Reprinted from
`Volume 19, Number 13
`
`July 1, 2001
`
`Phase I and Pharmacologic Study of OSI-774, an Epidermal Growth
`Factor Receptor Tyrosine Kinase Inhibitor, in Patients With
`Advanced Solid Malignancies
`
`ASCO_JCO
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`APOTEX EX. 1031-001
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`

`

`P h a s e I a n d P h a r m a c o l o g i c S t u d y o f O S I - 7 7 4 , a n
`E p i d e r m a l G r o w t h F a c t o r R e c e p t o r T y r o s i n e K i n a s e
`I n h i b i t o r , i n P a t i e n t s W i t h A d v a n c e d S o l i d M a l i g n a n c i e s
`
`By Manuel Hidalgo, Lillian L. Siu, John Nemunaitis, Jinee Rizzo, Lisa A. Hammond, Chris Takimoto, S. Gail Eckhardt,
`Anthony Tolcher, Carolyn D. Britten, Louis Denis, Karen Ferrante, Daniel D. Von Hoff, Sandra Silberman, and Eric K. Rowinsky
`
`Purpose: To assess the feasibility of administering
`OSI-774, to recommend a dose on a protracted, contin-
`uous daily schedule, to characterize its pharmacoki-
`netic behavior, and to acquire preliminary evidence of
`anticancer activity.
`PatientsandMethods: Patients with advanced solid
`malignancies were treated with escalating doses of
`OSI-774 in three study parts (A to C) to evaluate pro-
`gressively longer treatment intervals. Part A patients
`received OSI-774 25 to 100 mg once daily, for 3 days
`each week, for 3 weeks every 4 weeks. Part B patients
`received OSI-774 doses ranging from 50 to 200 mg
`given once daily for 3 weeks every 4 weeks to establish
`the maximum tolerated dose (MTD). In part C, patients
`received this MTD on a continuous, uninterrupted sched-
`ule. The pharmacokinetics of OSI-774 and its O-de-
`methylated metabolite, OSI-420, were characterized.
`Results: Forty patients received a total of 123 28-
`day courses of OSI-774. No severe toxicities precluded
`dose escalation of OSI-774 from 25 to 100 mg/d in part
`A. In part B, the incidence of severe diarrhea and/or
`
`THE EPIDERMAL GROWTH factor receptor (EGFR),
`
`a type I receptor tyrosine kinase (TK) involved in the
`regulation of cellular differentiation and proliferation, is
`highly expressed by many types of human cancer and is a
`rational strategic target for anticancer therapeutic develop-
`ment.1-3 The receptor is composed of three major domains:
`an extracellular ligand-binding domain, a transmembrane
`lipophilic segment, and a cytoplasmic protein TK domain.1
`After binding of epidermal growth factor (EGF), transform-
`ing growth factor alpha, and other activating ligands, the
`EGFR undergoes dimerization, which, in turn, activates the
`intrinsic protein TK via intermolecular autophosphorylation
`within its cytoplasmic domain. The tyrosine autophospho-
`rylated region functions as a binding site for cytoplasmic
`messenger proteins, which thereby initiate a series of signals
`from the cytoplasm to the nucleus that culminate in DNA
`synthesis and cell division.2
`The notion of targeting EGFR TK as a therapeutic
`strategy against cancer is supported by experimental evi-
`dence indicating that
`the dysregulation of the EGFR-
`mediated signal
`transduction pathways plays a role in
`tumorigenesis and cancer cell proliferation.3 The clinical
`relevance of this strategy is further substantiated by the
`overexpression of EGFR in head and neck, breast, brain,
`lung, cervical, bladder, gastrointestinal, renal, and other
`
`cutaneous toxicity was unacceptably high at OSI-774
`doses exceeding 150 mg/d. Uninterrupted, daily ad-
`ministration of OSI-774 150 mg/d represented the MTD
`on a protracted daily schedule. The pharmacokinetics
`of OSI-774 were dose independent; repetitive daily
`treatment did not result in drug accumulation (at 150
`mg/d [average]: minimum steady-state plasma con-
`centration, 1.20 ⴞ 0.62 ␮g/mL; clearance rate, 6.33 ⴞ
`6.41 L/h; elimination half-life, 24.4 ⴞ 14.6 hours; vol-
`ume of distribution, 136. 4 ⴞ 93.1 L; area under the
`plasma concentration-time curve for OSI-420 relative to
`OSI-774, 0.12 ⴞ 0.12 ␮g/h/mL).
`Conclusion: The recommended dose for disease-di-
`rected studies of OSI-774 administered orally on a daily,
`continuous, uninterrupted schedule is 150 mg/d. OSI-774
`was well tolerated, and several patients with epidermoid
`malignancies demonstrated either antitumor activity or
`relatively long periods of stable disease. The precise con-
`tribution of OSI-774 to these effects is not known.
`J Clin Oncol 19:3267-3279. © 2001 by American
`SocietyofClinicalOncology.
`
`epithelial malignancies and the evidence indicating that
`EGFR overexpression is a determinant of tumor aggressive-
`ness.3 EGFR activation can be inhibited by anti-EGFR anti-
`bodies, which block binding of endogenous ligands, as well as
`
`From the Institute for Drug Development, Cancer Therapy and
`Research Center, and the University of Texas Health Science Center at
`San Antonio, San Antonio, TX; U.S. Oncology, and Baylor University
`Medical Center, Dallas, TX; and Pfizer Pharmaceuticals, Inc, Groton, CT.
`Submitted November 30, 2000; accepted March 27, 2001.
`Supported in part by a grant from Pfizer, Inc. Some patients were
`treated at the Frederic C. Barter General Clinical Research Unit of the
`Audie Murphy Veterans Administration Hospital, which is supported
`by NIH grant no. MO1 RR01346. M.H. is supported in part by grant no.
`PF 97 52273279 from the Ministerio de Educacion y Cultura, Spain,
`and is the recipient of a National Cancer Institute-European Organi-
`zation for Research and Treatment of Cancer Fellowship Award.
`OSI-774 (OSI Pharmaceuticals, Uniondale, NY) was formerly known as
`CP-358,774 (Pfizer Global Research and Development, Groton, CT).
`Presented in part at the Thirty-Fifth Annual Meeting of the American
`Society of Clinical Oncology, Atlanta, GA, May 15-19, 1999.
`Address reprint requests to Manuel Hidalgo, MD, Department of
`Medicine, The University of Texas Health Science Center at San
`Antonio, 7703 Floyd Curl Dr, Mail Code 7884, San Antonio, TX
`78229; email: manuelh@oncology.uthscsa.edu.
`© 2001 by American Society of Clinical Oncology.
`0732-183X/01/1913-3267
`
`Journal of Clinical Oncology, Vol 19, No 13 (July 1), 2001: pp 3267-3279
`
`3267
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`APOTEX EX. 1031-002
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`3268
`
`Fig 1. Structure of OSI-774.
`
`small molecules, which results in the inhibition of downstream
`components of the EGFR pathway.4 Because the activity and
`function of EGFR TK are necessary for receptor signaling, the
`development of specific small molecules that inhibit EGFR TK
`represents a logical therapeutic approach.5,6
`Recently several classes of agents have been shown to be
`highly selective for EGFR TK and possess impressive
`preclinical activity against tumors that express EGFR.5,6
`Nanomolar concentrations of
`the quinazoline OSI-774
`([6,7-bis(2-methoxy-ethoxy)-quinazolin-4-yl]-[3-ethylphe-
`nyl]amine; OSI Pharmaceuticals, Uniondale, NY; formerly
`known as CP-358,774; Pfizer Pharmaceuticals, Inc, Groton,
`CT) (Fig 1) inhibit the activity of purified EGFR TK and
`EGFR autophosphorylation in intact tumor cells, with 50%
`inhibitory concentration values of 2 and 20 nmol/L, respec-
`tively.6 OSI-774 is 1,000-fold more potent against EGFR
`TK than most other human kinases, including c-src and
`insulin receptor TK.6 In studies of Fisher rat embryo
`fibroblasts, nanomolar concentrations of OSI-774 inhibited
`cell division induced by EGF, whereas 1,000-fold higher
`drug concentrations were required to block cell division
`stimulated by other growth factors. Nanomolar concentra-
`tions of OSI-774 also were demonstrated to inhibit growth
`of various EGFR-expressing cancers in vitro, which is
`associated with cell cycle arrest in G1 and apoptosis.6 In
`addition, treatment of mice that bear human HN5 head and
`neck carcinoma xenografts with OSI-774 profoundly inhib-
`ited tumor growth, with a 50% effective dose of 9.2
`mg/kg/d.7 In HN5 xenografts, OSI-774 inhibited intratu-
`moral EGFR autophosphorylation with 50% effective doses
`of 9.2 and 9.9 mg/kg after intraperitoneal and oral admin-
`istration, respectively. Maximum (90%) inhibition of EGFR
`autophosphorylation was evident 1 hour after administration
`of 100 mg/kg orally, EGFR autophosphorylation was re-
`duced by 75% to 85% for at least 12 hours, and complete
`recovery was noted by 24 hours after treatment.7
`Toxicology studies in both rodents and dogs revealed
`negligible toxicity after protracted daily administration of
`OSI-774 doses up to 15 mg/kg/d. At higher doses, emesis,
`
`HIDALGO ET AL
`
`gastric distention, and transient elevations in serum biliru-
`bin occurred. Two of eight dogs treated with protracted
`daily OSI-774 doses in excess of 50 mg/kg/d developed
`edema, ulceration, and perforation of the cornea, and three
`developed azotemia. In preclinical pharmacology studies,
`OSI-774 was highly bioavailable (approximately 80%), and
`peak plasma concentrations were achieved within 2 hours
`after oral treatment. The agent also was demonstrated to be
`highly bound to plasma proteins (90% to 95%) and predom-
`inantly metabolized by the P450 microsomal isoenzyme
`CYP1C to anO-demethylated active metabolite (OSI-420).
`Dose-dependent pharmacokinetics were noted at OSI-774
`doses that ranged from 2 to 50 mg/kg/d, but nonlinear
`pharmacokinetics were observed at higher doses.8
`OSI-774 also was administered to healthy human volun-
`teers before evaluations in cancer patients.8 Administration
`of a wide range of single oral doses of OSI-774 (10 to 1,000
`mg) resulted in mild to moderate toxicities,
`including
`headache and a mild, diffuse, erythematous rash at the
`highest dose. However, all patients treated continuously
`with OSI-774 400 mg/d,
`in two divided daily doses,
`developed a severe papulopustular dermatitis that involved
`the face, scalp, chest, back, and arms. As a result, treatment
`was discontinued in all patients after a maximum of nine
`doses. The rash resolved slowly thereafter. Other,
`less
`profound effects included diarrhea, mucositis, and transient
`elevation of hepatic transaminases. Pharmacokinetic studies
`revealed drug accumulation with protracted daily treatment,
`which was not predicted from single-dose studies.
`The novel mechanism of action of OSI-774 as an EGFR
`TK inhibitor, and its impressive preclinical antitumor activ-
`ity, served as the impetus for its clinical development. This
`phase I and pharmacokinetic study sought to evaluate the
`feasibility of oral OSI-774 administration on a protracted,
`continuous daily schedule to patients with advanced solid
`malignancies. The principal objectives of the study were to
`(1) determine the principal toxicities of OSI-774 adminis-
`tered on a protracted, continuous daily schedule, (2) deter-
`mine the maximum tolerated dose (MTD) and recommend a
`dose for subsequent disease-specific evaluations, (3) char-
`acterize the pharmacokinetic behavior of OSI-774, and (4)
`seek preliminary evidence of antitumor activity.
`
`PATIENTS AND METHODS
`
`Eligibility
`Patients with histologically documented advanced solid malignan-
`cies refractory to conventional
`therapy or for whom no effective
`therapy existed were candidates for this study. Eligibility criteria also
`included the following: (a) age 18 years or older; (b) Karnofsky
`performance status higher than 70% (capable of self-care); (c) life
`expectancy greater than 12 weeks; (d) no previous chemotherapy,
`
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`APOTEX EX. 1031-003
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`PHASE I TRIAL OF OSI-774
`
`radiation therapy, or major surgical procedures within 4 weeks of entry
`onto the study; (e) adequate hematopoietic (absolute neutrophil count
`ⱖ 1,500/␮L, hemoglobin level ⱖ 9.0 g/dL, and platelet count ⱖ
`100,000/␮L), hepatic (total bilirubin concentration ⱕ 1.5 mg/dL; AST
`and ALT levels ⱕ two times the upper normal limit [ⱕ five times the
`upper normal limit for patients with liver metastases]), and renal
`(creatinine concentration ⱕ 1.5 mg/dL or creatinine clearance ⱖ 50
`mL/min) functions; (f) no active infection or other coexisting medical
`problems of sufficient severity to limit compliance with the study; and
`(g) no malabsorption syndrome or any other disorder that would affect
`gastrointestinal absorption. All patients gave written informed consent
`in accordance with federal and institutional guidelines before treatment.
`Drug Administration
`OSI-774 was supplied by Pfizer Research Institute (Groton, CT) as
`25-mg and 100-mg tablets. The agent was administered orally with at
`least 200 mL of water in the morning before breakfast. Subjects fasted
`for at least 2 hours before and 1 hour after treatment. The study was
`conducted in three parts in successive cohorts of new patients; the main
`objective was assessment of feasibility and toxicities, as well as
`determination of a recommended phase II dose of OSI-774 adminis-
`tered daily on a continuous, uninterrupted daily schedule.
`In the first part of the study (part A), OSI-774 was administered daily
`for 3 days each week for 3 weeks. Courses were repeated every 4
`weeks. The starting dose of OSI-774 was 25 mg, which was equivalent
`to one third of the toxic dose low of a single dose of the agent in healthy
`volunteers. At least three new patients each were treated successively
`with OSI-744 at dose levels of 25 mg/d, 50 mg/d, and 100 mg/d. In part
`B of the study, patients were treated with OSI-774 daily for 3 weeks
`every 4 weeks. In course 1 only, patients received a single dose of the
`agent on day 1, which was followed by a 2-day washout period for
`pharmacokinetic studies. Patients resumed treatment on day 4. The
`starting dose of OSI-744 in part B was to be 50 mg, provided that 100
`mg was determined to be safe in part A. Dose escalation was to proceed
`in cohorts of at least three patients each in increments of 100%,
`spanning doses levels of 50 mg/d, 100 mg/d, and 200 mg/d.
`Thereafter, dose escalation was to be in increments of 200 mg/d in
`separate cohorts of new patients. Intermediate dose levels could be
`established to more precisely define the MTD. Once the MTD was
`established in part B, patients were treated with OSI-774 at this dose
`on a continuous, uninterrupted schedule (part C), and each 28-day
`period was considered one course of treatment. There was to be no
`further dose escalation in part C.
`Successive cohorts of at least three patients were treated at each dose
`level in parts A, B, and C. If any patient experienced dose-limiting
`toxicity (DLT) during course 1, as many as three additional patients
`were treated. The MTD was defined as the highest dose level at which
`less than two of six new patients experienced DLT in course 1. DLT
`was defined as any grade 3 or 4 hematologic or nonhematologic
`toxicity, because the principal study objective was to evaluate the
`feasibility of protracted daily administration. Nausea, vomiting, and
`diarrhea of grade 3 severity were considered DLT, provided that
`patients had received optimal antiemetics and antidiarrheal premedica-
`tion and management, and elevations of hepatic transaminases were a
`DLT if grade 3 toxicity lasted longer than 7 days. Toxicities were
`graded according to the National Cancer Institute common toxicity
`criteria (version 1.0). Patients who developed DLT could continue to
`receive treatment at a reduced dose-schedule level after recovery.
`Intraindividual escalation of the dose-schedule level was allowed,
`provided that
`the patient had completed at
`least
`two courses of
`treatment with either no or grade 1 toxicity,
`the patient did not
`
`3269
`
`experience disease progression, and the next higher dose level had
`previously been determined to be safe in accordance with the afore-
`mentioned criteria.
`
`Pretreatment and Follow-Up Studies
`Patient histories, including performance status, concomitant medi-
`cations, physical examinations, and routine laboratory evaluations,
`were obtained before treatment and weekly thereafter. Routine labora-
`tory evaluations included complete blood counts with differential,
`electrolytes, blood urea nitrogen, creatinine, glucose, total protein,
`albumin, calcium, phosphate, uric acid, alkaline phosphatase, total and
`direct bilirubin, AST, ALT, prothrombin and partial thromboplastin
`times, and urinalysis. ECGs, chest radiographs, and skin biopsies were
`performed before treatment and after the first course. Skin biopsies of
`affected areas also were performed in selected patients who developed
`cutaneous toxicity. For the routine skin biopsies, a 5-mm area of the
`back or underside of the upper arm was cleaned with an antiseptic and
`anesthetized with 2% lidocaine, and a 4-mm punch biopsy was
`obtained. The specimen was next embedded in an OCT gel (10.24%
`w/w polyvinylalcohol, 4.26% w/w polyethylenglycol, and 85.5% w/w
`non reactive ingredients), snap frozen in a methanol and dry ice bath,
`cut into 5-␮m sections, and assessed for epidermal proliferation and
`histopathologic changes by a dermatopathologist.
`Evaluations of measurable and assessable disease by appropriate
`radiologic studies, as well as an assessment of relevant tumor markers,
`were performed before treatment and after every other course. Patients
`were able to continue treatment in the absence of progressive disease.
`A complete response was scored if there was disappearance of all
`active disease on two measurements separated by a minimum period of
`4 weeks, and a partial response required at least a 50% reduction in the
`sum of the products of the bidimensional measurements of all lesions
`documented by two measurements at least 4 weeks apart. A concurrent
`increase in the size of any lesion by 25% or more, or the appearance of
`new lesions, was considered disease progression.
`
`Pharmacokinetic Sampling and Assay
`To study the pharmacokinetic behavior of OSI-774 and its O-
`demethylated metabolite (OSI-420), blood was sampled during the first
`course in all patients. The sampling scheme differed in parts A, B, and
`C of the study. In part A, in which OSI-774 was administered daily for
`3 days each week, blood was collected before treatment and at 2, 4, 6,
`8, 12, and 24 hours after treatment on day 1; before treatment and 2, 4,
`6, 8, and 12 hours after treatment on day 3; and before treatment on
`days 8 and 15. In part B, in which OSI-774 was administered daily for
`3 weeks every 4 weeks, except in course 1, in which there was a 2-day
`washout period after the first dose, blood was collected before
`treatment and 2, 4, 6, 8, 12, 24, 36, and 48 hours after treatment on day
`1; before treatment on days 4, 6, 11, and 18; and before treatment and
`2, 4, 6, 8, 12, 24, 36, and 48 hours after treatment on the last treatment
`day (day 24). In part C, in which OSI-774 was administered daily on a
`continuous schedule, blood was sampled before treatment and 2, 4, 6,
`8, 12, and 24 hours after treatment on day 1; before treatment on days
`8, 15, and 22; and before treatment and 2, 4, 6, 8, 12, and 24 hours after
`treatment on day 28. The samples were centrifuged at 3,000 rpm for 15
`minutes immediately after collection in a refrigerated centrifuge. Next,
`the plasma was transferred to a screw-capped polypropylene tube,
`which was frozen at ⫺80°C.
`Separation of the plasma samples for quantification of both OSI-774
`and OSI-420 was accomplished by reverse-phase high-performance
`liquid chromatography (HPLC) after extraction in our laboratory. After
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`APOTEX EX. 1031-004
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`3270
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`plasma samples were thawed to room temperature and vortexed, 100
`␮L of internal standard (OSI-597) solution was added to a 200-␮L
`aliquot of sample in a 15-mL polypropylene extraction tube, and 5.0
`mL of methyl t-butyl ether was added to each tube. The samples
`were then rotated for 10 minutes and centrifuged at 3,000 rpm for 5
`minutes. The supernatant was transferred to a clean 5-mL polypro-
`pylene tube and evaporated to dryness under a gentle stream of
`nitrogen at room temperature.
`The extracts were reconstituted with 200 ␮L of the mobile phase,
`filtered through an Alltech Microspin Nylon 66 microcentrifuge filter
`(Alltech Associates, Deerfield, IL), centrifuged at 6,000 rpm for 3
`minutes, and transferred to glass microinserts; 75 ␮L were then injected
`into the HPLC system. The HPLC system was equipped with a Waters
`model no. 515 isocratic solvent delivery pump (Waters Corporation,
`Milford, PA), a Waters model no. 717 refrigerated autosampler, a
`Waters model no. 2487 dual wavelength absorbance detector, and a
`Waters Symmetry (4.9 mm ⫻ 150 mm, 5 ␮m) C18 column. The flow
`rate was 1.0 mL/min, and an aliquot of 75 ␮L was injected for sample
`analysis. Detection of the compounds of interest was at 345 nm, and the
`data were collected by use of the Waters Millennium chromatography
`data collection software. OSI-774, OSI-420, and the internal standard
`were separated on a Waters Symmetry (4.9 mm ⫻ 150 mm, 5 ␮m) C18
`column. Samples were eluted isocratically at a flow rate of 1.0 mL/min
`with a mobile phase that consisted of acetonitrile and water (vol/vol,
`30/70; pH 2.40). Under these conditions,
`the retention times for
`OSI-774, OSI-420, and the internal standard were 3.2, 2.1, and 5.0
`minutes, respectively. Standard curves for OSI-774 and OSI-420 were
`prepared over a concentration range of 10.0 to 2,500 ng/mL by the
`addition of known amounts of OSI-774, OSI-420, and internal standard
`to appropriate volumes of human plasma. Plasma concentrations were
`determined by plotting the plasma OSI-774 and OSI-420 peak areas to
`that of the internal standard versus known concentrations. The lower
`limit of assay quantification, which was based on the extraction of
`200-␮L plasma samples, was 10 ng/mL for both OSI-774 and OSI-420.
`The performance of the assay was monitored using quality control
`(QC) samples at 20, 200, and 2,000 ng/mL. On each day of analysis,
`duplicate QC samples at each concentration were extracted and
`quantified along with patient samples. Each separate analysis was
`considered acceptable if two thirds of all QC samples were within 15%
`of the nominal concentration and at
`least one QC sample was
`acceptable at each concentration analyzed.
`Pharmacokinetic Analysis
`Individual plasma OSI-774 and OSI-420 concentration data from
`days 1 and 3 (part A), day 24 (part B), and day 28 (part C) were
`analyzed by noncompartmental methods analysis with the WINNonlin
`software program (Scientific Consultant, Apex, SC). The maximum
`plasma concentration (Cmax) and the time to the maximum plasma
`concentration (Tmax) were determined by inspection of the data. The
`area under the plasma concentration-time curve (AUC) from time 0 to
`24 hours (AUC0-24) was calculated by the linear trapezoidal rule. The
`AUC was extrapolated to infinity (AUC0-⬁) by dividing the last
`measured concentration by the terminal rate constant (␭z), which was
`determined from the log-linear fit of the terminal portion of the drug
`concentration-time curve. The oral clearance (Cl/F) was determined by
`dividing the dose by the AUC; the elimination half-life (t1/2) was
`calculated by dividing 0.693 by ␭z; and the apparent volume of
`distribution was calculated by dividing CL/F by ␭z. The accumulation
`ratio was calculated as the ratio of AUC0-24 during the dosing interval
`to the AUC0-⬁ after the first dose. The plasma OSI-774 minimum
`steady-state concentration (Css,min) was equivalent to the average of the
`
`HIDALGO ET AL
`
`pretreatment drug concentrations from days 8 to 28. Intersubject
`variability in the pharmacokinetic parameter estimates was expressed
`in terms of the coefficient of variation percentage. The extent of
`conversion of OSI-774 to OSI-420 (metabolic ratio) was determined by
`dividing the AUC0-24 of the metabolite by the AUC0-24 AUC of the
`parent compound.
`Pharmacokinetic parameters were characterized by use of descriptive
`statistics. The nonparametric statistical
`test for several unrelated
`(Kruskal-Wallis one-way analysis of variance [ANOVA]) or related
`(Wilcoxon matched-pairs signed-rank test) parameters was used to
`determine whether the pharmacokinetics of OSI-774 were dose or time
`dependent. Relationships between drug dose and indices that reflect
`drug exposure (Cmax, AUC, and Css,min) were evaluated with the
`Kruskal-Wallis one-way ANOVA test. The extent of drug exposure as
`determined by Cmax, AUC, and Css,min was compared among patients
`with various grades of toxicity with the use of nonparametric statistical
`tests for two (Mann-Whitney U test) or several (Kruskal-Wallis
`one-way ANOVA) independent samples.
`
`Immunohistochemistry
`to
`Paraffin-embedded tissues from tumor biopsies were sent
`IMPATH (Los Angeles, CA) for evaluation of EGFR expression.
`Immunohistochemical studies on formalin-fixed paraffin-embedded
`specimens were performed by use of an indirect immunoperoxidase
`method. Slides were heated to 60°C, deparaffinized in xylene,
`rehydrated in graded alcohols, and rinsed with tap water. The tissues
`were then subjected to epitope retrieval by treatment with proteinase
`K (Dako, Carpinteria, CA) for 15 minutes at room temperature.
`After the slides were washed with phosphate-buffered saline (PBS)
`(Amresco, Solon, OH), endogenous peroxidase activity was blocked
`using peroxidase block (Dako Envision Plus System) for 5 minutes
`followed by three consecutive rinses with PBS. The sections were
`then incubated with one of two anti-EGFR antibodies including
`clone H1 (Dako) at a concentration of 5.81 ␮g/mL or clone 31G7
`(Zymed, Inc, San Francisco, CA) at a concentration of 1.5 ␮g/mL.
`Murine immunoglobulin G1 antibodies from Dako and Sigma
`Chemical Co (St Louis, MO) were used as negative reagent controls
`for the clone H1 and clone 31G7 anti-EGFR antibodies, respec-
`tively. All antibodies were diluted in primary antibody diluent
`(Research Genetics, Huntsville, AL). Slides were incubated for 30
`minutes at room temperature, then washed three times in PBS. The
`sections were then incubated with labeled polymer (Dako Envision
`Plus System) for 30 minutes at room temperature and washed three
`times in PBS. The peroxidase reaction was visualized after the
`sections were incubated for 1 minute with 3,3-diaminobenzidine-
`tetrahydrochloride solution (Dako). The slides were then thoroughly
`washed with tap water, counterstained with a modified Harris
`hematoxylin (Fisher Scientific, Fairlawn, NJ), dipped in 0.25% acid
`alcohol, and treated with 0.2% ammonia. Subsequently, the sections
`were dehydrated through graded alcohols and treated with xylene,
`and finally coverslips were applied. EGFR expression was assessed
`by use of the following scoring procedure: 0, no staining; 1⫹,
`weakly positive staining of tumor cells; 2⫹, moderately positive
`staining of tumor cells; and 3⫹, strong positive staining of tumor
`cells. Positive staining was scored if stain was visualized within the
`cytoplasm and/or the cytoplasmic rim. In addition, the intensity of
`staining and the percentage of tumor cell that stained positive for
`EGFR also were recorded.
`
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`PHASE I TRIAL OF OSI-774
`
`Table 1. Patient Characteristics
`
`Characteristic
`
`No. of patients
`No. of fully assessable patients
`Sex, male/female
`Age, years
`Median
`Range
`ECOG performance status
`0
`1
`Prior therapy
`Chemotherapy only
`Chemotherapy and radiation
`Hormonal therapy
`Immunotherapy
`None
`Tumor type
`Colorectal
`Breast
`Kidney
`Non–small-cell lung
`Ovary
`Gastric
`Head and neck
`Prostate
`Anal, cervical, pancreas, hepatocellular
`
`No. of Patients
`
`40
`39
`21/19
`
`57
`31-78
`
`18
`22
`
`29
`8
`1
`1
`1
`
`9
`5
`7
`4
`3
`2
`3
`3
`1 each
`
`Abbreviation: ECOG, Eastern Cooperative Oncology Group.
`
`RESULTS
`
`General
`Forty patients, whose pertinent characteristics are listed
`in Table 1, received a total of 123 28-day courses of
`OSI-774. The median number of courses administered per
`patient was two (range, 1 to 20⫹), and a single patient was
`in treatment at the time of this report. One patient, who was
`taken off study as a result of the development of pneumonia
`after 7 days of treatment with OSI-774 150 mg/d in study
`part C, was not considered fully assessable. Thirty-seven
`patients had received cytotoxic therapy previously, includ-
`ing 29 patients who had been treated with chemotherapy
`alone and eight patients who had been treated with both
`chemotherapy and radiation. The numbers of patients and
`courses administered as a function of the dose-schedule
`levels are listed in Table 2. Dose reduction and escalation
`were performed in five patients each, and four patients were
`treated with OSI-774 in two study parts.
`In study part A (OSI-774 once daily for 3 days each
`week, for 3 weeks every 4 weeks), no DLT occurred among
`three groups of new patients treated with doses of 25, 50,
`and 100 mg/d. In study part B (OSI-774 once daily for 3
`weeks every 4 weeks), four groups of new patients were
`treated with 50, 100, 150, and 200 mg/d of OSI-774.
`
`3271
`
`Because one of three patients treated at the 200-mg/d dose
`level experienced DLT during course 1, three additional
`patients were treated. Overall, three patients experienced
`DLT during course 1,
`including grade 3 diarrhea (one
`patient) and grade 4 diarrhea (one patient), and another
`patient developed a grade 2 but clinically intolerable acne-
`iform rash. To evaluate whether the administration of the
`agent on a divided daily dose schedule would permit further
`dose escalation, the next group of patients was treated with
`OSI-774 at a dose of 100 mg twice daily for 3 weeks every
`4 weeks. This strategy was not successful, however, as one
`of three new patients in course 1 developed a grade 2 but
`clinically intolerable acneiform rash, which was considered
`dose limiting. Three additional patients were then treated
`with OSI-774 at the dose level of 150 mg once daily, and
`they experienced no DLT. In the final part of the study (part
`C), one of 12 new patients developed DLT (grade 3
`mucositis) after treatment with OSI-774 150 mg/d on a
`continuous once-daily dose schedule, which was considered
`the MTD.
`
`Toxicity
`The rates of the principal toxic effects of OSI-774 as a
`function of dose-schedule level are listed in Table 3.
`Diarrhea and skin rash were the principal
`toxicities of
`OSI-774, and they precluded treatment at doses higher than
`150 mg/d on an uninterrupted protracted oral schedule.
`
`Diarrhea
`Both the incidence and severity of diarrhea generally
`seemed related to the schedule and dose of OSI-774.
`Although there were no episodes of diarrhea among nine
`patients treated with 20 courses of OSI-774 on the intermit-
`tent schedule evaluated in part A, 18 patients experienced
`38 episodes of diarrhea during treatment on the protracted
`schedules evaluated in parts B and C. In part B, in which
`patients were treated with OSI-774 doses that ranged from
`50 to 200 mg/d for 3 weeks every 4 weeks, the incidence of
`patients who experienced diarrhea increased with higher
`doses of OSI-774. Diarrhea was not noted among five
`patients who received nine courses at the 50-mg/d dose
`level (part B); however, 25%, 86%, and 67% of patients
`treated with OSI-774 at doses of 100, 150, and 200 mg/d,
`respectively, experienced diarrhea.
`Most episodes of diarrhea were mild to moderate (grade
`1 to 2) in severity. In general, the onset of this toxicity
`occurred during weeks 3 to 4 of course 1, and symptoms
`were either self limited or managed successfully with
`nonspecific, symptomatic measures such as loperamide.
`OSI-774 was not discontinued for mild to moderate diar-
`rhea, which usually did not worsen with cumulative treat-
`
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`
`APOTEX EX. 1031-006
`
`

`

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`
`Study Part
`
`Dose Level (mg/d)
`
`New
`(assessable)
`
`Escalated to
`This Dose
`
`Reduced to
`This Dose
`
`Table 2. Dose Escalation Scheme
`
`No. of Patients
`
`A*
`
`B*
`
`B†
`C*
`Total
`
`25
`50
`100
`50
`100
`150
`200
`200 (100 mg bid)
`150
`
`3
`3
`3
`3
`3
`3
`6
`3
`13 (12)
`40 (39)
`
`Abbreviation: DLT, dose-limiting toxicity.
`*Once daily.
`†Twice daily administration.
`
`0
`1
`0
`2
`1
`0
`0
`0
`2
`
`0
`0
`0
`0
`0
`4
`0
`0
`0
`
`HIDALGO ET AL
`
`No. of Patients With DLT/
`Total No. of Patients in
`Course 1 (all courses)
`
`0/3 (0/3)
`0/3 (0/3)
`0/3 (0/3)
`0/3 (0/3)
`0/3 (0/3)
`0/3 (0/3)
`2/6 (2/6)
`1/3 (1/3)
`1/12 (1/12)
`
`Total
`
`3
`3
`3
`5
`4
`7
`6
`3
`15
`
`No. of
`Courses
`
`7
`6
`7
`9
`6
`19
`11
`4
`54
`123
`
`ment. Severe (grade 3 to 4) diarrhea occurred in only two
`patients; they were treated with OSI-774 200 mg/d in study
`part B. The first patient, a 69-year-old woman with breast
`cancer and liver metastases, developed grade 4 diarrhea and
`abdominal discomfort on day 18 of her first course. The
`event was characterized by eight to 10 watery stools daily,
`which resulted in dehydration that required treatment with
`parenteral fluids. This toxicity resolved 3 days after OSI-
`774 was discontinued. The second patient, a 49-year-old
`woman with ovarian carcinoma, developed grade 3 diarrhea
`on day 8 of course 1, which worsened to a grade 4 event on
`day 13 despite treatment with loperamide. At peak toxicity