Phase I and pharmacokinetic study of CCI-779, a novel cytostatic cell-cycle inhibitor, in combination with 5-fluorouracil and leucovorin in patients with advanced solid tumors

C. J. A. Punt1,+, J. Boni2, U. Bruntsch3, M. Peters1 and C. Thielert4

1 University Medical Center, St Radboud Nijmegen, The Netherlands; 2 Wyeth Research, Collegeville, PA, USA; 3 Klinikum Nürnberg, Germany; 4 Wyeth Oncology, Munich, Germany

Received 21 October 2002; revised 16 January 2003; accepted 20 February 2003


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background:

CCI-779 is a novel ester of the immunosuppressive agent sirolimus that exerts cytostatic effects by the inhibition of the translation of cell-cycle regulatory proteins. We investigated the maximum tolerated dose (MTD) and pharmacokinetics (PK) of CCI-779 in combination with leucovorin (LV) and 5-fluorouracil (5-FU) in patients with advanced solid tumors.

Patients and methods:

Patients were treated with LV at 200 mg/m2 as a 1-h i.v. infusion directly followed by continuous 24-h i.v. infusion of 5-FU, in the first patient at 2000 mg/m2 and in subsequent patients at 2600 mg/m2. CCI-779 was administered directly prior to LV as a 30-min i.v. infusion at a starting dose of 15 mg/m2 beginning at day 8 and escalated in subsequent cohorts of patients. One cycle consisted of six weekly administrations followed by 1 week of rest. Blood samples were drawn to assess PK of CCI-779 as well as its effect on steady-state 5-FU exposures.

Results:

Twenty-eight patients entered the study, the majority having tumor types for which 5-FU is used as a treatment. CCI-779 doses of 15, 25, 45 and 75 mg/m2 were investigated. Skin toxicity (rash) was prominent at all dose levels examined. Stomatitis was the dose-limiting toxicity (DLT) for 75 mg/m2 doses of CCI-779. Subsequently the cohort at 45 mg/m2 was expanded to a total of 15 patients, and at this dose level two treatment-related deaths occurred due to mucositis with bowel perforation. Based on the toxicities observed, it was decided to discontinue the study. Partial responses were observed in three patients with gastrointestinal tumors. No pharmacokinetic interaction between CCI-779 and 5-FU was observed.

Conclusions:

The safety profiles of CCI-779 and 5-FU/LV suggest an overlap of drug-related toxicities, and the administration of these drugs at these doses and schedule resulted in unacceptable toxicity and therefore cannot be recommended. If CCI-779 is to be used in combination with 5-FU/LV, other doses or schedules of administration will need to be explored.

Key words: CCI-779, 5-fluorouracil, pharmacokinetics, phase I study, rapamycin, sirolimus, solid tumors


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
CCI-779 is an ester of the immunosuppressive agent rapamycin (sirolimus) and inhibits the mammalian target of rapamycin (mTOR) [1]. It has demonstrated significant inhibitory effects both in vitro and in vivo. Its cytostatic properties result from the inhibition of translation of several key proteins that regulate the G1 phase of the cell cycle [2, 3]. Similar to rapamycin, CCI-779 is hypothesized to form a complex with the intracellular cytoplasmic protein FK-506 binding protein-12 (FKBP) that binds to mTOR, resulting in inhibition of key signaling pathways involved in the G1 phase of the cell cycle. A phase I study with CCI-779 given weekly as a single agent was completed, and preliminary results showed that doses ≤220 mg/m2/week were feasible, with clinical activity in renal and breast cancer [4]. Preliminary safety results from this and other studies with CCI-779 at 25, 75 or 250 mg given as a weekly i.v. infusion show that skin rash/acne, mucositis, asthenia, nausea, thrombocytopenia, hyperglycemia, hypertriglyceridemia, hypokalaemia and depression may occur as toxicities, usually as grade 1 or 2 [46]. In humans, sirolimus is expected to be a significant metabolite in the circulation. Sirolimus is extensively partitioned into red blood cells, with a mean (range) blood/plasma ratio of ~36 in renal allograft patients [7, 8]. It is extensively metabolized via intestinal and hepatic microsomal CYP3A to hydroxylated and demethylated products that are predominantly excreted in the feces [9, 10]. In stable human transplant recipients, the mean apparent steady-state volume of distribution in whole blood is approximately 1.7 l/kg, mean clearance is approximately 29 ml/h/kg, and terminal half-life is approximately 62 h [11].

5-Fluorouracil (5-FU) is an antineoplastic antimetabolite which is frequently used as palliative treatment for patients with carcinoma of the colon, rectum, breast, stomach and pancreas. The pharmacokinetics (PK) of 5-FU have been relatively well characterized [12, 13]. Depending on the dose and schedule, its main toxicities are diarrhea, stomatitis and leukopenia. With continuous i.v. infusion, hand–foot syndrome is frequently observed.

Based on the hypothesis that cytostatic agents are likely to be more effective with other chemotherapeutic agents in patients with solid tumors, we initiated a phase I study of CCI-779 in combination with 5-FU and leucovorin (LV). The PK of CCI-779 and its major metabolite, sirolimus, are described. In addition, the potential for CCI-779 to influence the disposition of 5-FU is examined.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
Eligibility criteria included histological evidence of advanced solid tumor, refractory to standard therapy or no standard therapy available; or a tumor type for which 5-FU/LV was appropriate therapy; at least 4 weeks since any previous antitumor therapy; age ≥18 years; neutrophil count ≥1.5 x 109/l; platelets ≥100 x 109/l; hemoglobin ≥5.6 mmol/l; serum creatinine <200 µmol/l; bilirubin <26 µmol/l; aspartate aminotransferase and alanine aminotransferase <3 x upper limit of normal (or <5 x in case of liver metastases); cholesterol <9 mmol/l; triglycerides <3.4 mmol/l; Eastern Cooperative Oncology Group (ECOG) performance status 0–2; and written informed consent. Patients with symptomatic brain metastases, serious concomitant conditions, or pregnant/nursing females were excluded. The study was approved by the local review boards of the three participating institutions.

Study objectives
The primary objectives of the study were to assess the safety and tolerability of CCI-779 when combined with 5-FU/LV and to identify its maximum tolerated dose (MTD) in this group of patients. The secondary objectives were to determine the PK and clinical efficacy of CCI-779 and 5-FU with this schedule. The protocol allowed the inclusion of additional patients at MTD in order to expand the experience at this dose level.

Treatment
A tunneled central venous catheter for 5-FU infusion was inserted in all patients prior to the start of the study. Treatment was given in the ambulatory setting except for the days of PK sampling. Patients received LV administered at 200 mg/m2 as a 1-h i.v. infusion, directly followed by a continuous 24-h i.v. infusion of 5-FU by ambulatory pump. The first patient at one of the institutions received 5-FU at a dose of 2000 mg/m2 as requested by one of the local review boards. Subsequent patients received 5-FU at 2600 mg/m2, which has been used frequently for the treatment of colorectal cancer [14, 15]. CCI-779 (Wyeth Research, PA, USA) was administered immediately prior to LV as a 30-min i.v. infusion by pump at a starting dose of 15 mg/m2 beginning at day 8 and escalated in subsequent cohorts of three to six patients. The starting dose of CCI-779 was based on preliminary results of a phase I single-agent study in which CCI-779 was administered on a weekly schedule to patients with cancer [4]. The starting dose of 15 mg/m2 was well below (~25%) the dose achieved at the time that the single-agent phase I study was simultaneously ongoing, and was neither associated with dose-limiting toxicities nor with National Cancer Institute (NCI) toxicity greater than grade 2.

CCI-779 25 mg/ml ethanolic concentrate was diluted 10-fold using a special PEG (polyethylene glycol)/polysorbate diluent, and was administered protected from light.

One cycle consisted of six weekly administrations followed by 1 week of rest. NCI-common toxicity criteria and World Health Organization (WHO) response criteria were used. Dose-limiting toxicities (DLT) were defined as grade 3–4 non-hematological toxicity (excluding nausea/vomiting, unless optimal antiemetic treatment was given, and rise in serum triglycerides, if <17 mmol/l and recovery had occurred by the next cycle), grade 4 thrombocytopenia, grade 4 uncomplicated neutropenia ≥5 days, febrile neutropenia, or treatment delay ≥2 weeks due to any toxicity. In the absence of DLT as determined by the evaluation of at least three patients at that dose level and at least a total of 9 weeks of completed treatment with all three drugs, CCI-779 was to be escalated following a modified Fibonacci scheme. Intrapatient dose escalation was not allowed. After the occurrence of DLT at a particular dose level in the first three patients, the cohort was to be expanded to six patients. The MTD was defined as the dose level at which one or two of six patients experienced DLT. Patients were followed weekly for toxicity and every 2 months for response. The dose of 5-FU was to be reduced upon the occurrence of grade 3 (25% reduction) or grade 4 (50% reduction) hematological toxicity and grade 2 (25% reduction) or grade 3 (50% reduction) non-hematological toxicity. The dose of CCI-779 was to be reduced to 33% upon the occurrence of grade 4 hematological toxicity or grade 3 non-hematological toxicity. Patients were to be withdrawn from the study when grade 4 non-hematological toxicity occurred. No prophylactic medication was used except for the administration of clemastine 2 mg i.v. for the prevention of allergic skin reactions beginning with the expanded 45 mg/m2 cohort.

Bioanalytical methodology
During week 1 (5-FU alone) and weeks 2 and 4 (5-FU with CCI-779), venous blood sampling (3 ml per sample) to measure 5-FU in plasma was performed during 5-FU infusion at 0 (pre-dose), 0.5, 1, 1.5, 2, 3, 5.5–6, 8 and 24 (end of infusion) h. During weeks 2 and 4, venous blood samples (3 ml each for CCI-779 and sirolimus metabolite) were drawn at 0 (pre-dose), 0.25, 0.5 (end of infusion), 1.5, 3.5, 8, 24, 48, 96 and 168 h.

The bioanalytical method used to measure CCI-779 concentrations was performed and validated at Taylor Technology, Inc. (Princeton, NJ, USA). The method employs a liquid chromatography, tandem mass spectrometry (LC/MS/MS) procedure with deuterated internal standard and is validated through the quantitation range of 0.25–100 ng/ml using 1 ml of whole blood. During validation, inter- and intra-day variations were <5% coefficient of variation (CV) and biases were <9.4%. At the lower limit of quantitation (0.25 ng/ml), variability was 7.6% and bias was <2.4%. CCI-779 exhibited adequate room-temperature stability in whole blood (25 h at 25°C), in autosampler extracts (66 h at 25°C and 8 days at –80°C), and after three freeze/thaw cycles. Unpublished data from long-term studies indicate that CCI-779 stability in whole blood is adequate when stored at –70°C. In addition, no interference was observed in blank blood or blood spiked with 100 ng/ml of sirolimus.

The bioanalytical method used to measure sirolimus in this study also employs an LC/MS/MS procedure. The method was performed at Taylor Technology, Inc. and validated through the quantitation range of 0.1–100 ng/ml using 1 ml of whole blood. Collectively, inter- and intra-day variabilities of quality control samples measured during validation were <12.7% and biases were <11.3%. Sirolimus is stable in whole blood when stored at –70°C.

Concentrations of 5-FU were measured in plasma using a validated GC/MS/MS (gas chromatography, tandem mass spectrometry) assay. The 5-FU assay was performed at Northwest Bioanalytical (NWB; Salt Lake City, UT, USA). The bioanalytical method exhibited a linear range from plasma of 1–100 ng/ml. Inter- and intra-assay variability of quality control samples measured during validation were <7% and biases were <13%. 5-FU is stable in plasma when stored at –20°C.

Pharmacokinetic methodology
All PK data analyses were performed by the Department of Clinical Pharmacology and Pharmacokinetics at Wyeth Research. Concentrations of CCI-779, sirolimus and 5-FU were analyzed using a noncompartmental modeling approach [16]. The PK parameters for CCI-779 and sirolimus include peak concentration (Cmax), time to Cmax (tmax), lambda ({lambda}), terminal half-life (t1/2), area under the curve through infinity (AUC), clearance (Cl), volume of distribution at steady state (Vss) and AUC ratio (week 4/week 2). For sirolimus, the apparent clearance (Cl/fm) and apparent Vss/fm (where fm is the unknown fraction of parent drug metabolized to form sirolimus), and the AUC ratio (sirolimus/CCI-779 and week 4/week 2) were determined. For 5-FU, the average concentration during infusion (Cmean) was also determined. Derivation of the PK parameters was performed using statistical analysis software.

Statistical analysis of PK parameters includes summary representation for the 15, 25, 45 and 75 mg/m2 dose treatments, and an analysis of variance on log-transformed data with dose treatment and patient as statistical factors. In addition, the ratio [test (week 2 or 4)/reference (week 1)] of least-squares geometric means was determined and the corresponding 90% confidence intervals.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
A total of 28 patients entered the study, 24 in Nijmegen, three in Nürnberg and one in Munich. Patient characteristics are shown in Table 1. The majority of patients had tumors for which 5-FU is commonly used as a treatment and 13 had not received any prior systemic treatment for advanced disease.


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Table 1. Patient characteristics
 
Toxicity
Twenty-seven patients were evaluable for toxicity. Grade 1–2 skin toxicity consisting of maculopapular rash was a prominent finding and occurred in 67% of patients at all dose levels. This was usually asymptomatic and resolved either spontaneously or following treatment. A variety of agents, primarily topical, were used. Histopathological examination of skin lesions performed in four patients confirmed a diagnosis of nonspecific chronic dermatitis compatible with an allergic drug reaction. Other frequent grade 1–2 toxicities consisted of epistaxis (in 78% of patients), mucositis (89%), anorexia (59%), asthenia (74%), nausea (56%) and fever (52%). The most prominent grade 2–4 toxicities included mucositis (usually limited to stomatitis), asthenia, diarrhea, nausea/vomiting, rash, anemia and hyperglycemia, and are summarized in Table 2. At a CCI-779 dose of 75 mg/m2, five of six patients experienced toxicities necessitating dose delays and dose reductions or discontinuations. In four of six patients at that dose level, grade 2 mucositis (stomatitis) was observed, which recurred in one patient upon 5-FU dose reduction. Therefore, despite the fact that no formal DLTs were observed, it was concluded that this dose was not feasible and the previous dose of 45 mg/m2 was expanded by 11 patients. Subsequently two deaths occurred, both due to bowel perforation which was accompanied in one patient with neutropenic sepsis. Both patients experienced grade 2–3 stomatitis as well as grade 2–3 diarrhea at the time of this event. In one patient a postmortem examination located the perforation in a bowel area affected by mucositis and not by tumor. This patient with cholangiocarcinoma metastatic to the lungs, bone, omentum and mesenterium had chronic active mucositis of the oral cavity as well as the bowel, without signs of mycosis, with focal transmural purulent inflammation of the rectosigmoid with two perforation sites as well as purulent peritonitis with ascites. The second patient presented with neutropenic sepsis together with acute abdominal signs and symptoms most compatible with bowel perforation. Another patient with regional recurrence of head and neck cancer died as a result of carotid artery bleeding due to a rapidly occurring almost complete regression of tumor. This event was not considered specifically related to the study drugs.


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Table 2. Drug-related grade 2–4 toxicities
 
Pharmacokinetics
Whole blood and plasma samples were obtained from 27 patients. Patient demography indicates that eight (30%) patients were female, mean age was 56 years, and mean weight was 72 kg. Following a 30-min i.v. infusion of CCI-779, sirolimus was the major observed metabolite in whole blood (Figure 1). Peak concentrations and total exposure to both CCI-779 and sirolimus increased in a dose-related but sub-proportional fashion (Table 3). Following the week 2 (first) dose of CCI-779, mean volume of distribution exceeded total body water, ranging from 260 to 361 l. Clearance of CCI-779 from whole blood was nonlinear and increased in a dose-related manner from 12.7 l/h (15 mg/m2 dose) to 41.5 l/h (75 mg/m2 dose). This finding of non-linearity explains the generally significant treatment effects across doses observed for the various parameters. Inter-patient variability in clearance for a given dose was modest (CV = 17% to 27%). Values of AUC ratio (week 4 /week 2) were near unity and indicated that little or no accumulation was apparent with multiple (weekly) treatments. Mean t1/2 ranged from 14 to 18 h.



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Figure 1. Mean (standard deviation) concentrations of CCI-779 and sirolimus in whole blood following a single 45 mg/m2 i.v. dose of CCI-779. Data reflect profiles for CCI-779 (circles, solid line) and sirolimus (squares, dotted line).

 

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Table 3. Mean pharmacokinetic parameters of CCI-779 in whole blood (treatment week 2)
 
For sirolimus, mean Cmax values were approximately 10- to 16-fold lower than respective values of CCI-779 (Table 4). Measurable concentrations of sirolimus were detectable early (by 15 min following the start of CCI-779 infusion) and, as expected, exhibited tmax values that were markedly variable. Decline following peak concentration appeared to follow mono-exponential decay. The contribution of sirolimus metabolite to total exposure was substantial, albeit variable, with mean AUC ratio (sirolimus/CCI-779) values ranging from ~2 to 5 during week 2, and from 3 to 8 during week 4. As with CCI-779, sirolimus/AUC ratio (week 4/week 2) was near unity, indicating little or no accumulation with multiple treatments. Mean t1/2 ranged from 46 to 53 h and exhibited modest variability (CV = 5% to 30%).


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Table 4. Mean pharmacokinetic parameters of sirolimus in whole blood (treatment week 2)
 
For each patient, concentrations of 5-FU were measured during continuous i.v. infusion. Values between the 1 and 8 h time points were chosen to represent the mean steady-state concentrations for 5-FU (Table 5). Given the inability to draw samples between 8 and 24 h and the expectation that variability in steady-state exposure to 5-FU could increase due to diurnal factors [17], the 24-h concentration values were not included in calculation of mean values. Statistical analysis of mean concentrations of 5-FU did exhibit a significant difference (P <0.0002); however, a consistent dose-treatment effect during CCI-779 treatment weeks 2 and 4 was not apparent (Figures 2 and 3). Mean clearance of 5-FU observed in the absence (week 1) and presence (weeks 2 and 4) of CCI-779 ranged from 300 to 346 l/h, was not significantly different, and exhibited a moderate degree of inter-patient variability (CV = 33% to 43%). While the ratios of least-squares geometric means lie within 80–125% of nominal value, the 90% confidence intervals around these means is somewhat more variable (Table 6). Collectively, the data suggest that CCI-779 does not exert a consistent, clinically relevant effect on exposure to 5-FU.


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Table 5. Mean concentrations and pharmacokinetic parameters of 5-fluorouracil in plasma
 


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Figure 2. Individual patient Cmean concentrations of 5-FU in plasma during a 2600 mg/m2 dose over a 24-h i.v. infusion period in the absence (week 1) and presence (weeks 2 and 4) of CCI-779. Data reflect patients receiving the 15 (circles), 25 (squares), 45 (triangles) and 75 (diamonds) mg/m2 doses, respectively.

 


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Figure 3. Individual patient clearance of 5-FU in plasma during a 2600 mg/m2 dose over a 24-h i.v. infusion period in the absence (week 1) and presence (weeks 2 and 4) of CCI-779. Data reflect patients receiving the 15 (circles), 25 (squares), 45 (triangles) and 75 (diamonds) mg/m2 doses, respectively.

 

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Table 6. Ratio of least-squares geometric means of 5-fluorouracil
 
Tumor responses
Twenty-six patients were evaluable for response (one patient never received CCI-779 and one patient did not have evaluable disease parameters). Three patients, with colorectal cancer, gastric cancer and gallbladder cancer, achieved confirmed partial responses lasting for 7.4, 24.5+ and 6.0 months, respectively. For the 14 patients with either partial response (three) or stable disease (11), the median duration before disease progression was 5.2 months [95% confidence interval (CI) 4.0–10.8 months]. For all 26 evaluable patients, the median time to tumor progression was 3.2 months (95% CI 1.5–5.2 months).


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
In this phase I study, we demonstrated that the combination of CCI-779 and 5-FU at the doses and schedule used is not feasible. Stomatitis/mucositis occurred at all dose levels and was found to be the DLT, resulting in fatal bowel perforation in two patients. Mucositis has also been observed with CCI-779 when given as a single agent [46]. The incidence of grade 3–4 stomatitis/mucositis with this schedule of 5-FU/LV is approximately 5% [14] which has been confirmed in subsequent larger studies. Because of these serious events as well as the fact that stomatitis/mucositis occurred at various dose levels, an interaction between CCI-779 and infusional 5-FU/LV could not be excluded and the study was terminated due to the unpredictable nature of these events. Since all patients in whom a clinical response, including prolonged disease stabilization, was achieved had tumor types that are 5-FU-sensitive, it is difficult to assess the relative contribution of CCI-779 to this result.

Following i.v. administration, the PK of CCI-779 in whole blood were non-linear and exhibited increasing clearance with increasing dose. This phenomenon is thought to be a result of saturable specific binding of CCI-779 to FKBP in the red blood cell. Sirolimus was a major metabolite, was present early after the start of dose administration, and decreased in an apparently mono-exponential fashion. The longer half-life of sirolimus relative to CCI-779 is an important determinant of the high parent/metabolite ratios observed with CCI-779.

The PK of 5-FU varies according to dose and schedule of administration. Clearance of 5-FU is reported to be much faster with continuous i.v. infusion than with bolus administration. For 24-h infusion and a dose of 2000–2600 mg/m2, a mean clearance of ~8 µM or ~780–1040 ng/ml has been reported. This is somewhat higher than the values reported herein, but the difference may be accounted for by diurnal increases in exposure not characterized in the present study. Notwithstanding, generally comparable exposures of 5-FU were observed between reference and test treatments, and these exposures were associated with moderate variability.

In conclusion, the administration of CCI-779 and 5-FU at these doses and schedule resulted in unacceptable toxicity. Three clinical responses were observed and these were for tumors for which 5-FU is often used as a treatment so that the contribution of CCI-779 to these responses is unknown. However, CCI-779 has been shown to be generally well-tolerated and antitumor activity in renal cell carcinoma and breast cancer has been observed [5, 6]. Thus, studies to determine the appropriate use of CCI-779 for the treatment of solid tumors need to continue. If CCI-779 is to be used in combination with 5-FU/LV, other doses or schedules of administration will need to be explored.


    Acknowledgements
 
The study was supported by Wyeth GmbH (Munich, Germany). Dr A. R. Hanauske (Onkologische Tagesklinik, Munich, Germany) also contibuted to this study. The authors thank the data managers from the IKO Trial Office (Nijmegen, The Netherlands) for their excellent support and Ms Lisa Salacinski for the PK programming support.


    Footnotes
 
+ Correspondence to: Dr C. J. A. Punt, Department of Medical Oncology, University Medical Center, St Radboud, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Tel: +31-24-3615215; Fax: +31-24-3540788; E-mail: c.punt{at}onco.umcn.nl Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
1. Huang S, Houghton PJ. Inhibitors of mammalian target of rapamycin as novel antitumor agents: from bench to clinic. Curr Opin Investig Drugs 2002; 3: 295–304.[Medline]

2. Neshat MS, Mellinghoff IK, Tran C et al. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci USA 2001; 98: 10314–10319.[Abstract/Free Full Text]

3. Yu K, Toral-Barza L, Discafani C et al. mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer. Endocr Relat Cancer 2001; 8: 249–258.[Abstract/Free Full Text]

4. Raymond E, Alexandre J, Depenbrock H et al. CCI-779, a rapamycin analog with antitumor activity: a phase I study utilizing a weekly schedule. Proc Am Soc Clin Oncol 2000; 19: 187a (Abstr 728).

5. Atkins MB, Hidalgo M, Stadler W et al. A randomized double-blind phase II study of intravenous CCI-779 administrated weekly to patients with advanced renal cell carcinoma. Proc Am Soc Clin Oncol 2002; 21: 10a (Abstr 36).

6. Chan S, Johnston S, Scheulen ME et al. First report: a phase 2 study of the safety and activity of CCI-779 for patients with locally advanced or metastatic breast cancer failing prior chemotherapy. Proc Am Soc Clin Oncol 2002; 21: 44a (Abstr 175).

7. Brattstrom C, Sawe J, Jansson B et al. Pharmacokinetics and safety of single oral doses of sirolimus (rapamycin) in healthy male volunteers. Ther Drug Mon 2000; 22: 537–544.[CrossRef][ISI]

8. Tejani A, Alexander S, Kohaut E et al. Safety and pharmacokinetic profile of ascending single doses of oral liquid sirolimus (rapamycin) in pediatric patients with stable chronic renal failure. Transplantation 1999; 67: S125 (Abstr).

9. Lampen A, Zhang Y, Hackbarth I et al. Metabolism and transport of the macrolide immunosuppressant sirolimus in the small intestine. J Pharmacol Exp Ther 1998; 285: 1104–1112.[Abstract/Free Full Text]

10. Sattler M, Guengerich FP, Yun C-H et al. Cytochrome P-450 3A enzymes are responsible for biotransformation of FK506 and rapamycin in man and rat. Drug Metab Dispos 1992; 20: 753–761.[Abstract]

11. Zimmerman JJ, Kahan BD. Pharmacokinetics of sirolimus in stable renal transplant patients after multiple oral dose administration. J Clin Pharmacol 1997; 37: 405–415.[Abstract/Free Full Text]

12. Heggie GD, Sommadossi JP, Cross DS et al. Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma. Cancer Res 1987: 47; 2203–2206.[Abstract]

13. Schathorn A, Kühl M. Clinical pharmacokinetics of fluorouracil and folinic acid. Semin Oncol 1992; 19 (Suppl 3): 82–92.[ISI][Medline]

14. Ardalan B, Chua L, Tian E et al. A phase II study of weekly 24-hour infusion with high-dose fluorouracil with leucovorin in colorectal carcinoma. J Clin Oncol 1991; 9: 625–630.[Abstract]

15. Weh HJ, Wilke HJ, Dierlamm J et al. Weekly therapy with folinic acid (FA) and high-dose 5-fluorouracil (5-FU) 24-hour infusion in pretreated patients with metastatic colorectal carcinoma. Ann Oncol 1994; 5: 233–237.[Abstract]

16. Jusko WJ. Guidelines for collection and analysis of pharmacokinetic data. In Evans WE, Schentag JJ, Jusko WJ (eds): Applied Pharmacokinetics. Principles of Therapeutic Drug Monitoring, 3rd edition. Spokane: Applied Therapeutics 1992; 1–43.

17. Petit E, Milano G, Levi F et al. Circadian rhythm—varying plasma concentration of 5-fluorouracil during a five-day continuous venous infusion at a constant rate in cancer patients. Cancer Res 1988; 48: 1676.[Abstract]