A Phase I and Pharmacokinetic Study of Exisulind and Docetaxel in Patients with Advanced Solid Tumors

Vol. 10, 7229–7237, November 1, 2004 Clinical Cancer Research 7229

Samir E. Witta,1 Daniel L. Gustafson,1 of exisulind and the development of grade 2 or greater A. Scott Pierson,1 Alexander Menter,1 toxicities. One third of patients maintained stable disease for 1 1 3 to 12 cycles, but no objective responses were observed. Scott N. Holden, Michele Basche, 2 1 1 Conclusions: The combination of docetaxel (36 mg/m , Martha Persky, Cindy L. O’Bryant, weekly) and exisulind (500 mg/d) was reasonably well toler- 1 1 1 Chan Zeng, Anna Baron, Michael E. Long, ated, and it is undergoing phase II testing in patients with 2 1 1 Amy Gibbs, Karen Kelly, Paul A. Bunn, Jr., non–small-cell lung cancer. Daniel C. Chan,1 Patrick Pallansch,2 and S. Gail Eckhardt1 INTRODUCTION 1University of Colorado Health Sciences Center, Denver, Colorado; and 2OSI Pharmaceuticals, Inc., Melville, New York Currently, the induction of apoptosis in malignant cells is undergoing intense investigation as a therapeutic strategy for cancer patients. Exisulind (sulindac sulfone, FGN-1, Aptosyn), ABSTRACT a proapoptotic drug, is a sulfone metabolite of the nonsteroidal Purpose: Exisulind (sulindac sulfone, FGN-1, Aptosyn) anti-inflammatory drug (NSAID) sulindac. Exisulind promotes is a sulindac metabolite that induces apoptosis via inhibition programmed cell death by inhibiting cyclic GMP (cGMP)- of cyclic GMP-phosphodiesterase. This agent demonstrated phosphodiesterases 2 and 5 that are often overexpressed in a tumor growth inhibition in rodent models of colon, breast, variety of cancers (1). This inhibition results in sustained ele- prostate, and lung carcinogenesis. In an orthotopic model of vation of cGMP, which leads to the activation of c-GMP– human non–small-cell lung cancer, the combination of ex- dependent protein kinase G (PKG). Activation of PKG promotes isulind and docetaxel prolonged survival in athymic nude the proteasomal degradation of ␤-catenin and activation of c-Jun rats, forming the basis of this phase I combination study. NH2-terminal kinase (JNK), leading to apoptosis (2). Interest- Experimental Design: This study evaluated the toxicity ingly, exisulind exhibits broad antitumor activity independent of and pharmacokinetics of combining exisulind (150–250 mg) cyclooxygenase-1, cyclooxygenase-2, p53, and bcl-2 inhibition given orally twice daily and docetaxel (30–36 mg/m2)ad- (3). The proapoptotic effect of exisulind differs from the ben- ministered intravenously on days 1, 8, and 15 of a 4-week zylamide analogs of sulindac, OSI-461 (formerly CP461) and cycle. OSIP486 821 (formerly CP248), by having no effect on micro- Results: Twenty patients with a range of advanced solid tubule polymerization (4, 5). In rodent models, exisulind dem- tumors (median age, 59 years; age range, 35–77 years; me- onstrated tumor growth inhibition of colon, bladder, breast, dian performance status, 1) received a total of 70 courses. prostate, and lung cancers (6–8). Observed adverse events were mild to moderate, and there Based on the sulindac data in familial adenomatous polyp- was no dose-limiting toxicity at any level. Grade 3 gastroin- osis (FAP), there was initial interest in developing exisulind as testinal toxicities were present in 10 of the 70 cycles (10%) a chemopreventive agent. In one phase I trial, exisulind was and included nausea, vomiting, dyspepsia, and elevated al- administered twice daily for 6 months to 20 patients with FAP kaline phosphatase. Neutropenia was present in four cycles who had undergone subtotal colectomies (9). Dose-limiting in patients treated with a docetaxel dose of 36 mg/m2. Phar- toxicity (DLT) was observed at the 800 mg/d dose level and macokinetic analysis did not demonstrate a clear effect of consisted of National Cancer Institute Common Toxicity Crite- exisulind on docetaxel pharmacokinetics and vice versa. Re- ria (CTC) grade 3 reversible hepatic transaminitis. Side effects lationships were evident between the plasma concentration were common at all dose levels and included CTC grade 1 or 2 hepatic transaminase elevations, nausea, vomiting, dyspepsia, abdominal pain, diarrhea, and headache. The maximum tolerated dose (MTD) was determined to be 300 mg twice a day in Received 9/8/03; revised 5/3/04; accepted 7/15/04. patients with subtotal colectomies. There was no significant Grant support: OSI Pharmaceuticals, Inc. succesor-in-interest to Cell effect on polyp number or cellular proliferation, although there Pathway, Inc. (Melville, NY) and Aventis Pharmaceuticals Inc. (Bridge- was a trend toward increased apoptosis in polyps removed from water, NJ). patients treated at the MTD. A randomized, double-blind, pla- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked cebo-controlled 12-month trial in 73 patients with FAP demon- advertisement in accordance with 18 U.S.C. Section 1734 solely to strated a 25% reduction in the formation of new polyps in the indicate this fact. exisulind-treated group that was not statistically significant (10). Requests for reprints: S. Gail Eckhardt, University of Colorado Health However, during a 12-month extension, patients taking placebo Sciences Center, Division of Oncology, 4200 East 9th Avenue B171, Denver, CO 80262. Phone: 303-315-0882; Fax: 303-315-1035; E-mail: who crossed over to exisulind and those who continued exisu- [email protected]. lind experienced statistically significant decreases in new polyp ©2004 American Association for Cancer Research. formation (11).

Exisulind was also studied alone as an anticancer agent. In effective therapy existed were eligible for this study. Other one phase I study, exisulind was administered to 15 heavily eligibility criteria included the following: (a) age Ն 18 years; pretreated patients with non–small-cell lung cancer [NSCLC (b) Southwestern Oncology Group performance status of Յ1; (12)]. The MTD of exisulind was 250 mg twice daily (500 (c) no prescription or over-the-counter NSAIDs for 2 weeks mg/d). Gastrointestinal discomfort and elevated liver function before enrollment (patients taking a cumulative monthly dose of tests were dose-limiting. There were no objective responses; aspirin of Ͻ3,250 mg for cardiovascular prevention were not however, two patients maintained stable disease for 14 and 15 excluded from the study); (d) life expectancy Ն 3 months; (e)no weeks, respectively. In a randomized, placebo-controlled trial in chemotherapy or investigational agents within 4 weeks of study patients with prostate cancer, exisulind (500 mg/d) or placebo entry or within 6 weeks of study entry for nitrosoureas or was administered to 96 men with increasing prostate-specific mitomycin C; (f) adequate hematopoietic (absolute neutrophil antigen (PSA) after radical prostatectomy (13). Exisulind sig- count Ն 1,500/␮L, hemoglobin Ն 9.0 g/dL, platelet count Ͼ nificantly suppressed the rise in PSA overall and lengthened the 100,000/␮L), hepatic [total bilirubin Յ the institutional upper interval to doubling of the PSA in high-risk patients. As with the limit of normal (ULN); aspartate aminotransferase (AST) and prior studies, the most common toxicities included gastrointes- alanine aminotransferase (ALT) Յ 2.5ϫ ULN and alkaline tinal symptoms (e.g., dyspepsia), increased transaminases, and phosphatase Յ ULN, or alkaline phosphatase Յ 4ϫ the ULN if nausea. transaminases were Յ ULN, or transaminases Յ 1.5ϫ ULN and Based on the novel mechanism of action of exisulind, there alkaline phosphatase Յ 2.5ϫ ULN)], and renal (serum creati- was interest in conducting combination studies with cytotoxic nine concentration Յ ULN) functions; (g) no palliative radiation agents that have complementary effects on tumor cells. Do- therapy for the prior 2 weeks; and (h) resolution of all previous cetaxel (Taxotere; Aventis Pharmaceuticals Inc., Bridgewater, therapy-related toxicity. Patients with uncontrolled brain metas- NJ) and paclitaxel, both semisynthetic taxanes, inhibit tumor tases (rapidly evolving neurologic symptoms or metastases that cell growth through microtubule stabilization. Interfering with needed specific treatment before systemic chemotherapy) and microtubules leads to activation of the JNK/stress-activated those with significant medical conditions (e.g., uncontrolled protein kinase signaling pathway, resulting in apoptosis (14). hypertension, heart disease, or diabetes mellitus) were excluded. Thus, the hypothesis was that docetaxel, paclitaxel, and exisu- Female patients of child-bearing age were required to have a lind converge at JNK/stress-activated protein kinase activation negative pregnancy test before study entry and were required to and could combine to potentiate apoptosis. This was assessed in be on adequate birth control while on study. Informed consent vitro when exisulind was combined with paclitaxel and demon- was obtained according to federal and institutional guidelines. strated synergistic effects against six lung cancer cell lines Drug Administration. Exisulind was started on day 3 of regardless of drug resistance phenotype (15). These effects were the first treatment cycle and continued without interruption then studied in vivo. In an orthotopic model of NSCLC (A549) throughout the remainder of the study period. The starting dose in athymic nude rats, the efficacy of exisulind was studied alone was 150 mg orally twice daily, with subsequent cohorts receiv- and in combination with docetaxel (16). One week after tumor ing 200 mg (cohort 2) and 250 mg twice daily (cohorts 3 and 4). implantation, rats were given either (a) oral exisulind at 25 or 50 The dose-escalation increments of exisulind were based on prior mg/kg/d, (b) docetaxel given intraperitoneally at 2.5 or 5 mg/kg weekly for 4 weeks, or (c) exisulind and docetaxel in four studies, with the MTD of 250 mg twice daily (BID) designated combinations of the doses listed above. After 80 days, the as the target dose for the combination, whereas 150 mg BID combination arm of exisulind (50 mg/kg/d) with docetaxel (5 (60% of single-agent MTD) was thought to be a safe starting mg/kg) resulted in a significant improvement of survival rate dose. Docetaxel was administered intravenously over 30 min- (60%) compared with control animals (P Ͻ 0.0004) or animals utes on days 1, 8, and 15 of a 28-day treatment cycle. Patients treated with either drug alone. received 8 mg of dexamethasone intravenously before do- 2 Based on these encouraging preclinical results, a phase I cetaxel. The dose of docetaxel was 30 mg/m /week, except for 2 dose-escalation and pharmacokinetic study of exisulind and the fourth and final cohort, which received 36 mg/m /week. docetaxel was initiated in patients with advanced solid tumors Patients could receive docetaxel and exisulind concurrently for using weekly intravenous docetaxel and daily oral exisulind. a maximum of six cycles. Patients who demonstrated disease The main objectives of this study were as follows: (a)to stability in the absence of intolerable toxicity were allowed to characterize the toxicities of docetaxel and exisulind when ad- remain on single-agent exisulind after six cycles of the combi- ministered concurrently over a 4-week cycle; (b) to determine nation. Toxicities were graded according to the National Cancer the MTD and recommended dose for subsequent phase II trials; Institute CTC Version 2.0. DLT was defined as grade 4 neutro- (c) to characterize the pharmacokinetic behavior of exisulind penia for Ͼ5 days, grade 4 neutropenia complicated by fever, and docetaxel in combination in addition to describing any drug grade 4 thrombocytopenia, or grade 3 nonhematologic toxicity interactions; and (d) to seek preliminary evidence of antitumor (excluding alopecia or nausea and vomiting in the absence of activity of this combination in patients with advanced solid optimal medical management) occurring in the first cycle. Pa- malignancies. tients were prohibited from taking NSAIDS (other than that described in Patient Selection) for the duration of the study. The dose-escalation scheme was to proceed with three PATIENTS AND METHODS patients per cohort until one patient experienced DLT, in which Patient Selection. Patients with biopsy-confirmed solid case three additional patients were added to the cohort. If more malignancies refractory to standard therapy or for whom no than one of six patients in a given cohort experienced DLT, the

MTD was exceeded, and the prior dose level was declared the MS). The development of a LC/MS/MS assay for docetaxel was MTD. A minimum of six patients were to be treated at the MTD. necessitated by the fact that later sampling points (8–24 hours) If an elevated bilirubin or any grade 3 hepatotoxicity oc- were often lower than the limit of quantitation for the HPLC/ curred, exisulind and docetaxel dosing was interrupted. When ultraviolet-based assay. The LC/MS/MS-based assay had a the patient had recovered to a grade 1 toxicity, exisulind could lower limit of quantitation of about 10-fold lower than any of be restarted at the next lower dose level, and the docetaxel dose the samples collected at the later time points, and thus full was reduced by 25%. The dose of docetaxel was also reduced by pharmacokinetic time courses could be evaluated. The initial 25% for an elevated ALT to 1.6ϫ the ULN, and by one dose docetaxel analysis was performed by HPLC with ultraviolet level for any grade 3 or greater hematologic toxicity. detection following solid-phase extraction from plasma as de- Exisulind was supplied by OSI Pharmaceuticals Inc., scribed previously (17). The LC/MS/MS assay developed and succesor-in-interest to Cell Pathways Inc. (Melville, NY) as used in this study has been detailed in a separate report (18). In 100- or 150-mg gelatin capsules. Docetaxel was purchased brief, quantitative analyses were performed with a PE Sciex commercially from Aventis Pharmaceuticals Inc. API-3000 triple quadrupole mass spectrometer with a turbo Pretreatment and Follow-up Studies. Complete medi- ionspray source, interfaced with a PE Sciex 200 Autosampler cal histories, physical examinations, concurrent medication pro- and HPLC system (Applied Biosystems, Framingham, MA). files, assessments of performance status, and routine laboratory The liquid chromatography system consisted of a Luna 5-cm studies were performed before study initiation and weekly dur- C-18 column (2-mm internal diameter; Phenomenex, Torrance, ing treatment. Routine laboratory studies included a complete CA), and the mobile phase was isocratic 90% acetonitrile in 10 blood count, differential white blood cell count, electrolytes, mmol/L ammonium acetate. The flow rate was 200 ␮L/min, and blood urea nitrogen, serum creatinine, glucose, total protein, the injection volume was 20 ␮L. The mass spectrometry settings albumin, calcium, phosphate, uric acid, lactate dehydrogenase, used were as follows: turbo ion spray temperature of 300°C, alkaline phosphatase, total bilirubin, ALT, and AST. A urinal- needle voltage of 3,500 V, declustering potential of 25 V, focus ysis and a serum pregnancy test (as appropriate) were performed plate of 175 V, collision energy of 20 V, needle position of 7,

before treatment. Pretreatment studies also included an electro- and collision gas (N2) density set at 10. The instrument was cardiogram, relevant radiographic studies to evaluate all meas- operated in the selective reaction monitoring mode (positive urable and assessable sites of disease, and an assessment of all ion), monitoring the ion transitions from m/z 808 to 226 for relevant tumor markers. Patient adherence was monitored by pill docetaxel and m/z 854 to 286 for paclitaxel (internal standard). counts and dosing diaries. Radiographic evaluations for disease The peaks coeluted at 1.6 minutes with a total analysis time of status assessment were repeated after every other course. A 4 minutes. Blank pooled plasma samples were spiked with complete response was defined as the disappearance of all docetaxel (LKT Laboratories, St. Paul, MN) and paclitaxel measurable and assessable disease for at least two measure- (internal standard; Sigma, St. Louis, MO) to yield final concen- ments performed at least 4 weeks apart without worsening of trations in plasma ranging from 0.25 to 1,000 nmol/L and disease-related symptomatology or declining performance sta- extracted as described previously to generate a standard curve. tus. A partial response required at least a 50% reduction in the After the development of the LC/MS/MS assay, docetaxel sum of the product of the bidimensional measurements of all was determined in samples using the method described above lesions documented by at least two measurements separated by (18). The accuracy of the docetaxel assay was determined by a minimum of 4 weeks. Any increase in the size of a lesion by preparation of standard plasma samples at 7.5, 25, and 50 Ն25% or the appearance of a new lesion was considered disease nmol/L. The accuracy and precision % relative standard devia- progression. Patients continued on treatment in the absence of tion (%RSD) observed were 94.4 Ϯ 3.8% at 7.5 nmol/L, 97.3 Ϯ disease progression or intolerable toxicity. 2.0% at 25 nmol/L, and 98.9 Ϯ 2.0% at 50 nmol/L. Exisulind Pharmacokinetic Sampling and Assay. To study the concentrations in plasma were determined by HPLC with de- pharmacokinetics of docetaxel, whole blood samples were ob- tection at 329 nm using sulindac as an internal standard as tained from an indwelling venous catheter distant from the site described previously (19). The intra- and interday variability for of drug infusion. On days 1 and 8 of the first treatment course, the exisulind assay was 3.4% and 4.7%, respectively, as deter- samples were collected before the infusion; at the end of the mined in spiked plasma samples. infusion; at 5, 10, 20, and 30 minutes after the end of the infu- Pharmacokinetic/Pharmacodynamic Analysis. Individual sion; and at 1, 2, 4, 8, 24, and 48 h after the end of the infusion. plasma concentration time-course measurements for docetaxel The 48 hour sample was drawn before the administration of were analyzed by noncompartmental analysis using WinNonlin exisulind on day 3. Exisulind concentrations were measured at Version 3.0 (Pharsight Corp., Mountain View, CA). Area under baseline and then measured weekly, before dosing, on days 8, the concentration-time curve (AUC) extrapolated to infinity

15, and 22 of course 1 and on day 1 of course 2. The samples (AUCinf) was calculated using the linear trapezoidal method to were collected in tubes containing EDTA, inverted several the last desired point (24 hours) and then dividing the last ␭ times, and immediately placed on ice. Within 15 minutes of measured concentration by the terminal rate constant ( z), blood collection, samples were centrifuged at room temperature which was determined from the slope of the linear, terminal Ϫ ␭ to separate plasma and then frozen at 70°C until needed for phase of the curve. The terminal half-life (t1/2 ) was calculated ␭ analysis. based on the terminal rate constant by 0.693/ z. The AUCinf and ␭ Plasma was assayed for docetaxel by high-performance t1/2 values are only reported for those patients for whom the liquid chromatography (HPLC) with ultraviolet detection and by docetaxel concentration was above the lower limit of quantita- liquid chromatography-tandem mass spectrometry (LC/MS/ tion for the assay at 24 hours, allowing for a full set of values for

Table 1 Patient characteristics which included three grade 1 elevations and one grade 2 eleva- Characteristic No. tion. Other gastrointestinal toxicities included anorexia, nausea, No. of patients 20 Total no. of assessable courses 70 vomiting, dyspepsia, diarrhea, and epigastic pain. The majority No. of courses/patient of patients experienced some degree of nausea, vomiting, and Median 2 anorexia. Overall, 24% of courses were accompanied by mild to Range 1–12 moderate nausea. Two patients experienced three courses of Sex grade 3 nausea. Vomiting occurred during 15 of 70 courses Male 12 Female 8 (21%), with two patients experiencing two courses (3%) of Age (y) grade 3 vomiting. No patients required hospitalization for symp- Median 59 tom control, and most were adequately managed with standard Range 35–77 antiemetic therapy. Anorexia was generally mild (grade 1–2), ECOG performance status 04occurring in 14 of the 70 courses (20%), although one patient 115did experience grade 3 toxicity that improved without dose 21modification. Diarrhea occurred in 14 of 70 courses (20%), with Previous treatment grade 2 diarrhea occurring in four patients for a total of 4 Chemotherapy courses. Dyspepsia was evident in 13 of 70 courses (19%), and Ͻ3 prior regimens 13 Ͼ3 prior regimens 7 epigastric pain was encountered in 10 of 70 courses (25%). Radiotherapy 14 Symptoms were generally relieved with proton pump inhibitor None 1 administration. One patient developed biliary pancreatitis during Tumor type the second course of treatment and required hospitalization. This Colorectal 3 NSCLC 3 patient exhibited the highest AUC and lowest clearance of Unknown primary 2 docetaxel and was taken off study for grade 3 toxicity. Two Other* 12 patients received exisulind for 10 and 12 cycles. One of the Abbreviation: ECOG, Eastern Cooperative Oncology Group. patients developed peptic ulcer disease after receiving 10 cycles, * Other tumor types include one each of renal cancer, gastric whereas the other patient did not develop any cumulative tox- cancer, prostate cancer, breast cancer, adenoid cystic cancer, nasopha- icity. None of the grade 3 gastrointestinal toxicities occurred in ryngeal carcinoma, melanoma, small-cell lung cancer, neuroendocrine the first cycle and hence were not considered dose-limiting. cancer, hepatocellular, leiomyosarcoma, and carcinoid. Hematologic Toxicity. Grade 1 and 2 anemia was the most common hematologic toxicity that resulted from the combination. Fourty-four of 70 (63%) courses in the study were accompanied by mild to moderate anemia. The anemia appeared parameter calculations. The relationship between toxicity and slightly more severe in the group treated with the highest dose ␹2 exposure to exisulind was assessed using a test, in which the of docetaxel (36 mg/m2). Three patients experienced four sample size of 19 evaluable patients achieved 86% power to courses (6%) of neutropenia. One of the three patients required detect an effect size of 0.7 with 1 degree of freedom and a dose modification of the docetaxel secondary to grade 2 and 3 significance level of 0.05. neutropenia. All of the patients with documented neutropenia initially received the highest dose of docetaxel (36 mg/m2/ RESULTS week). Only one patient experienced two courses of grade 1 Twenty patients received 70 courses of treatment with thrombocytopenia, which did not require dose modification. exisulind and docetaxel. Patient characteristics are depicted in Other Toxicities. Fatigue was the other principal (occur- Table 1. All but one patient had received prior therapy, and the ring in Ͼ20% of courses) toxicity of exisulind and docetaxel in majority of patients had a performance status of 0 or 1. A wide variety of tumor types were represented among the 20 patients. Table 2 depicts the dose-escalation scheme. Ten courses of Table 2 Dose-escalation scheme treatment did not include docetaxel because two patients had received six cycles of combination therapy. All patients were Exisulind Docetaxel No. of No. of 2 evaluable for toxicity, and there were no DLTs at any level. No (mg BID) (mg/m /wk) patients cycles patients required dose modification of exisulind secondary to 150 30 3 13* toxicity, but two patients required dose reduction of docetaxel 200 30 3 11† 250 30 8 24‡ due to grade 3 neutropenia and grade 3 asthenia, respectively. 250 36 6 22§ Gastrointestinal Toxicity. Seventeen of 70 courses Total 20 70 (24%) were accompanied by mild to moderate AST elevations * Four courses were administered without docetaxel because the (Table 3). Mild to moderate elevations of ALT or alkaline patient had received six cycles of combination therapy. phosphatase were evident in 15 of 70 courses (21%) each. Only † Six courses were administered without docetaxel because the one of the patients who developed elevation of alkaline phos- patient had received six cycles of combination therapy. ‡ One patient had docetaxel dose reduced to 22.5 mg/m2 for one phatase had metastatic disease to the bone. All but one of the course secondary to grade 3 diarrhea. patients with AST or ALT elevations was receiving 500 mg/d § One patient in this cohort had two courses with lower doses of exisulind. Serum bilirubin was normal in all but four courses, docetaxel secondary to grade 2 and 3 myelosuppression.

Table 3 Gastrointestinal toxicities of exisulind and docetaxel Elevated AP Elevated AST Nausea Vomiting Dyspepsia Epigastric pain Exisulind Docetaxel No. of courses (mg/d) (mg/m2) (patients) G1 G2 G3 G1 G2 G3 G1 G2 G3 G1 G2 G3 G1 G2 G3 G1 G2 G3 300 30 13 (3) 0 0 0 0 0 0 3 2 1 2 1 0 0 0 0 1 2 1 400 30 11 (3) 0 0 0 1 0 0 4 0 2 3 1 2 2 3 2 0 1 0 500 30 24 (8) 7 3 1 9 1 0 2 2 0 3 0 1 0 0 0 0 3 0 500 36 22 (6) 1 4 0 6 0 0 4 0 0 2 0 0 4 2 0 1 1 0 G1–G3 represent National Cancer Institute CTC grades 1–3. The number of cycles in which each toxicity was observed is indicated in the body of the table. Abbreviation: AP, alkaline phosphatase.

this study. Fatigue was reported in 34 of 70 courses (49%) and Fig. 4. There was no relationship between the plasma concen- was not dose-related. Three patients experienced grade 3 fa- trations of exisulind and the administered dose. Inter- and in- tigue, which generally corresponded to a decline in Eastern trapatient variability was substantial. Interpatient variability Cooperative Oncology Group performance status and disease among the exisulind trough concentrations at the 150-, 200-, and progression. One patient experienced a grade 4 anaphylactic 250-mg dose levels was 38.5%, 31.5%, and 63.8%, respectively, reaction several minutes after receiving the docetaxel infusion, whereas the intrapatient variability was 36.7 Ϯ 20.0% across all which responded well to steroids and antihistamines. Although dose levels. There did not appear to be any impact of either dose there was an apparent temporal correlation with the docetaxel of docetaxel on the plasma concentrations of exisulind (data not infusion, it was noted that this patient also had the highest shown). plasma exisulind concentration at the time of the docetaxel Pharmacodynamic Analysis. Interestingly, there was a infusion. significant correlation between the gastrointestinal toxicities Antitumor Activity. No partial or complete responses observed and exisulind plasma concentrations (P ϭ 0.0108; were observed during the study period. Seven patients main- Tables 5 and 6). Independent of the exisulind dose, patients who tained stable disease for 3 to 10 cycles of therapy. One patient demonstrated higher plasma concentrations of exisulind experi- with prostate cancer and another with carcinoid remained on enced more severe gastrointestinal toxicities such as epigastric study with stable disease for 12 and 10 cycles, respectively. pain, dyspepsia, nausea, vomiting, and an increase in alkaline Pharmacokinetic Studies. Pharmacokinetic samples for phosphatase from baseline. Among the nine patients whose docetaxel were obtained from 19 patients on day 1 and 18 mean concentrations of exisulind exceeded 15 ␮mol/L, seven patients on day 8. Docetaxel pharmacokinetic parameters were experienced grade 2 or greater toxicities related to the gastro- analyzed using noncompartmental methods (Table 4). Mean intestinal tract, one had a grade 3 anaphylactic reaction, and one plasma concentration-time profiles on days 1 and 8 after admin- experienced grade 2 tinnitus. Of 10 patients with mean plasma 2 istration of docetaxel doses of 30 and 36 mg/m as a 30-minute concentrations of Ͻ15 ␮mol/L, 3 patients had grade 2 or greater intravenous infusion are shown in Fig. 1. The pharmacokinetic gastrointestinal toxicities. There was no relationship between profile of docetaxel was compared on days 1 and 8 for patients the increased plasma concentrations of exisulind and pretreat- 2 receiving 30 and 36 mg/m doses of docetaxel (Figs. 1 and 2). ment renal function, pretreatment hepatic function, or adminis- ␭ The AUCinf and t1/2 values are displayed in Figs. 2 and 3, tered dose of exisulind. respectively, and the mean values were not statistically different between days 1 and 8 using Student’s t test. The pharmacokinetic samples for exisulind were obtained DISCUSSION from 18 patients on day 8, 17 patients on day 15, 15 patients on Exisulind is a selective proapoptotic agent that inhibits day 22, and 17 patients on day 1 of cycle 2. Mean steady-state cGMP-phosphodiesterases 2 and 5, resulting in a sustained concentrations of exisulind at all dose levels are illustrated in increase in cGMP and activation of PKG (1). Apoptosis by this

Table 4 Docetaxel pharmacokinetic parameters as calculated using noncompartmental methods Treatment

Exisulind Docetaxel No. of Cmax (SD) AUC (SD) CL (SD) Vss (SD) t1/2 (SD) (mg/d) (mg/m2) Day patients (nmol/L) (nmol/L ϫ h) (L/h/m2) (L/m2) (h) 300 30 1 3 770 (144) 855 (76) 54 (17) 377 (220) 12 (6) 8 67 (6) 276 (159) 201 (115) 1315 (97) 8 (4) 400 30 1 3 542 (85) 757 (107) 50 (8) 519 (128) 13 (2) 8 227 (197) 476 (318) 128 (84) 1199 (326) 12 (6) 500 30 1 7 996 (305) 1220 (680) 38 (15) 211 (81) 10 (3) 8 1278 (843) 1458 (1084) 43 (33) 177 (73) 10 (4) 500 36 1 6 797 (301) 958 (311) 52 (19) 460 (273) 12 (5) 8 770 (344) 910 (481) 66 (35) 330 (254) 10 (10)

Abbreviations: Cmax, maximum concentration; CL, clearance; Vss, volume at steady state.

trointestinal in nature and included mild to moderate nausea, vomiting, abdominal pain, diarrhea, anorexia, dyspepsia, and elevated liver function enzymes. Neutropenia was encountered in four cycles in three patients treated with the highest dose of docetaxel (36 mg/m2), requiring dose reduction in one patient. Therefore, the cohort combining a lower dose of docetaxel (30 mg/m2) was also expanded and evaluated. At the third dose level (30 mg/m2 docetaxel and 500 mg/d exisulind), no patient experienced neutropenia. Anemia was present in the majority of patients but was only mild to moderate in severity. Based on these results, the recommended phase II dose for the combination is 250 mg BID of exisulind and 36 mg/m2 docetaxel weekly for 3 weeks every 4 weeks, with reduction of docetaxel to 30 mg/m2/week, if required for myelosuppression.

Fig. 1 Docetaxel plasma concentration versus time data for 30 (A) and 36 mg/m2 (B) doses on day 1 (open symbols) and day 8 (closed symbols). Points represent the mean Ϯ SD of measured values for individuals at that time after dosing.

agent is preceded by the proteasomal degradation of ␤-catenin and activation of JNK (2). Preclinical studies demonstrated synergistic effects when exisulind was combined with docetaxel or paclitaxel against human lung and prostate cancer cell lines in vitro (15). One hypothesis for the observed synergy was that both agents converge at the activation of JNK to promote apoptosis (2, 14). In an orthotopic model of NSCLC in athymic nude rats, an increase in apoptosis and survival was demonstrated when exisulind was combined with docetaxel, compared with either drug given alone (16). This phase I and pharmaco- logical study was based on these promising in vivo results and designed to evaluate the feasibility and safety of the adminis-

tration of twice-daily exisulind and weekly docetaxel in patients Fig. 2 Comparison of AUCinf on days 1 and 8 for patients receiving 30 2 with advanced solid tumors. (A) and 36 mg/m (B) docetaxel. Points represent individual calculated AUCinf values with matched symbols for each patient. The mean AUCinf The results of this study demonstrate that the combination values on days 1 and 8, represented by the horizontal line (error bars, of exisulind and docetaxel is tolerable, and no DLTs occurred SD), are not different between days 1 and 8 as determined by Student’s during the first cycle. The principal toxicities noted were gas- t test.

23). In combination with capecitabine, the 125-mg BID (250 mg/d) dose of exisulind was recommended for phase II testing because only 7 of 13 patients could tolerate the higher dose of 250 mg BID of exisulind. Interestingly, in this study, the DLTs were characteristic of capecitabine and did not appear to be more prominent with the combination (22). However, at the higher dose of exisulind, the rate of grade 2/3 nausea or vomiting was 21%, whereas in the present study, it was only 11% at the 250 mg BID dose levels of exisulind. These results imply that there may be somewhat synergistic effects between exisulind and other oral agents, such as capecitabine, that are associated with similar gastrointestinal toxicities. The mean plasma concentrations of exisulind at the highest dose level ranged from 5 to 47 ␮mol/L and were not affected by docetaxel dosing. These concentrations are substantially lower

than the range of IC50 values that have been reported in previous in vitro studies of exisulind against human tumor cell lines, which ranged from 100 to 300 ␮mol/L (15, 24, 25). Exisulind plasma concentrations displayed substantial inter- and intrapatient variability. There was no relationship between the plasma concentration of exisulind and the administered dose or any other pretreatment condition including renal or hepatic function, weight, height, age, or gender. Interestingly, the majority of exisulind undergoes biliary excretion with no evidence of me- tabolism by the cytochrome P450 isoenzyme system, which can be a major source of interpatient variability (19, 26). Thus, the etiology of this variability may be related to hepatobiliary transporters that have also been implicated in the gastrointestinal toxic effects of a range of agents including NSAIDS, irinotecan,

and methotrexate (27, 28). By contrast, the mean Cmax concentrations of docetaxel achieved at the 30 and 36 mg/m2 dose levels ranged from 67 to 1,278 nmol/L, which was significantly ␭ Fig. 3 Comparison of terminal half-lives (t1/2 ) on days 1 and 8 for higher than the IC50 (2.5 nmol/L) reported in the preclinical patients receiving 30 or 36 mg/m2 docetaxel. Points represent individual ␭ calculated t1/2 values with matching symbols for individual patients. ␭ The mean t1/2 values on days 1 and 8, represented by the horizontal line (error bars, SD), are not different between days 1 and 8 as determined by Student’s t test.

Gastrointestinal toxicities have been reported in previous studies with exisulind. In studies in patients with FAP, prostate cancer, or lung cancer, the predominant toxicities were elevation of liver function enzymes, nausea, and vomiting (9, 12, 13). In this trial, they were common at all dose levels but were mostly of mild to moderate severity. Grade 1 and 2 elevation of ALT, AST, and alkaline phosphatase accompanied the higher doses of exisulind, whereas nausea, vomiting, dyspepsia, and abdominal pain were present at all dose levels. Grade 3 gastrointestinal toxicities occurred in 5 of the 19 patients at some point during therapy. One patient developed pancreatitis during the second course of treatment, and an abdominal sonogram demonstrated borderline dilation of the extrahepatic bile ducts without evidence of cholelithiasis. Previous studies have reported a chole- retic effect of sulindac and exisulind (20, 21), whereas only 2 of 45 patients with prostate cancer treated with exisulind devel- Fig. 4 Plasma exisulind levels in patients receiving 150-, 200-, and Points oped a biliary event (13). Exisulind has been evaluated in 250-mg BID doses. represent average steady-state trough levels for each individual patient. The mean values for each dose group, combination with several chemotherapeutic agents, including represented by the horizontal line (error bars, SD), are not different capecitabine and docetaxel on an every 3-week schedule (22, between dose groups as determined by analysis of variance.

Table 5 Exisulind concentration (␮mol/L) and CTC grade of toxicities related to treatment Patient Mean exisulind Epigastric Nausea/ Increase in Other toxicity Cohort no. concentration (SD) pain/dyspepsia vomiting AP grade (grade) 1 1 30 (7) 3 1 1 2 13 (5) 1 3 23 (9) 2 3 Diarrhea (3) 2 4 18 (16) 3 3 2 5 9 (4) 3 1 2 6 15 (4) 3 7 22 (7) 2 2 3 8 29 (2) 2 3 2 Lipase (4) 3 9 15 (2) 1 3 16 10 (6) 3 3 18 38 (11) 2 1 1 Hearing (2) 3 19 15 (4) Reflux (3) 3 20 47 (8) Anaphylaxis (4) 4 10 11 (3) 1 1 4 11 26 (6) 2 1 4 12 26 (17) 2 2 4 13 7 (4) 1 4 14 9 (4) 2 4 15 5 (3) 1 1 Abbreviation: AP, alkaline phosphatase.

studies of docetaxel and exisulind against the A549 NSCLC cell types studied. However, prior studies of the combination of line (16). A more sensitive LC/MS/MS method was developed exisulind and docetaxel/carboplatin or capecitabine have to assess plasma concentrations of docetaxel on a weekly sched- yielded response rates of 45% and 16% in patients with un- ule, and no apparent impact of exisulind was noted between treated metastatic NSCLC and anthracycline- and taxane-refrac- days 1 and 8 on the pharmacokinetics of docetaxel, when tory metastatic breast cancer, respectively (22, 29). These results exisulind would have achieved steady-state concentrations. suggest that combinations of exisulind and chemotherapeutic Surprisingly, a significant relationship (P ϭ 0.0016) was agents result in objective responses that are at least consistent established between the mean plasma concentrations of exisu- with that expected with chemotherapy alone. Any additional lind and the occurrence of grade 2 or greater toxicities, which benefit of exisulind may only be detected in randomized studies were predominantly gastrointestinal in nature. Although at first in these diseases. However, the fact that single-agent exisulind glance, this may appear to be a statistical anomaly, these effects, inhibited the increase in PSA overall and prolonged PSA dou- in conjunction with the interpatient variability observed, may bling time in men after prostatectomy, compared with placebo, relate to the biliary excretion of exisulind and the hepatobiliary indicates that at least in prostate cancer, the agent has independ- transport system. Clearly, future studies of exisulind and struc- ent anticancer effects (13). turally related analogs should incorporate studies to assess pa- In summary, this phase I study demonstrated that exisulind tients for single-nucleotide polymorphisms that may impact on and docetaxel may be safely administered in combination at full the function of hepatobiliary transport proteins. single-agent doses of each drug. Docetaxel (36 mg/m2, weekly) In this phase I study, no objective responses were observed and exisulind (500 mg/d) were reasonably well tolerated and are with the combination of exisulind and docetaxel, although a recommended for appropriate disease-directed studies. Future third of patients maintained stable disease for 3 to 12 cycles and studies of exisulind and other analogs, such as OSI-461, should two patients (one with prostate cancer and one with carcinoid) incorporate pharmacogenomic studies to assess the impact of remained on study for 10 and 12 cycles. Several factors could be genetic polymorphisms on the toxicity and pharmacology of responsible for this, including the inability to achieve exisulind these agents. Based on this phase I study and the preclinical data plasma concentrations of 100 to 300 ␮mol/L, the extensive prior demonstrating the additive effects of exisulind and docetaxel on therapy received by the patients, and the variability of tumor A549 NSCLC cells in vivo, a phase II study using this combination was initiated in patients with NSCLC.

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Samir E. Witta, Daniel L. Gustafson, A. Scott Pierson, et al.

Clin Cancer Res 2004;10:7229-7237.

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