F l a v o p i r i d o l , A N o v e l C y c l i n - D e p e n d e n t K i n a s e I n h i b i t o r ,
`i n M e t a s t a t i c R e n a l C a n c e r : A U n i v e r s i t y o f C h i c a g o P h a s e
`I I C o n s o r t i u m S t u d y
`
`By Walter M. Stadler, Nicholas J. Vogelzang, Robert Amato, Jeffery Sosman, David Taber,
`David Liebowitz, and Everett E. Vokes
`
`is the first cyclin-dependent
`Purpose: Flavopiridol
`kinase (cdk) inhibitor to enter clinical trials. Serum
`levels of flavopiridol obtained during phase I studies
`were sufficient to inhibit in vitro cancer cell growth.
`Because responses were observed in kidney cancer
`patients in the phase I trials, we performed a phase II
`trial of flavopiridol in this patient population.
`Patients and Methods: Thirty-five minimally pre-
`treated patients were accrued using a standard two-step
`mechanism. Flavopiridol (50 mg/m2/d) was adminis-
`tered by continuous infusion for 72 hours every 2 weeks,
`and response was evaluated every 8 weeks. Peripheral
`blood mononuclear cells (PBMCs) were collected at base-
`line, at completion of drug infusion, and on day 7 of the
`first therapy cycle, and cell cycle parameters after phyto-
`hemagglutinin and interleukin-2 stimulation were as-
`sessed.
`Results: There were two objective responses (re-
`
`sponse rate 5 6%, 95% confidence interval, 1% to 20%).
`The most common toxicities were asthenia, occurring in
`83% of patients (grade 3 or 4 in 9%), and diarrhea,
`occurring in 77% of patients (grade 3 or 4 in 20%). Also,
`nine patients (26%) experienced grade 3 or 4 vascular
`thrombotic events, including one myocardial infarction,
`two transient neurologic ischemic attacks, four deep
`venous thrombosis, and two pulmonary emboli. Cell
`cycle studies did not reveal any effect of flavopiridol on
`stimulated PBMCs.
`Conclusion: Flavopiridol, at the dose and schedule
`administered in this trial, is ineffective in metastatic
`renal cancer. In addition to the diarrhea observed in
`phase I studies, we also observed a higher incidence of
`asthenia and serious vascular thrombotic events than
`expected.
`J Clin Oncol 18:371-375. © 2000 by American
`SocietyofClinicalOncology.
`
`THE MAMMALIAN CELL cycle is driven by series of
`
`cyclins which bind and activate a number of cyclin-
`dependent kinases (cdks).1 Negative control of cdk activity
`is in part accomplished by small protein inhibitors, includ-
`ing p21, p27, p15, and p16.1 These proteins are frequently
`mutated or dysregulated in human malignancies. This ob-
`servation, as well as the conservation of cdk inhibitors
`throughout evolutionary history, makes small molecule
`inhibitors of these enzymes attractive putative anticancer
`compounds. One of the first cdk inhibitors to undergo
`clinical trials is flavopiridol (NSC 649890). Initial in vitro
`studies revealed that it leads to growth inhibition, G2/M
`arrest, and cytotoxicity in breast carcinoma cells by inhib-
`iting the cdc2 cdk.2-4 A subsequent study showed that
`flavopiridol also inhibits cdk2 and cdk4, which can lead to
`G1/S arrest under certain conditions.5 Although initial in
`vitro evaluation suggested that flavopiridol induces only
`cell cycle arrest in proliferating cells, additional publica-
`tions revealed that it is cytotoxic to some resting carci-
`noma cells and can induce apoptosis in others.6-11 Fla-
`vopiridol is also toxic to resting as well as proliferating
`endothelial cells in culture, suggesting that it may have
`some antiangiogenic properties as well.12 Finally, pre-
`liminary data from our own laboratory suggests that
`flavopiridol may be equally effective in cells overex-
`pressing the MDR1 multidrug resistance gene,11 a com-
`mon abnormality in renal carcinoma.13
`
`These in vitro evaluations, along with animal studies with
`rodents
`bearing murine
`and
`human
`tumor
`xeno-
`grafts,6,10,14,15 suggested that prolonged exposure to fla-
`vopiridol was necessary to observe its therapeutic efficacy.
`Thus, two phase I studies evaluated a 72-hour continuous-
`infusion schedule.16,17 Although the dose-limiting toxicity
`was secretory diarrhea, other organ toxicity was mild and
`the diarrhea could be controlled with standard measures.
`The recommended phase II dose was 50 mg/m2/d for 72
`hours every 2 weeks. At this dose, the median plasma
`flavopiridol concentration was 271 nmol/L, a concentration
`sufficient to inhibit cdk activity in vitro. Finally, in one of
`the phase I trials, 19 patients with refractory renal carci-
`noma were entered, and one partial and one minor response
`
`From the University of Chicago and University of Illinois, Chicago,
`IL; M.D. Anderson Cancer Center, Houston, TX; and Michiana
`Hematology/Oncology, South Bend, IN.
`Submitted April 12, 1999; accepted August 25, 1999.
`Supported in part by National Cancer
`Institute grant no.
`UO1CA63187-01.
`Address reprint requests to Walter M. Stadler, MD, Section Hema-
`tology/Oncology, 5841 S. Maryland, MC2115, Chicago, IL 60637;
`email wmstadle@mcis.bsd.uchicago.edu.
`© 2000 by American Society of Clinical Oncology.
`0732-183X/00/1802-371
`
`Journal of Clinical Oncology, Vol 18, No 2 (January), 2000: pp 371-375
`
`371
`
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`372
`
`were observed.16 Thus, we initiated a phase II trial of
`flavopiridol in patients with refractory renal carcinoma.
`
`PATIENTS AND METHODS
`
`Patients
`
`This study was conducted through the University of Chicago phase
`II consortium in collaboration with the M.D. Anderson Cancer Center.
`Patients were required to have metastatic or unresectable renal carci-
`noma clearly measurable on radiologic or physical examination. One
`prior immunotherapy regimen but no prior chemotherapy was allowed.
`The requirements for normal organ function included WBC $ 3,000/
`mL, platelets $ 100,000/mL, hemoglobin $ 9.0 g/dL, total bilirubin #
`1 5 mg/dL, transaminases # 2.5 times the upper limit of normal, and
`creatinine # 2.0 mg/dL (or estimated creatinine clearance $ 60
`mL/min). Patients were required to have a World Health Organization
`performance status of 0 to 2 and no other history of invasive
`malignancy in the past 5 years. CNS metastases were allowed, provided
`that patients had adequate local therapy and did not require systemic
`steroids. All patients signed a written informed consent approved by
`individual institutional review boards. Thirty-five patients were ac-
`crued from October 1997 to May 1998 through the following institu-
`tions: University of Chicago (14 patients); M.D. Anderson Cancer
`Center (eight patients); University of Illinois (six patients); Michiana
`Hematology/Oncology (four patients); Fort Wayne Medical Center
`(two patients); and Weiss Memorial Hospital (one patient).
`
`Therapy and Dose Modifications
`
`Flavopiridol was supplied by the Division of Cancer Treatment and
`Diagnosis, National Cancer Institute and was administered as a
`continuous 72-hour infusion via a central venous catheter at a dose of
`50 mg/m2/d every 2 weeks. Drug was reconstituted in benzyl alcohol
`preserved saline at a final concentration of 0.25 to 2.25 mg/mL in
`polyvinylchloride bags and administered via an ambulatory pump.
`Patients experiencing grade 3 or greater diarrhea had flavopiridol
`infusion halted, and loperamide or diphenoxylate and atropine therapy
`was initiated. If diarrhea continued, octreotide was begun. Any grade 3
`or grade 4 toxicity led to dose reductions of 25% or 50%, respectively,
`in subsequent cycles.
`
`Patient Follow-Up and Study End Points
`
`The primary objective of this study was to determine the objective
`response rate of patients with metastatic renal carcinoma treated with
`flavopiridol. A secondary clinical objective was to determine flavopiri-
`dol’s toxicity in this patient population. Response evaluations were
`performed every 8 weeks. Complete response was defined as complete
`disappearance of all radiologic and clinical signs of cancer maintained
`for at least 4 weeks. Partial response was defined as a 50% reduction
`from baseline in the sum of the products of two perpendicular
`diameters of all measured lesions, without the appearance of new
`lesions, also maintained for at least 4 weeks. Progressive disease was
`defined as 25% increase from baseline in the sum of the products of two
`perpendicular diameters of all measured lesions or the appearance of
`new lesions. All other situations were defined as stable disease. Patients
`discontinued therapy when there was disease progression, which
`included the need for palliative radiotherapy or other systemic antineo-
`plastic therapy. Other withdrawal criteria included interruption of
`therapy for greater than 2 weeks for any cause, intolerable or unre-
`solved adverse events, or patient decision.
`
`STADLER ET AL
`
`Biologic Monitoring
`
`Additional secondary objectives of this trial included pharmacoki-
`netic monitoring and assessment of flavopiridol’s effect on in vitro cell
`cycle parameters in stimulated peripheral-blood mononuclear cells
`(PBMCs). Pharmacokinetic monitoring and correlation with response
`and toxicity are objectives in ongoing trials of flavopiridol in colon
`cancer and non-Hodgkin’s lymphoma as well and, therefore, will be
`reported separately. For the cell cycle study, PBMCs were isolated
`before beginning therapy, 71 hours after the start of infusion, and on
`day 7 of cycle 1 only. Collected cells were frozen for viability at
`280 C in RPMI 1640 media with 10% fetal bovine serum and 10%
`dimethyl sulfoxide and then batch analyzed. To study the effect of
`flavopiridol on cell cycle kinetics, 1.0 3 106 PBMCs were grown in
`RPMI 1640 with 10% fetal bovine serum and 2 mmol/L L-glutamine
`and stimulated with phytohemagglutinin A (PHA) 10 mg/mL and
`interleukin-2 (IL-2) 8 ng/mL. After 72 or 96 hours of stimulation,
`bromodeoxyuridine (10 mmol/L) was added to the media. Cells were
`harvested, washed in phosphate buffered saline, and fixed with 4%
`paraformaldehyde. PBMCs were then stained with 10 mg/mL pro-
`pidium iodide, 100 mg/mL RNAse A and 0.1% Triton X-100 for 30
`minutes at room temperature. Thereafter, cells were stained for 30
`minutes with fluorescein isothiocyanate– conjugated antibody to bro-
`modeoxyuridine. Cell cycle distributions and apoptotic fractions were
`determined using ModFit LT v2.0 software (Verity Software House,
`Inc, Topsham, ME) after analysis on a FACScan flow cytometer
`(Becton Dickinson, Franklin Lakes, NJ). Because no effects on cell
`cycle parameters were noted in the first six patients (see Results),
`additional patient samples were not analyzed.
`
`Statistical Considerations
`
`Accrual proceeded using a standard two-stage mechanism. The first
`stage was designed to accrue 14 patients, with a second stage designed
`to accrue an additional 21 patients if one or more patients responded in
`the first stage. The regimen would be considered worthy of further
`study if four or more of 35 patients (.11%) experienced an objective
`response. This design yields a 0.91 or greater probability of accepting
`the regimen if the true response rate is at least 20% and a 0.91 or greater
`probability of rejecting the regimen if the true response rate is less than
`5%. It also gives a 0.49 or greater probability of stopping early if the
`flavopiridol response rate in this population is less than 5%. Survival
`was assessed using the Kaplan-Meier method. All survival data was
`censured as of December 1, 1998.
`
`RESULTS
`
`Table 1 lists the patient characteristics. One response was
`observed in the first 14 patients. Thus, accrual proceeded
`through the second stage, and a total of 35 patients were
`entered. As a group, the enrolled patients were predicted to
`have a good prognosis.18 Thirty-one patients (89%) had a
`World Health Organization performance status of 0 or 1.
`The median time since diagnosis of metastatic disease was
`14 months; 12 patients (34%) had only one site of metastatic
`disease, and 10 patients (29%) had lung only or lymph node
`only disease. One patient refused clinical and radiologic
`follow-up and was, therefore, not assessable for response
`but was assessable for toxicity. All other patients were
`considered assessable for toxicity and response. Among the
`
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`FLAVOPIRIDOL IN METASTATIC RENAL CANCER
`
`373
`
`Table 1. Patient Characteristics
`
`
`
`Characteristic
`
`No. of Patients
`
`58
`29-76
`
`22
`
`22
`1-389
`
`14
`2-28
`
`Total
`Patients eligible for response evaluation
`Sex
`Male
`Female
`Age, years
`Median
`Range
`WHO performance status*
`0
`1
`2
`Prior nephrectomy
`Time since nephrectomy, months
`Median
`Range
`Time since metastatic disease diagnosis, months
`Median
`Range
`Prior therapy
`Immunotherapy
`Radiotherapy
`Resection metastatic site
`No. of metastatic sites
`1
`2
`$3
`Sites of metastatic disease
`Lung only
`Lymph node only
`Liver (with or without other sites)
`Bone (with or without other sites)
`
`35
`34
`
`24
`11
`
`19
`12
`4
`
`21
`8
`8
`
`12
`10
`13
`
`7
`3
`9
`3
`
`Abbreviation: WHO, World Health Organization.
`*Median performance status 5 0.
`
`34 patients assessable for response, there were two partial
`responders, for an overall response rate of 6% (95%
`confidence interval, 1% to 20%.) Fig 1 depicts event-free
`and overall survival with events defined as progressive
`disease or toxicity requiring discontinuation of therapy. The
`median event-free survival was 9 weeks, and the median
`overall survival was 48 weeks. As of this report, three
`patients remain on study at 311, 321, and 571 weeks.
`Table 2 lists the observed toxic events. There was
`essentially no myelosuppression and no evidence of renal,
`neurologic, or hepatic toxicity either. The most common
`toxicity was asthenia and/or fatigue, which occurred in 83%
`of patients. In the majority of patients, it was mild and did
`not influence therapy, but it was graded as severe by three
`patients. Diarrhea was also a common toxicity despite the
`use of prophylactic antidiarrheal medication at the first sign
`of loose stools and was considered severe in seven patients
`(20%). Twenty-four of 35 patients required some antidiar-
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Fig 1. Overall and event free survival with events defined as progressive
`disease or toxicity requiring discontinuation of drug therapy. Dotted lines
`reflect 95% confidence intervals, and tick marks represent censured patients.
`
`rheal medication during therapy. This included loperamide
`and/or diphenoxylate and atropine in 22 patients and addi-
`tional octreotide in four patients. Seven patients required
`intravenous fluids for control of fluid and electrolyte abnor-
`malities secondary to diarrhea. Nausea was also significant
`in a few patients. A number of patients were noted to have
`hyperglycemia during therapy. All patients with grade 2 or
`3 hyperglycemia had known diabetes and the relationship to
`flavopiridol is questionable.
`The most serious adverse events were arterial and venous
`thrombotic events. Three arterial events were noted. The
`first was a patient who experienced a nonfatal myocardial
`infarction on day 10 of the study and received no further
`flavopiridol. A second event occurred in a patient with a
`history of transient ischemic attacks, who was admitted on
`the last day of the third flavopiridol infusion with an episode
`of confusion that was similar to previous transient ischemic
`
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`374
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`Table 2.
`
`Toxicity
`
`Patients With Toxicity (no.)
`
`Event
`
`Grade 1
`
`Grade 2
`
`Grade 3
`
`Grade 4
`
`Neutropenia
`Thrombocytopenia
`Hemoglobin
`Hyperglycemia
`Asthenia/fatigue
`Nausea/vomiting
`Diarrhea
`Arterial vascular
`Venous vascular
`
`0
`6
`17
`8
`17
`9
`9
`0
`0
`
`0
`0
`11
`2
`9
`6
`11
`0
`0
`
`0
`0
`0
`5
`3
`1
`6
`1
`4
`
`0
`0
`0
`0
`0
`0
`1
`2
`2
`
`NOTE. Other grade 3/4 toxicities included two episodes of peripheral
`edema, one episode of catheter related infection, and one episode of elevated
`alkaline phosphatase. All these events were interpreted as being most likely
`unrelated to flavopiridol.
`
`attack episodes. Brain magnetic resonance imaging revealed
`only chronic small vessel disease. He was placed on
`warfarin, but experienced several additional transient con-
`fusional episodes that were not further evaluated. A final
`patient experienced transient vision loss and scotoma in one
`eye, with a normal ophthalmologic examination. This pa-
`tient underwent a dose reduction for concomitant grade 3
`diarrhea and was treated for an additional 6 months without
`recurrent symptoms. The relationship of each of these
`arterial events to flavopiridol was unclear.
`Six patients experienced deep venous thromboses during
`therapy. Four patients had documented extremity thrombo-
`ses, two in the lower extremities and two associated with a
`permanent intravenous access device in the upper extremi-
`ties. One of the latter patients remained on study and
`developed a second thrombosis in the upper extremity
`despite the use of warfarin. Two patients experienced
`probable pulmonary emboli. One patient was diagnosed
`after a computed tomography scan of the chest performed
`for disease re-evaluation. The second was in a patient who
`developed sudden chest pain and shortness of breath on day
`3 of therapy and died that same day with a cardiopulmonary
`arrest.
`In vitro cell cycle studies were performed on stimulated
`PBMCs collected at baseline, 71 hours into the infusion, and
`on day 7 of cycle 1. It was hypothesized that cells collected
`at the end of the flavopiridol infusion would be inhibited
`from entering the cell cycle when stimulated by PHA and
`IL-2. There was no difference in cell cycle parameters of
`PHA/IL-2–stimulated lymphocytes collected at baseline or
`72 or 96 hours after beginning the flavopiridol infusion in
`the first six enrolled patients. Thus, further patient samples
`were not analyzed.
`
`STADLER ET AL
`
`DISCUSSION
`
`We have conducted a phase II evaluation of the cdk
`inhibitor flavopiridol in patients with refractory renal carci-
`noma. This trial confirmed the phase I data; there was little
`hematologic, hepatic, or renal toxicity, but grade 3 or 4
`diarrhea was common and occurred in 20% of patients. The
`diarrhea did not always respond to opiate antidiarrheal
`medications, and some patients required octreotide and
`subsequent flavopiridol dose reduction. Interestingly, the
`most common toxicity was asthenia/fatigue, which was
`experienced by 83% of patients. Although this was also
`noted in the phase I trial, it seemed to be more distressing to
`the presumably better functioning patients in this study. A
`serious potential toxicity that was not appreciated in the
`phase I study was arterial and venous thromboses. Although
`the arterial events were not considered drug related by the
`treating physicians and although deep venous thromboses
`and pulmonary emboli are certainly common in patients
`with metastatic renal carcinoma, the frequency with which
`these events were observed was greater than we experienced
`in other trials with renal cancer patients. For example, in
`ongoing phase I and II studies using continuous-infusion
`fluorouracil and gemcitabine, we have observed no catheter-
`associated thromboses in more than 70 renal carcinoma
`patients (unpublished data). Thus, additional clinical trials
`with flavopiridol may need to consider the etiology and
`prevention of these events. Different doses and/or schedules
`may also need to be considered to decrease the incidence of
`toxic events, especially if prolonged administration is an-
`ticipated (see below).
`We attempted to detect the cell cycle inhibitory effect of
`flavopiridol by in vitro stimulation of PBMCs collected
`during drug infusion. The lack of observed effect may be
`caused by freezing the cells before analysis, but, more
`likely, it is because of the observation that cells need to be
`continuously exposed to flavopiridol in order for cell cycle
`arrest to become manifest.2 Thus, cells that are exposed to
`71 hours of flavopiridol and then undergo ex-vivo growth
`stimulation do not experience any permanent cell cycle
`perturbations.
`Despite encouraging preclinical data and suggestive data
`from the phase I study, little activity, as determined by
`objective responses, was observed in this population. In
`vitro, flavopiridol causes growth arrest in almost all cell
`types, but apoptosis and cytotoxicity are rapidly induced in
`only a few, usually hematopoietic cell lines.2,6,8-11 In these
`cases, apoptosis occurs within 24 hours of flavopiridol
`exposure at doses less than 1 mmol/L.6,9 Although apoptosis
`can be observed in other cell types, it usually requires
`flavopiridol doses and exposure times that are above plasma
`
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`FLAVOPIRIDOL IN METASTATIC RENAL CANCER
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`375
`
`levels achieved in the phase I study at the recommended
`phase II dose. Thus, such cells, which likely includes most
`renal carcinomas, will experience only a transient cell cycle
`arrest. This would not be expected to lead to objective tumor
`shrinkage. Such an effect may lead to slowing of tumor
`growth, but this trial was not designed to detect this event.
`Although the prolonged survival in this patient cohort may
`be encouraging, this was a good prognosis cohort in whom
`a more prolonged survival would be expected even in the
`absence of therapy.18
`Recently, it has been demonstrated that in vitro combi-
`nation standard chemotherapy and flavopiridol increases
`apoptosis and may be synergistic.19,20 Whether flavopiridol
`could enhance the minimal activity of fluorouracil in renal
`cancer21 or the activity of IL-2, whose mechanism of action
`
`is immune stimulation, remains to be determined. Thus,
`flavopiridol at this dose and schedule does not lead to
`significant objective responses in renal cancer patients. To
`determine whether prolonged administration at lower doses
`could lead to disease stabilization with minimal toxicity or
`whether combination therapy could be useful would require
`additional trials.
`
`ACKNOWLEDGMENT
`
`We thank J. Wright, MD, of the National Cancer Institute for
`protocol design advice; P. Schumm for statistical advice; S. Krauss,
`MD, D. Prow, MD, T. Gabrys, MD, R. Ansari, MD, L. Pagliano, MD,
`and J. Gibbons, MD, for enrolling patients; M. Dumas, RN, and S.
`Flickinger, RN, for protocol management and support; A. Lin for
`performing cell cycle studies; R. Arrieta for data management support;
`and the Kidney Cancer Association for patient referral.
`
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`Copyright © 2016 American Society of Clinical Oncology. All rights reserved.
`
`NOVARTIS EXHIBIT 2050
`Breckenridge v. Novartis, IPR 2017-01592
`Page 5 of 5
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