monolayer system or, by logical extension, in vivo. Reliable in vitro-in vivo extrapolation seems to require using the elementary rate constants rather than the Michaelis-Menten steady-state parameters.

P-glycoprotein, a human multidrug resistance transporter, has been extensively studied due to its importance to human health and disease. In order to understand transport kinetics via P-gp, confluent cell monolayers overexpressing P-gp are widely used. The purpose of this study is to obtain the mass action elementary rate constants for P-gp's transport and to functionally characterize members of P-gp's network, i.e., other transporters that transport P-gp substrates in hMDR1-MDCKII confluent cell monolayers and are essential to the net substrate flux. Transport of a range of concentrations of amprenavir, loperamide, quinidine and digoxin across the confluent monolayer of cells was measured in both directions, apical to basolateral and basolateral to apical. We developed a global optimization algorithm using the Particle Swarm method that can simultaneously fit all datasets to yield accurate and exhaustive fits of these elementary rate constants. The statistical sensitivity of the fitted values was determined by using 24 identical replicate fits, yielding simple averages and standard deviations for all of the kinetic parameters, including the efflux active P-gp surface density. Digoxin required additional basolateral and apical transporters, while loperamide required just a basolateral tranporter. The data were better fit by assuming bidirectional transporters, rather than active importers, suggesting that they are not MRP or active OATP transporters. The P-gp efflux rate constants for quinidine and digoxin were about 3-fold smaller than reported ATP hydrolysis rate constants from P-gp proteoliposomes. This suggests a roughly 3∶1 stoichiometry between ATP hydrolysis and P-gp transport for these two drugs. The fitted values of the elementary rate constants for these P-gp substrates support the hypotheses that the selective pressures on P-gp are to maintain a broad substrate range and to keep xenobiotics out of the cytosol, but not out of the apical membrane.

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