A Pharmacodynamic Study of Docetaxel in Combination with the P-glycoprotein Antagonist Tariquidar (XR9576) in Patients with Lung, Ovarian, and Cervical Cancer

Resistance to anticancer therapy remains a major problem in the treatment of cancer, highlighted in recent years by the onset of drug resistance in tumors treated with molecularly targeted agents. Drug resistance occurs via a myriad of mechanisms—including pharmacologic resistance whereby a drug fails to be activated or is rapidly excreted or inactivated; resistance at the level of the target through loss or mutation of the target; hypoxia and cell survival mechanisms; and transport-mediated resistance due to decreased drug influx and/or increased efflux, effecting reduced intracellular drug accumulation. P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) transporter family, can confer resistance to a large number of functionally and chemically distinct cytotoxic compounds. Pgp, encoded by the MDR-1 (ABCB1) gene, is an energy-dependent efflux pump that lowers the intracellular concentrations of many chemotherapeutic agents (1, 2). It has been hypothesized that Pgp inhibition could serve an important role in previously treated and naive tumors overexpressing the transporter. Despite correlation of Pgp expression with poor outcome in multiple settings, this hypothesis has not been confirmed clinically.

Many early phase I/II studies attempting Pgp inhibition used first-generation, nonspecific Pgp inhibitors such as verapamil, dexverapamil, tamoxifen, quinidine, and cyclosporine. Results from these studies proved disappointing and failed to show an improvement in overall drug efficacy, primarily attributed to poor potency (3). In addition, trials included heavily pretreated patients, without documented Pgp expression in tumors. The interpretation of these early studies was further hampered by a lack of randomization to prove efficacy.

Second-generation agents with increased potency were subsequently developed including PSC833 (valspodar), VX-710 (biricodar), and GF120918 (elacridar) (4–7). Data from clinical trials involving second-generation agents, particularly with valspodar, were similarly disappointing. Drug–drug interactions involving CYP3A4 inhibition required cytotoxic drug dose reduction due to a decrease in chemotherapeutic drug clearance, resulting in increased exposure. Several trials showed increased toxicity in the experimental arm (7–9). These investigations led to the development of third-generation Pgp inhibitors including tariquidar (XR9576), zosuquidar (LY335979), and laniquidar (R101933), with increased specificity and potency, and fewer pharmacokinetic interactions (10).

Tariquidar is a third-generation Pgp antagonist. It is an anthranilic acid–based drug that potently inhibits Pgp-mediated drug efflux (11–14). At low nanomolar concentrations, the compound restores the sensitivity of resistant human tumor cell lines to cytotoxic agents including anthracyclines, vinca alkaloids, and taxanes. Its duration of action is superior to previous Pgp inhibitors, with CD56⁺ cells in patients showing continued inhibition up to 48 hours after administration (14, 15). Although tariquidar had minimal toxicity in early testing (14, 16), two large randomized multicenter trials in lung cancer closed early due to toxicity in the experimental arms. It was therefore decided that further safety testing was warranted. The primary goal of this study was to evaluate the effects of tariquidar on docetaxel pharmacokinetics and to establish whether tariquidar at the administered dose modulates Pgp in tumors of the patients enrolled. Additional safety data were to be collected in the setting of combined therapy. Trials combining other Pgp modulators with taxanes have shown inhibition of drug clearance, requiring decreased taxane dosage (10). In contrast, previous studies with tariquidar suggested minimal pharmacokinetic interactions with doxorubicin, paclitaxel, and vinorelbine, which were clinically insignificant relative to natural intrapatient variability in pharmacokinetics (14–17). We anticipated that the docetaxel dose in the combination would not require a reduction because 75 mg/m² provided a margin of safety as a dose that is routinely used and better tolerated than the FDA-approved dose of 100 mg/m² (18, 19). The pharmacokinetic portion of the study used a 2-period crossover design and was analyzed accordingly.

Forty-eight patients were enrolled onto the trial, which was reviewed and approved by the National Cancer Institute's Institutional Review Board. Verbal and written consent was obtained from all patients. Enrollment criteria included age older than 18 years, an Eastern Cooperative Oncology Group (ECOG) performance status of 0–2, and a life expectancy greater than 3 months. Eligibility required histologic or cytologic confirmation of lung, cervical, or ovarian cancer, at least 1 prior standard treatment regimen, and no known standard therapy capable of extending life expectancy.

Suitable candidates required a platelet count ≥90,000/mL, absolute granulocyte count ≥1,500/mL, serum creatinine ≤1.5 mg/dL (or if >1.5, a measured 24-hour creatinine clearance ≥50 mL/min), aspartate transaminase (AST) and alanine transaminase (ALT) ≤2.5 × NL, and bilirubin ≤1.5 × NL (in patients with clinical evidence of Gilbert's disease, ≤3 × NL). Patients were also required to be ≥4 weeks from prior radiation or chemotherapy, >2 weeks from hormonal therapy, and >4 weeks from prior experimental therapy. Patients receiving agents that had major interactions with the CYP3A4 drug metabolizing system and that could not be discontinued were not allowed on study. Furthermore, patients with untreated brain metastases (or local treatment of brain metastases within the last 6 months) were excluded.

Tariquidar was supplied by Xenova Ltd. and docetaxel by the Warren G. Magnuson Clinical Center Pharmacy. This was an open-label pharmacokinetic/pharmacodynamic trial. To generate equivalent pharmacokinetic data, 40 mg/m² docetaxel was administered on both day 1 and 8 of cycle 1, and patients were randomized to receive tariquidar 150 mg on either day 1 or day 8 of cycle 1 (Supplementary Fig. 1). Tariquidar was given intravenously over 40 minutes before initiation of the 1-hour docetaxel infusion. Tariquidar was administered alone on or about day 22 (week 4 of cycle 1) to allow sestamibi imaging, uncomplicated by docetaxel administration. From cycle 2 and onward, 75 mg/m² docetaxel was administered every 21 days in combination with a single 150-mg tariquidar dose. Growth factor support was allowed in cycles 2 and beyond, as clinically indicated. If the nadir ANC was ≥1,000, the nadir platelet count was ≥50,000, and no grade 3/4 nonhematologic toxicities were observed after 75 mg/m² docetaxel, the dose could be increased to 90 mg/m² in subsequent cycles, provided no delay was required to start the next cycle.

Pharmacokinetic and pharmacodynamic studies were done in cycle 1, in which docetaxel was administered on days 1 and 8. Measurement of Pgp-mediated rhodamine 123 transport in CD56⁺ cells was done as described (20). Whole blood was obtained from patients prior to treatment (pre) and 24 hours and 48 hours after the start of the tariquidar infusion. Rhodamine 123 (final concentration 0.5 μg/mL) was added to aliquots of blood in the presence or absence of 3 μg/mL of the Pgp inhibitor valspodar (PSC 833); 1 aliquot was incubated without rhodamine to assess autofluorescence. Aliquots were incubated for 30 minutes at 37°C after which mononuclear cells were separated by density gradient. Aliquots were then washed with cold PBS, divided and either held at 4°C or resuspended in rhodamine-free complete medium (phenol red–free Richter's medium with 10% fetal calf serum) continuing with or without valspodar, and allowed to incubate for an additional 1 hour at 37°C. All aliquots were then washed and stained with phycoerythin-labeled anti-CD56 antibody (BD Biosciences) at 4°C. Multiparameter flow cytometry was conducted on a FACSort flow cytometer (BD Biosciences) equipped with an argon laser. Intracellular rhodamine fluorescence was calculated in CD56⁺ cells using the FlowJo analysis program (v 6.4.7; Tree Star Inc.).

A secondary objective of this study was to establish whether tariquidar (150 mg) modulates Pgp in tumors of patients. Evaluation was done by ^99mTc-sestamibi (Cardiolyte) scanning in conjunction with a solitary tariquidar dose administered in the fourth week of cycle 1. A baseline ^99mTc-sestamibi scan was obtained before the administration of tariquidar. A minimum of 48 hours later, on or about day 22, a single dose of tariquidar was administered, followed by a second ^99mTc-sestamibi scan. Cycle 1 was considered complete at day 28; subsequent cycles were 21 days in duration.

Blood samples were collected in tubes containing sodium heparin as an anticoagulant on days 1 and 8. Samples were obtained before administration, 1 hour after the start of the docetaxel infusion (several minutes prior to the end of infusion), and at 1 hour 5 minutes, 1 hour 15 minutes, 1 hour 30 minutes, 1 hour 45 minutes, 3 hours, 5 hours, 7 hours, 12 hours, and 24 hours. Samples were centrifuged for 5 minutes at 1,200 × g. The plasma supernatant was stored at −80°C until analysis.

All samples were analyzed using an analytical assay validated for the measurement of docetaxel in human plasma. Briefly, 100 μL of plasma was transferred to a glass centrifuge tube, and 1 mL of methyl tert-butyl ether containing the internal standard paclitaxel was added. After vortex mixing and centrifugation, the supernatant layer was collected and evaporated. The residue was reconstituted with a mixture of methanol/0.1% formic acid (v/v 60:40), of which 5-μL solution was injected into the Acquity UPLC (Waters Corp). Mass analysis was achieved by a Quattro Permier Triple Quadrupole Mass Spectrometer (Waters Corp) using electrospray ionization. The compounds were separated on a Symmetry Shield Rp18 column (2.1 × 50 mm, 3.5 μm) using a mobile phase consisting of methanol (B)/0.1% formic acid (A) at a flow rate of 0.2 mL/min. Initial condition, 40% B was gradually increased to 65% within the first 4 minutes of gradient run and then held for 3 minutes before it was set to the initial condition. The total run time was 8 minutes. Two ion transitions were monitored: docetaxel, 808.5 → 527.3 m/z; and paclitaxel, 854.4 → 569.1, m/z. Assay range was 1 to 1,000 ng/mL. Accuracy and precision of 3 concentrations of quality control samples ranged from 98% to 104.3% and 0% to 3.2%.

Noncompartmental pharmacokinetic data analysis was done using WinNonLin, v.5.2. The maximum plasma concentration (C_max) was the observed value. The area under the concentration-time curve (AUC) from time zero to 24 hours after the start of the docetaxel infusion (AUC_0–24) was calculated using the linear trapezoidal method. Nonparametric statistical methods for analyzing data from 2-period crossover trials were used (21). Specifically, tests were first conducted to determine whether there was a period or carryover effect (comparing days 1 and 8) and a residual effect, before testing whether there was a difference between docetaxel parameters when administered with and without tariquidar. As the parameters presented in this report did not exhibit a significant period effect, the difference in the parameter values for docetaxel with tariquidar minus those without tariquidar were determined by a Wilcoxon signed-rank test. P values less than 0.05 were considered to be statistically significant. For all data sets, geometric mean and 95% CIs were calculated using GraphPad Prism.

Table 1 summarizes the tumor types, performance status, age, and prior therapies of the 48 patients enrolled on the trial. The 3 main tumor types were ovarian (38%), cervical (29%), and lung (29%). A protocol amendment allowed inclusion of patients with renal cell carcinoma (RCC) who had previously been treated with either sunitinib or sorafenib and had been deemed ineligible for IL-2 therapy. Only 2 patients (4%) with RCC were enrolled before reaching the accrual ceiling of the patient population originally planned. All patients had an ECOG performance status score of 2 or better. All patients had received at least 1 prior cytotoxic chemotherapy regimen before commencing on study. The mean number of prior regimens was 4.8 and median 4. Approximately 50% of patients had received at least 3 prior regimens prior to enrollment and 29% had received 6 or greater (range, 6–16) prior treatments.

All patients received 40 mg/m² docetaxel on days 1 and 8, and 150 mg tariquidar randomized to either day 1 or day 8. This schedule was selected to allow detection of the effects of tariquidar on pharmacokinetics when only 8 days separated the 2 sampling periods, and using a dose of docetaxel that would be safe when repeated in a short interval. Although nonlinearity cannot be excluded, it was not expected on the basis of previously published data on docetaxel pharmacokinetics (22, 23). Cycle 2 commenced after completion of the pharmacokinetic and pharmacodynamic studies in cycle 1. In this and subsequent cycles, 75 mg/m² of docetaxel was administered in combination with a single 150-mg tariquidar dose. Granulocyte colony-stimulating factor (G-CSF) was allowable by the protocol after cycle 1 and it was used if clinically indicated on the basis of toxicity or on prior history of pelvic irradiation. Doses were reduced per protocol for toxicity. Although 75 mg/m² was considered an adequate dose, some patients had minimal to no toxicity and the protocol allowed dose increases. Among the 187 cycles administered in cycles 2 and beyond, 160 doses were administered at 75 mg/m² (86%), 11 doses at 60 mg/m² (6%), 1 dose at 40 mg/m² (<1%), 14 doses at 90 mg/m² (7%), and 1 dose at 100 mg/m² (<1%). A total number of 235 cycles of tariquidar in combination with docetaxel were administered to 48 patients with a median of 4 cycles per patient, a mean of 4.9 cycles, and a range of 1 to 26. Five subjects received more than 9 cycles and 1 received 26 cycles.

One of the primary objectives of this study was to assess the safety and toxicity profile of the 2-drug combination. In general, the combination was well tolerated. Two patients (4%) among the 48 receiving 40 mg/m² docetaxel in cycle 1 experienced grade 3 febrile neutropenia and received docetaxel dose reductions in cycle 2 to 60 and 40 mg/m², respectively. One patient had received extensive prior pelvic radiotherapy and the other had extensive liver involvement with preexisting liver dysfunction that became apparent after commencing on study. Table 2 outlines grade 3 or 4 toxicities seen during cycles 1 and 2 (n = 48 and 40, respectively). Ten percent of patients had grade 4 neutropenia in cycle 1, which increased to 48% in cycle 2. G-CSF was frequently used in subsequent cycles; grade 3/4 neutropenia was observed in 41% of 126 cycles (cycles 2–6), as shown in Supplementary Table 1. The rates of grade 3 anemia and thrombocytopenia were relatively low (8% and 2%, respectively). Nonhematologic toxicities overall were generally grade 1 or 2, with the exceptions being grade 3 fatigue (6%), hypoalbuminemia (4%), and hyponatremia (4%) (Supplementary Table 1). An uncommon toxicity observed in this study was docetaxel-induced epiphora secondary to canalicular stenosis. Fifteen patients (31%) developed epiphora, of which 10 had grade 1, 3 patients grade 2, and 2 patients grade 3. No one discontinued therapy for epiphora; 4 patients required ophthalmologic evaluation, and 3 were managed with bicanalicular silicone intubation or dacryocystorhinostomy to prevent stenosis.

As CD56⁺ peripheral blood mononuclear cells express high endogenous levels of Pgp, inhibition of Pgp-mediated rhodamine 123 efflux from CD56⁺ cells has been used to determine efficacy of Pgp inhibition in clinical trials (20, 24). As shown in Figure 1A (upper row, left panel), rhodamine 123 fluorescence was assessed in CD56⁺ cells from a patient after a 30-minute loading period in the absence (solid line, control histogram) or presence of exogenously added valspodar (dashed line, PSC histogram), which fully inhibits Pgp-mediated rhodamine transport in this assay. The remaining panels represent cells in which a 60-minute efflux period followed, continuing the cells without or with exogenous valspodar (to generate efflux and PSC/efflux histograms). We obtained the difference between the control and PSC histograms and the difference between the efflux and PSC/efflux histograms at each time point for each patient (a representative set of assays is shown in the remaining panels of Figure 1A: prior to (Pre), or 24 hours and 48 hours after the start of the tariquidar infusion). Overlap of efflux and PSC/efflux histograms reflects complete inhibition of Pgp by the tariquidar administered to the patient. Boxplots of the difference between control and PSC histograms and efflux and PSC/efflux histograms in 41 patients are shown in Figure 1B and C, respectively. Although rhodamine efflux from CD56⁺ cells was significantly decreased because of tariquidar administration at the 24 and 48 hour time points compared with Pre levels, inhibition appeared more variable at the 48 hour time point.

Sestamibi results were available in 35 (73%) of the 48 patients and were analyzed as previously described (13, 14). No statistically significant increase in exposure, measured as AUC up to 3 hours (AUC_0–3) was noted for cardiac or lung tissue (2-tailed Student's t test). Figure 2 presents a boxplot analysis of the sestamibi AUC monitored from 0 to 3 hours (AUC_0–3) in heart, lung, liver, and visible tumors in the presence or absence of tariquidar. A statistically significant change in AUC was detected in both liver and visualized tumor tissue (both P < 0.001). When the AUC_0–3 was evaluated as a percentage change and normalized to the AUC_0–3 of the heart (nAUC_0–3), an increased nAUC_0–3 for the liver was observed ranging from 5.8% to 252% over the pretariquidar scan. The median increase in liver uptake was 82.2%, whereas the median increase in visualized tumors was 12.4%, ranging from −7.2% to 24.1%. The results also showed that actual liver AUC_0–3 was much larger than the AUC_0–3 of the tumors. Post–tariquidar-treated liver AUC_0–3 ranged from 406 to 2,094 min cpm/px/mCi, whereas the AUC_0–3 of the tumor measured from 141 to 233 min cpm/px/mCi.

Examining the lung cancer cohort, in which 10 of 13 patients with lung cancer had visible lesions on sestamibi imaging, lung tumors in 8 of the 10 showed an increased nAUC ranging from 12% to 24.1%. In addition to the 3 patients with lung masses on computed tomographic (CT) scan in whom no tumor sestamibi uptake could be identified, many patients showed a mixed pattern, with some lesions showing sestamibi uptake whereas others did not. Figure 3A shows a large supraclavicular mass on CT that shows sestamibi uptake after tariquidar, whereas Figure 3B shows a sestamibi scan with mixed results in which a large peripheral right lung lesion is sestamibi negative. Supplementary Figure 2 shows a composite graph of sestamibi uptake levels over time in a single patient in the liver, heart, and left and right lung tumors pre- and post–tariquidar treatment.

Twenty-four hour pharmacokinetics were evaluable in a total of 45 patients, for a total of 76 cycles; 37 with tariquidar and 39 without. Thirty-one patients had evaluable pharmacokinetics on both C1D1 and C1D8, allowing for the assessment of the effect of tariquidar on docetaxel disposition. A comparison of exposure (AUC_0–24 and C_max) was done. As shown in Supplementary Table 2, no significant difference in docetaxel disposition was observed on the basis of pairwise comparison with and without tariquidar, though substantial interindividual variability was observed. Variability can be seen in Figure 4A and B, which graphically display the pairwise comparison of these pharmacokinetic parameters. The docetaxel exposure ratios with and without tariquidar are shown in Figure 4C. The geometric mean ratios for docetaxel C_max and AUC were 0.907 (95% CI: 0.697–1.18) and 1.07 (95% CI: 0.922–1.25). Similarly, no effect of sequence was observed by comparison of docetaxel clearance when administered as a single agent on either C1D1 or C1D8, as shown in Figure 4D.

In total, 4 partial responses (PR) were seen (8%). Three PRs measuring 40%, 57%, and 67% reduction in tumor size by RECIST criteria were independently verified in patients with heavily pretreated non–small cell lung cancer. One PR measuring 30% was seen in a patient with advanced ovarian cancer who had received 5 previous regimens. Although 11 (23%) patients had received prior docetaxel, none of the 4 patients with measurable response had received prior docetaxel. Sixteen patients showed stable disease (33%) ranging from 13 to 38 weeks. Stable disease as per RECIST criteria was attributed only after cycle 4 (12 weeks) of treatment. Of those with stable disease, 3 patients had evidence of prolonged clinical benefit of greater than 8 months lasting 32, 34, and 38 weeks, respectively. Twenty patients (42%) had progressive disease and 8 patients (17%) were considered not evaluable. Among these, 5 patients refused to return for further therapy and restaging after receiving cycle 1. Three patients developed complicating disease or intercurrent illness and were withdrawn from the trial for medical reasons.

This report summarizes a pharmacodynamic study of docetaxel in combination with the Pgp inhibitor tarquidar. We determined that a docetaxel dose of 75 mg/m² every 3 weeks can be safely administered with a single 150-mg dose of tariquidar. Pharmacodynamic studies confirmed complete inhibition of Pgp in circulating mononuclear cells. ^99mTc-sestamibi was used as a surrogate to evaluate drug accumulation in normal and tumor tissues and confirmed increased accumulation both in liver and in a subset of tumors. No significant difference in docetaxel disposition was observed on the basis of pairwise comparison with and without tariquidar. However, pharmacogenetic studies are currently underway to elucidate the cause of the interindividual and intraindividual variability observed. Although not a primary endpoint, clinical benefit was noted in a number of patients, including partial remissions in 4 tumors (3 NSCLC and 1 ovarian carcinoma).

The toxicities seen in this study were acceptable and were usual for the side effects seen with docetaxel. As this was a heavily pretreated patient population, G-CSF was used to support granulocyte counts, such that although grade 3/4 neutropenia was observed in 41% of 126 cycles, only 6 episodes of febrile neutropenia were reported. At present there is no evidence to suggest that Pgp inhibition via tariquidar in normal tissues leads to enhanced toxicity. Possibly, the only toxicity observed that may have been related to the use of docetaxel in combination with tariquidar was epiphora secondary to canalicular stenosis. This is a recognized, but relatively rare side effect of taxane therapy that is seen more commonly in patients who receive weekly docetaxel (33%–50%) than in those receiving 3 weekly docetaxel (11%), where it has been reported as conjunctivitis (25–27). Thus, these data suggest that a therapeutic window does exist to improve tumor tissue uptake without increased toxicity due to increased uptake in normal cells, particularly in bone marrow cells.

An issue that is still not proven is whether Pgp inhibition can actually increase concentrations of anticancer agents in tumor tissue in the clinical setting. Inhibition of rhodamine efflux in circulating CD56⁺ cells is a surrogate for Pgp inhibition in normal cells. As shown in Figure 1, CD56⁺ cells from all patients showed retention of rhodamine 24 hours after intravenous dosing of tariquidar with persistence to 48 hours in most. This is consistent with prior studies showing retention of rhodamine in CD56⁺ cells following the inhibitors valspodar, tariquidar, or CBT-1 (14, 20, 28, 29). However, the major limitation of the CD56⁺ assay is that it reflects Pgp inhibition in the blood and not in the tumor.

As a strategy to evaluate Pgp inhibition in normal and tumor tissues, imaging with the radionuclide cardiac imaging agent ^99mTc-sestamibi was included in this study. This γ-emitting organotechnetium complex is a substrate for the Pgp efflux pump (30, 31). Cardiac tissue does not significantly express Pgp and therefore tends to accumulate and retain sestamibi. In tissues that express Pgp, such as the liver, kidney, and some tumors, retention of sestamibi increases in the presence of Pgp antagonists (13). Similar results have been observed with another radiotracer, ^99mTc-tetrofosmin, which is also FDA approved for cardiac imaging. In lung cancer, radiotracer uptake has been correlated with response to treatment in small, single-institution analyses, where striking interindividual differences in ^99mTc-sestamibi or ^99mTc-tetrofosmin uptake were reported with the absence of imaging uptake indicative of poor response to chemotherapy (32–38). A recent meta-analysis concluded that ^99mTc-sestamibi scanning, particularly the initial uptake, can be used as a preselection technique to differentiate likely chemotherapy responders (38). Although our baseline sestamibi studies do show striking differences between patients, there were not enough patients in the lung cancer subset to evaluate correlation between sestamibi imaging and response.

Apart from basal uptake, our study queried whether sestamibi retention was higher after tariquidar intake. Sestamibi results were available in 35 of the 48 patients, and an increased nAUC for the liver was noted, ranging from 5.8% to 252% after tariquidar intake. A modest although statistically significant 12% to 24% increase in sestamibi uptake was noted in visible lesions in 8 of 10 patients with lung cancer. We have previously recognized that quantitation by planar sestamibi imaging tends to record greater changes in AUC values for hepatic tissue than for tumor tissue (13). A previous study noted increases in nAUC_0–3 from −14% to +278% in liver and from 36% to 263% in tumors of 8 patients among 17 who had imageable tumors, with the most pronounced effects in patients with renal or adrenocortical cancer, both tumor types with known high expression of Pgp (13). High Pgp expression may explain the relatively better results in the prior study and in liver tissue, compared with lung tumors. Alternatively, Pgp may not be the most important arbiter of sestamibi accumulation in lung cancer. As sestamibi is a substrate of both Pgp and MRP1 transporters (39), it is tempting to conclude that the lack of a convincing tariquidar effect in the lung tumors in our patients is due to the confounding effect of another transporter, such as MRP1, with documented expression in lung cancer (40). The lack of effect of tariquidar on sestamibi retention could be explained by the presence of other transporters, including other ABC transporters, and also organic anion transporting polypeptides (OATP) that have been shown to modulate chemotherapeutic concentrations. Notably, tariquidar does not inhibit MRP1 (41) and ABCG2 has been shown not to transport sestamibi (42).

Another explanation for the lesser changes seen in tumor tissue than in the liver is that with planar imaging, the counts per pixel over a region of interest include all counts in that region coming from tissue lying along the axis perpendicular to the face of the gamma camera (ventral-dorsal), as well as scatter from surrounding pixels. When the liver is imaged, a large number of counts along this axis come from liver tissue as compared with the lung tumors visualized in this study, which usually only extended a few centimeters in any direction—thus, many of the counts in our tumor regions of interest (ROI) actually represented lung or other normal tissue. Second, scatter from surrounding pixels is usually greater in liver than that in tumor because of its size and high sestamibi uptake compared with tumors that are relatively smaller and have lower uptake of sestamibi. Tomographic imaging with attenuation correction should eliminate much of this problem, and we are currently exploring this using ^94mTc-sestamibi PET imaging.

A number of studies have addressed the question of whether Pgp expression in lung cancer is a determinant of clinical outcome. Although there is evidence that MDR-1 expression may correlate with outcome, particularly in SCLC (40, 43, 44), for NSCLC, both poor outcome (43, 45) and no impact on outcome (46, 47) have been reported. More recent studies have examined the effect of other ABC transporters such as MRP1 or ABCG2 (BCRP), and conflicting conclusions suggesting both poor outcome and no impact on outcome have been reported (47–49). Little work has been done examining the expression of other ABC transporters. One unique feature of tariquidar is that it has been shown to inhibit both Pgp- and ABCG2-mediated resistance in vitro, although higher concentrations are required to inhibit ABCG2. As there are no clinical surrogates for the inhibition of ABCG2, it is unclear whether clinically achievable tariquidar levels would inhibit ABCG2 activity in tumors. The variable uptake of sestamibi in tumor tissue, and the failure of tariquidar to make a dramatic difference, suggests that both functional studies assessing drug accumulation and studies characterizing transporters that affect drug concentrations are needed in lung cancer.

Although 4 patients with heavily pretreated cancer attained PRs, this trial was not designed to answer the question of whether Pgp inhibition provided clinical benefit—experience with Pgp inhibition trials to date has clearly shown that randomized designs are necessary to answer this question. Two double-blind, randomized, placebo-controlled, multicenter phase III studies in advanced non–small cell lung cancer combining tariquidar with carboplatin/paclitaxel or with vinorelbine closed early due to toxicity. This was ascribed to the combination of high-dose chemotherapeutics in a lung cancer population, with a pharmacodynamic effect on the bone marrow, rather than a pharmacokinetic interaction (50). Thus, the questions of whether drug accumulation could be increased in lung cancer or whether there could be an added clinical value in lung cancer were not answered.

We are now in the era of utilizing predictive and prognostic biomarkers to individualize patient therapy, and it is clear that drug resistance is too complex to be attributed to a single protein. Where it has been evaluated, tumor drug accumulation is far more variable than generally assumed. Imaging agents or other strategies are required to identify tumors in which drug accumulation is a critical determinant of treatment response. This trial highlights the need to determine whether Pgp, or other transporters such as MRP1 or ABCG2, or the OATPs, play a dominant role in drug uptake in clinical tumors. With the increasing number of novel, targeted agents recognized as substrates for Pgp or ABCG2-mediated efflux (imatinib, dasatinib, nilotinib, lapatinib, and sunitinib have all been shown to interact with these transporters), there is new impetus to answer this question. If we are to deliver personalized medicine, we need to know that drugs are penetrating into tumor tissue. Improved imaging modalities need to be developed so that they can be coupled with novel therapeutics. A functional imaging study with greater sensitivity than sestamibi, as shown in this study, could be used to aid in choosing between available therapeutics. As molecular profiling of patients tumors and delivering personalized therapies becomes the norm, it is clear that an improved understanding of the factors that affect drug accumulation in human cancer will be needed to deliver on the promise of exciting novel therapies.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. Government.

The authors thank their patients and National Institutes of Health (NIH) nursing and clinical staff for participation in the clinical trial. In addition, the authors also thank Victoria Luchenko and Zhirong Zhan for assistance with the pharmacodynamic endpoints.

This project has been funded in whole or in part with federal funds from the National Cancer Institute, NIH, under contract HHSN261200800001E (E.R.G.). This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.