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Clinical Cancer Research 14, 3477, June 1, 2008. doi: 10.1158/1078-0432.CCR-07-4521
© 2008 American Association for Cancer Research
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Cancer Therapy: Clinical

Oral Administration of Gemcitabine in Patients with Refractory Tumors: A Clinical and Pharmacologic Study

Stephan A. Veltkamp1,2, Robert S. Jansen3, Sophie Callies6, Dick Pluim1, Carla M. Visseren-Grul5, Hilde Rosing3, Susanne Kloeker-Rhoades5, Valerie A.M. Andre6, Jos H. Beijnen2,3,4, Christopher A. Slapak5 and Jan H.M. Schellens1,2,4

Authors' Affiliations: 1 Division of Experimental Therapy and 2 Department of Clinical Pharmacology, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital; 3 Department of Pharmacy and Pharmacology, The Netherlands Cancer Institute/Slotervaart Hospital, Amsterdam, the Netherlands; 4 Faculty of Science, Department of Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands; 5 Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis; and 6 Lilly Research Laboratories, Lilly Research Center, Eli Lilly and Company, Windlesham, United Kingdom

Requests for reprints: Sander Veltkamp, Division of Experimental Therapy, The Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands. Phone: 31-512-2047; Fax: 31-512-2050; E-mail: s.veltkamp{at}nki.nl.


   Abstract
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 Abstract
Patients and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Purpose: To determine the toxicity, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of oral gemcitabine (2',2'-difluorodeoxycytidine; dFdC) in patients with cancer.

Experimental Design: Patients with advanced or metastatic cancer refractory to standard therapy were eligible. Gemcitabine was administered p.o. starting at 1 mg once daily using dose escalation with three patients per dose level. Patients received one of two dosing schemes: (a) once daily dosing for 14 days of a 21-day cycle or (b) every other day dosing for 21 days of a 28-day cycle. Pharmacokinetics were assessed by measuring concentrations of dFdC and 2',2'-difluorodeoxyuridine (dFdU) in plasma and gemcitabine triphosphate in peripheral blood mononuclear cells, and pharmacodynamics by measuring the effect on T-cell proliferation.

Results: Thirty patients entered the study. Oral gemcitabine was generally well-tolerated. The maximum tolerated dose was not reached. Mainly moderate gastrointestinal toxicities occurred except for one patient who died after experiencing grade 4 hepatic failure during cycle two. One patient with a leiomyosarcoma had stable disease during 2 years and 7 months. Systemic exposure to dFdC was low with an estimated bioavailability of 10%. dFdC was highly converted to dFdU, probably via first pass metabolism and dFdU had a long terminal half-life (~89 h). Concentrations of dFdCTP in peripheral blood mononuclear cells were low, but high levels of gemcitabine triphosphate, the phosphorylated metabolite of dFdU, were detected.

Conclusions: Systemic exposure to oral gemcitabine was low due to extensive first-pass metabolism to dFdU. Moderate toxicity combined with hints of activity warrant further investigation of the concept of prolonged exposure to gemcitabine.

Gemcitabine (2',2'-difluorodeoxycytidine; dFdC) is used in the treatment of patients with various solid tumors (1, 2). It is currently formulated in an i.v. solution (Gemzar) and usually administered as 30-minute infusion at a dose of 1,000 to 1,250 mg/m2 on days 1 and 8 of a 21-day or days 1, 8, and 15 of a 28-day cycle. Gemcitabine is taken up into cells by nucleoside transporters (3) and is intracellularly phosphorylated to gemcitabine monophosphate by deoxycytidine kinase (EC 2.7.1.74) and subsequently converted by other kinases into its active diphosphate and triphosphate (dFdCTP) forms (1, 4). dFdCTP competes with the natural substrate dCTP for incorporation into DNA, thereby inhibiting DNA synthesis (5, 6) and blocking cells in the early S phase of the cell cycle (7). In addition, gemcitabine diphosphate inhibits ribonucleotide reductase (8) ultimately resulting into depletion of dCTP pools and enhanced incorporation of dFdCTP into DNA. Moreover, gemcitabine can potentiate its own cytotoxic effect via other pathways (6, 9). Besides, dFdC is extensively deaminated by cytidine deaminase (EC 3.5.4.5; ref. 9) into 2',2'-difluorodeoxyuridine (dFdU), which has been reported to be present mainly in liver of humans and kidney of mice (10). Clinical studies showed that dFdU has a long terminal half-life (t1/2, ~50 h) and is largely excreted in the urine (>90% of the dose; ref. 11).

Preclinical studies showed that the efficacy and tolerability of gemcitabine was highly dose schedule dependent (1, 12). Clinical studies suggested that protracted or continuous administration of i.v. gemcitabine results in higher antitumor activity in cancer patients (13, 14). dFdCTP levels have been measured in peripheral blood mononuclear cells (PBMC) and leukemic cells of patients treated with i.v. gemcitabine to investigate the pharmacokinetics of gemcitabine (15–17). Intracellular exposure to dFdCTP and its incorporation into DNA increased with the gemcitabine concentration (5) and dFdCTP accumulation correlated with gemcitabine cytotoxicity (18–20). Pharmacodynamics of gemcitabine can be explored by measuring the effect on T-cell proliferation, which was shown to be inhibited by gemcitabine at low nanomolar concentrations (21, 22).

It was postulated that continuous treatment with oral gemcitabine might be efficacious in human malignancies and that p.o. dosing would be more convenient for patients than i.v. administration. Oral gemcitabine showed antitumor activity against human colon, lung, and prostate tumor xenografts in mice after once daily dosing for 14 days (0.5-3 mg/kg) and every other day dosing for seven doses (1.25-10 mg/kg).7 Marginal hematologic toxicity and dose-responsive intestinal lesions and enteropathy were found in mice after single oral gemcitabine administration at high doses (23).

Based on the promising antitumor activity in several human tumor xenografts and the marginal hematologic toxicity in preclinical models, a phase I study was started to investigate the feasibility of oral gemcitabine at a continuous dosing schedule in patients with advanced solid tumors. The following dosing schedules were investigated: once daily dosing for 14 days of a 21-day cycle (part A) or every other day dosing for 21 days of a 28-day cycle (part B). Because of the observation of dose-limiting intestinal lesions in mice (23), a starting dose of 0.6 mg/m2/day corresponding to 1 mg/day was chosen for part A.

The objectives of this study were to determine the following: (a) the toxicity, tolerability, and maximum tolerated dose (MTD); (b) preliminary antitumor activity; and (c) the pharmacokinetics and pharmacodynamics of oral gemcitabine for both dosing schedules.


   Patients and Methods
Top
 Abstract
 Patients and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Eligibility. Patients with histologically or cytologically confirmed advanced and/or metastatic cancer for which no treatment of higher priority existed were eligible. Other eligibility criteria were as follows: age of ≥18 y, performance status of ≤2 on the Eastern Cooperative Oncology Group scale, and an estimated life expectancy of ≥3 mo. Previous chemotherapy, hormonal therapy, and radiotherapy had to be discontinued for at least 4 wk before study entry and 6 wk in the case of mitomycin-C or nitrosourea. Patients had to have adequate bone marrow function, defined as absolute neutrophil count of ≥1.5 x 109/L, platelets of ≥100 x 109/L, and hemoglobin of ≥5.6 mmol/L, and adequate renal and hepatic function defined as serum creatinine of ≤1.5 of the upper limit of normal (ULN), serum bilirubin of ≤1.5 times of the ULN, and alanine transaminase (ALT) and aspartate transaminase (AST) of ≤2.5 times of the ULN (or ≤5 of times the ULN in the case of tumor involvement in the liver). The study protocol was approved by the local Medical Ethics Committee and all patients had to give written informed consent.

Treatment plan and study design. Patients started with gemcitabine once daily for 14 d of a 21-day cycle (part A). Gemcitabine was p.o. administered as capsule(s) with one glass of tap water within 1 h after breakfast. Cohorts of three patients per dose level were used. A low flat starting dose of 1 mg and the introduction of 1 wk of rest were chosen based on the preclinical toxicology studies and criteria by DeGeorge et al (24). If no dose-limiting toxicity (DLT) was reported in the first three patients at any dose level, the dose was escalated. Dose levels were initially set at 2, 4, 8, 12, 15, and 20 mg. If one DLT occurred, three additional patients were enrolled at that dose level. DLTs were defined as any of the following events occurring during the first treatment cycle and related to study treatment: any grade 4 hematologic toxicity lasting longer than 5 d, or grade 3 or grade 4 nonhematologic toxicity (except for untreated nausea and vomiting). The MTD was defined as one dose level below the dose level at which two of six patients experienced a DLT. By protocol, part B was planned to start upon identification of the MTD in part A. In part B, patients were to receive oral gemcitabine every other day for 21 d of a 28-day cycle. The starting dose in part B was planned to be at the established MTD for part A because toxicity in part B was expected to be milder. Subsequently, dose escalation proceeded to the next dose levels. An additional six patients were planned to be treated at the dose level determined to be the MTD for part B.

Drug formulation. Gemcitabine (LY188011) was provided by Eli Lilly and Company as capsules containing gemcitabine as hydrochloride salt equivalent to 1, 5, or 15 mg of gemcitabine. The capsules were stored in the refrigerator at 2°C to 8°C.

Patient evaluation and follow-up. Pretreatment evaluation included a complete medical history and physical examination, vital signs, electrocardiogram, chest X-ray, hematology (hemoglobin, WBC, platelets, neutrophils, and lymphocytes), serum chemistry (bilirubin, alkaline phosphatase, AST, ALT, blood urea nitrogen, creatinine, calcium, glucose, total protein, albumin, sodium, and potassium), and pregnancy test (if indicated). Before each cycle, the physical examination and vital signs were repeated, hematology and serum chemistry were checked, and any concomitant medication was noted. During each cycle, hematology was checked on day 7 and 14 in part A and day 7, 14, and 21 in part B (±3 d). Serum chemistry was checked on day 7 in part A and day 14 in part B (±3 d). All toxicities were graded according to the Common Toxicity Criteria version 2.0. If hematologic toxicity (absolute neutrophil count is <1.0 x 109/L for 5 d or platelets are <50 x 109/L) or nonhematologic toxicity (grade 3/4, excluding nausea and vomiting) was observed within a cycle, the dose was reduced or was held until recovery and retreatment was allowed according to protocol. No new cycle of gemcitabine was allowed unless the absolute neutrophil count was ≥1.0 x 109/L and platelets were ≥100 x 109/L. If treatment was held for longer than 3 wk for toxicity reasons, the patient had to be removed from the study. Dose adjustments had to take into account the capsule strengths available (1, 5, and 15 mg), and therefore, dose reductions had to be rounded down to the nearest increment. Tumor assessments were done by radiological imaging and/or tumor measurement of palpable or visible examination at baseline and day 1 of every other cycle and were evaluated according to the RECIST criteria (25).

Analysis of dFdC and dFdU in plasma. Blood samples were collected during cycle 1 on day 1 and 2 at 0.5, 1, 2/3, 4, 6, 8, and 24 h after oral gemcitabine administration. On day 14 (part A) and 21 (part B) samples were collected at the following time points: predose, 0.5, 1, 2, 4, 6, 8, 24, 48 to 72 h, and 7 d (day 21 of 28; ± 1 day) after p.o. intake of gemcitabine. Blood samples of 3 mL of venous blood were drawn into sodium-heparinized tubes containing 500 µg tetrahydrouridine and immediately centrifuged at 4°C for 5 min at 1,500 x g. Plasma was collected and stored at –20°C until analysis. Analytic standards of dFdC and dFdU and [13C2][15N3]-dFdC and [13C4][15N2]-dFdU (internal standards) were obtained from Eli Lilly. Plasma samples were vortex mixed and centrifuged at 2,000 x g for 5 min, and 100 µL of plasma were transferred into a 2.0 mL well of a 96-wells plate. The same was done for the analytical standards, quality controls, and blank plasma. Three-hundred µL of Milli-Q were transferred to blank plasma and 300 µL of internal standards working solution were added to the samples. An OASIS column for solid-phase extraction was conditioned by 500 µL methanol and 500 µL Milli-Q. The samples with internal standards were mixed 5 times, transferred to the extraction plate, and washed with 500 µL Milli-Q. Then, dFdC, dFdU, and internal standards were eluted with 200 µL methanol. The eluate was collected and evaporated until dryness under nitrogen at 65°C for 45 min. Samples were reconstituted in 200 µL Milli-Q, vortex mixed, centrifuged at 2,000 x g for 5 min, and the supernatant was collected. Separation of the analytes was done by reversed phase high pressure liquid chromatography using an Ace 5 C18 HL column maintained at room temperature. The mobile phase consisted of a mixture of water (A) and methanol with 1% formic acid (B) using the following gradient: t = 0: 100% A; t = 0.25 min: 25% B; t = 2.0 min: 100% B; and t = 3.0 min: 100% A. The injection volume was 20 µL and the total run time was 4 min. The analytes were quantified using a tandem mass spectrometer with a turbo V ionspray source operating in the positive ion mode. The ionspray voltage was kept at 3.5 kV with a source temperature of 650°C. The following mass-to-charge (m/z) transitions were monitored: m/z 264 to 112 for dFdC, m/z 265 to 113 for dFdU, m/z 269 to 117 for [13C2][15N3]-dFdC, and m/z 271 to 119 for [13C4][15N2]-dFdU. The interassay accuracy for dFdC ranged from 0.1% to 6.5% and the interassay precision ranged from 2.0% to 3.7%, whereas for dFdU, the interassay accuracy ranged from –1.2% to 6.9% and the interassay precision ranged from 1.3% to 6.8%. The lower and upper limit of quantification of both dFdC and dFdU in human plasma were 0.5 and 500 ng/mL, respectively.

Analysis of dFdCTP in PBMCs. Blood samples were collected during cycle 1 on day 1 and 14 (part A) or 21 (part B) at 1, 4, 8, and 24 h and on day 21 (part A) or 28 (part B; ± 1 day) after p.o. administration of gemcitabine. Blood samples of 15 mL of venous blood were drawn in sodium-heparinized tubes, centrifuged at 4°C for 5 min at 1,500 x g, PBMCs were isolated, and dFdCTP concentrations were determined as described previously using our validated liquid chromatography-tandem mass spectrometry assay (26). The lower limit of quantification of dFdCTP in PBMCs was 0.277 ng/mg protein corresponding to 0.047 ng/106 cells (94 fmol per 106 cells).

To investigate whether dFdUTP, the triphosphate form of dFdU, was formed in PBMCs, the m/z transition of 503 to 159 was monitored. Blank human PBMCs were added to the reference compound dFdUTP and the response ratio (including ion suppression and mass spectrometry response) was determined. We compared the response ratio of dFdCTP to internal standards with dFdUTP to internal standards and used this factor to quantify the response ratios in the PBMC samples based on the dFdCTP to internal standards calibration curve. Internal standards, dFdCTP, and dFdUTP had similar elution times.

T-cell proliferation assay. An explorative functional T-cell proliferation assay was used to characterize the effect of the treatment on T-cell proliferation in patients as a pharmacodynamic surrogate marker for bone marrow toxicity and or activity of oral gemcitabine. T-cell proliferation index (PI) of both CD4+ and CD8+ T cells was determined during cycle 1. In brief, samples were taken on day 1 (predose), on day 14 (part A) and 21 (part B) 1 h after p.o. dosing, and on day 21 (part A) and 28 (part B; ±1 day). Samples of healthy volunteers were taken as a control. For each sample, 16 mL whole blood was collected in BD Vacutainer CPT tubes with sodium citrate, and after centrifugation, PBMCs were isolated and resuspended in medium. Cells were counted, and 0.5 x 106 PBMCs in 1 mL were plated in 9 wells of a 24-well plate; 3 wells were used for flow cytometry settings, 2 for internal positive control, and 4 wells for the samples (2 of these wells were precoated with anti-CD3 to stimulate T-cell proliferation). Cells were cultured for 3 d. Then, 10 µL of anti–CD4-FITC and anti–CD8-phycoerythrin antibody were added to 500 µL of the samples. After washing and centrifugation, cells were taken up in 450 µL washing buffer. A volume of 50 µL of flow count beads was added to all samples. Both CD4+ and CD8+ T cells were counted within 1 h using fluorescent-activated cell sorting. The PI was calculated by the ratio of cell number after stimulation and cell number without stimulation of proliferation.

Pharmacokinetics/pharmacodynamics analysis. The pharmacokinetic variables of dFdC and dFdU in plasma, and of dFdCTP and dFdUTP in PBMCs were determined by noncompartmental analysis using WinNonLin version 5.0.1. The area under the plasma concentration time curve up to 24 h (AUC0-24) was determined and up to the last measured concentration-time point extrapolated to infinity (AUC0-{infty}) using the trapezoidal method. The maximal observed drug concentration (Cmax) was obtained directly from the experimental data. The t1/2 was determined for dFdU. PI values of T cells were calculated as described above. The pharmacokinetic variables were reported as median and range. Statistical analysis was done with SPSS version 12.1.1. Paired Student's t tests were applied on the log-transformed values of the pharmacokinetic variables to investigate the differences between day 14 (part A) or 21 (part B) and day 1. ANOVA and Bonferoni post hoc tests were conducted to investigate any differences in PI between day 1, 14, and 21 (part A) and 1, 21, and 28 (part B). Differences were considered to be statistically significant at P value of <0.05.

A Bayesian analysis using WinBUGS version 1.4 was applied to the dFdC pharmacokinetic data. The model fitted was a two-compartment disposition pharmacokinetics model with first-order absorption rate constant. The distribution of the pharmacokinetics variables in the population was assumed to be log normal. Previously reported pharmacokinetics variables after i.v. gemcitabine 500 to 3,600 mg/m2 in 353 patients were used as informative priors for the total and intercompartmental clearance, and the central and peripheral volume of distribution (11, 27). These studies with i.v. gemcitabine 1,000 mg/m2 as 30 min infusion showed a median Cmax of dFdC of 40,000 ng/mL (range, 28,000-52,000 ng/mL) and indicated a dose and time-independent clearance of dFdC and dFdU. In addition, cytidine deaminase has a reported Km for gemcitabine of 290 ± 20 µmol/L corresponding to 76,850 ± 5,300 ng/mL (28). These data indicate no saturation of cytidine deaminase after i.v. administration of gemcitabine at clinical doses.


   Results
Top
 Abstract
 Patients and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Patient characteristics, dose-levels tested, and main drug-related toxicities. Thirty patients were treated in this study and their characteristics are presented in Table 1 . The following dose-levels of gemcitabine were tested: 1, 2, 4, 6, and 8 mg (part A) and 8, 12, 16, and 20 mg (part B). Oral gemcitabine was generally well-tolerated without any DLTs, except for one patient treated with 8 mg daily gemcitabine as described below. All patients were evaluable for safety. Toxicity data are presented in Table 2 . The most prominent drug-related nonhematologic toxicities were nausea (37% of patients) and vomiting (30% of patients). Hematologic toxicity was mild and did not exceed Common Toxicity Criteria grade 2.


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  		Table 1. Patient characteristics

 

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  		Table 2. Drug-related toxicities: maximum grade across all cycles per dosing regimen and dose level

 
In part A, a total of 39 drug related adverse events were experienced by 11 of 18 patients; 74% of these were ≤ grade 2. In part B, 19 adverse events were experienced by 6 of 12 patients; all ≤ grade 2 (Table 2). Of these 58 observed adverse events, 55% were grade 1, 28% were grade 2, and 17% were grade 3 and 4 (corresponding to 4 patients in part A). Gastrointestinal-related toxicities (i.e., vomiting, nausea, abdominal pain, constipation, diarrhea, and anorexia) corresponded to 53% and 50% of the grade 1 and grade 2 adverse events, respectively. In part A, 54% of the toxicities were gastrointestinal related of which 52% were grade 2/3, and in part B, 53% were gastrointestinal related of which 30% were grade 2/3.

Fourteen patients experienced one or more serious adverse events, of which only 4 patients in part A (1 at 1 mg, 2 at 4 mg, and 1 at 8 mg) experienced serious adverse events related to the study drug. For three of these patients, the nature of these serious adverse events were vomiting and nausea and were not considered DLTs. A 68-year-old male with an adenocarcinoma of unknown primary (ACUP) treated once daily with 8 mg oral gemcitabine was considered to have a toxic death. The first cycle was almost uneventful. However, 9 days after the initiation of the second cycle, he developed fever (temperature, 38.8°C), followed by the following DLTs: grade 4 liver and kidney failure with rapid steep increases in ALT (2,424 U/L) and AST (2,781 U/L), {gamma}-GT (237 U/L), lactate dehydrogenase (3,360 U/L), ureum (24, 6 mmol/L), and creatinine (200 µmol/L); grade 3 fatigue; nausea; anorexia; and grade 3 disseminated interstitial coagulation. All cultures were negative. Ultrasound of liver and kidney showed normal caliber of liver and biliary ducts, with however, an aberrant corticomedullar differentiation and a strongly reduced blood flow in the left kidney. Treatment with oral gemcitabine and comedication (simvastatin, irbesartan/hydrochloorthiazide, oxazepam, and omeprazol) were stopped, and the patient was given i.v prednisone 1 mg/kg. Despite this, his clinical situation deteriorated, and 5 days later, the patient died. Obduction revealed severe toxic induced liver necrosis, which was likely related to the study drug. Other possible causes for severe liver toxicity, such as viral infections (hepatitis B and C), liver metastasis, and alcohol abuse were excluded and an interaction between gemcitabine and comedication was unlikely.

Due to this lethal toxicity, the dose of the other patient on 8 mg daily treatment was deescalated to 4 mg, and this dose level was expanded by inclusion of 3 extra patients. Review of safety and pharmacokinetic data of the first 18 patients enrolled in five dose levels (1, 2, 4, 6, and 8 mg) in part A led to the pharmacologically driven decision to stop enrollment in part A in the absence of a MTD. The exposure to dFdC and dFdCTP was low and variable at all dose levels. It was expected that the dosing schedule in part B would allow higher doses of oral gemcitabine to be safely administered, possibly leading to increased dFdC exposure. Dose escalation in part B was carried out from 8 to 12, 16, up to 20 mg and stopped at that level in the absence of a MTD because of the low systemic exposure to dFdC and dFdCTP.

Response. Twenty-seven of the 30 patients were evaluable for response. The total number of cycles of therapy completed by all patients was 112. Seven patients (23%) had stable disease and 20 patients (67%) developed progressive disease. Of the 7 patients with stable disease, 5 subjects were treated in part A at the dose levels of 2 mg (n = 1), 4 mg (n = 2), 6 mg (n = 1), and 8 mg (n = 1), and 2 subjects were treated in part B at the dose levels of 12 mg (n = 1) and 16 mg (n = 1). The patients with stable disease had the following tumor types: leiomyosarcoma (1x; 39 cycles), cholangiocarcinoma (1x; 2 cycles), ovarian carcinoma (1x; 4 cycles), ACUP (2x; 2 and 2 cycles each), adenocarcinoma of the pancreas (1x; 8 cycles), and adenocarcinoma of the parathyroid gland (1x; 6 cycles). One female with advanced leiomyosarcoma who had been treated with doxorubicin and ifosfamide received 2 mg oral gemcitabine once daily for 14 days of a 3-weekly cycle. She tolerated the therapy well with only a few delays in the start of new treatment cycles due to 2- to 3-fold increased liver enzyme values that were also present before start of therapy [at start: AST, 66 U/L (ULN, 40 U/L); ALT, 124 U/L (ULN, 45 U/L)]. She had stable disease for 27 cycles of therapy and then had to switch to 5 mg every other day for 21 days of a 4-weekly cycle because the supply of 1 mg capsules was exhausted. She received another 12 cycles of therapy. After having stable disease for 39 cycles, the therapy was stopped because she developed increased liver enzyme values (AST, 180 U/L; ALT, 246 U/L), which was likely caused by oral gemcitabine. Treatment had to be held for >3 weeks as a result of the increased liver function tests, and the patient was required by protocol to stop study treatment. She was on study for 2 years and 7 months. Concentrations of dFdC and dFdU in plasma and levels of dFdCTP in PBMCs during cycle 1 were comparable with those of the other patients.

Pharmacokinetics/pharmacodynamics of gemcitabine. Blood sampling was done in all 30 patients. The pharmacokinetic variables of dFdC, dFdU, dFdCTP, and dFdUTP are shown in Table 3A and B . Concentration versus time profiles are depicted in Fig. 1 , and Cmax values are shown in Fig. 2 . Systemic exposure to dFdC was low at all dose levels due to extensive first-pass metabolism to dFdU, and no dose-dependent increase in exposure to dFdC was observed (Figs. 1 and 2). For dFdC, Cmax values are presented only because AUC and t1/2 could not be precisely calculated. Median bioavailability of gemcitabine was estimated at 10% (range, 5-17%). Because the study was terminated, it was decided not to give the patients a low dose of i.v. gemcitabine that was planned at the end of the study for calculation of the bioavailability of oral gemcitabine.


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  		Table 3.

 

Figure 1
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  		Fig. 1. Concentration versus time curves of dFdC (ng per mL) and dFdU (ng per mL) in plasma on day 1 (A) and 14 (B) and of dFdCTP in PBMCs (ng per mg protein) on day 1 (C) and 14 (D) after daily administration of oral gemcitabine. Points, mean on a semilogarithmic scale; bars, SD.

 

Figure 2
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  		Fig. 2. Cmax of dFdC, dFdU, dFdCTP, and dFdUTP in part A (A-C) and part B (D-F) on day 1 and day 14 (part A) and day 1 and 21 (part B) after oral administration of gemcitabine. Columns, mean; bars, SD.

 
The systemic exposure to dFdU increased with dose after single and multiple administration and showed accumulation after once daily and every other day dosing of oral gemcitabine (Figs. 1 and 2). This is explained by the long t1/2 of dFdU. As expected, accumulation of dFdU was approximately twice as high after once daily (~5- to 6- fold) compared with every other day dosing (~2.5- to 3-fold; Fig. 2).

From the pharmacokinetics of i.v. gemcitabine, it is known that almost all of the administered gemcitabine dose (>90%) is excreted into the urine as dFdU (11); hence, the fraction of the total gemcitabine dose that is converted into dFdU (Fm) is almost 1. The pharmacokinetics data for dFdU, after oral gemcitabine administration, support the estimation of an apparent dFdU clearance (Cl/(F*Fm) = Dose/AUC; F = bioavailability) of 0.5 L/h and of a t1/2 of 89 h. With Fm being close to 1, the apparent clearance of dFdU can be considered as Cl/F. After i.v. gemcitabine, the t1/2 of dFdU is ~50 hours and Cl/F is ~1 to 2 L/h. The differences in Cl/F and t1/2 of dFdU after oral and i.v. administration of gemcitabine show that first-pass metabolism is the rate-limiting step in the pharmacokinetics of dFdU after p.o. administration; hence, dFdC is metabolized in the liver and dFdU is slowly released into the systemic circulation. Thus, the pharmacokinetics of dFdU after p.o. administration of gemcitabine is indicative of a "flip-flop" phenomenon.

Concentrations of dFdCTP in PBMCs were detectable by our sensitive assay up to 24 hours after p.o. administration of low-dose gemcitabine. The Cmax of dFdCTP slightly increased with dose (Fig. 2) but showed high interpatient variability. The mean AUC of dFdCTP in PBMCs (part A+B) was 32 h*ng/mg protein (~12.8 h*pmol per 106 cells) after single p.o. administration of 8 mg gemcitabine.

We hypothesized that dFdUTP was formed after administration of gemcitabine. Indeed, a significant peak eluting just after the dFdCTP peak was detected and dFdUTP eluted at the same retention time as the peak in the patient samples. Exposure to dFdUTP was higher compared with dFdCTP at almost all dose levels. Cmax and AUC of dFdUTP increased with dose of oral gemcitabine (Table 3 and Fig. 2; dFdUTP levels were available for all patients, except for the patients at the 20 mg dose level). The mean value of the dose normalized AUC of dFdUTP (AUC dFdUTP/gemcitabine dose) for all dose levels in part A after single dosing on day 1 (11 h*ng/mg protein) and multiple dosing on day 14 (35 h*ng/mg protein) was significantly higher (P = 0.001) than the dose normalized AUC of dFdCTP on day 1 (4.0 h*ng/mg protein) and day 14 (6.2 h*ng/mg protein).

The PI values of CD4+ and CD8+ T cells showed high interpatient variability. We recently determined that in cultured T cells from healthy volunteers (n = 42), the mean PI values of CD4+ and CD8+ T cells were 3.6 ± 1.9 and 3.2 ± 1.8, respectively.8 In this study, no significant effects of oral gemcitabine on T-cell PI were found, consistent with the low hematologic toxicity and low systemic exposure to gemcitabine. In part A, for example, the mean PI of CD4+ cells altered from 3.4 ± 2.6 on day 1 (predose) to 3.0 ± 2.6 on day 14 and to 3.9 ± 3.5 on day 21 (P = 0.714).


   Discussion
Top
 Abstract
 Patients and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
In this clinical study, we administered gemcitabine p.o. to patients with advanced solid tumors for the first time.

Overall, oral gemcitabine at low dose levels was well-tolerated. The study was discontinued mainly because of the unfavorable pharmacokinetics of dFdC. More precisely, once daily administration for 14 days of oral gemcitabine at a dose of 8 mg (corresponding to a 225-fold lower dose compared with i.v. gemcitabine 1,000 mg/m2) resulted in the following mean systemic exposures: dFdC Cmax of 4.4 ng/mL (~5,700-fold lower compared with i.v. gemcitabine), dFdU AUC0-24 of 13.8 x 103 h*ng/mL (~25-fold lower compared with i.v. gemcitabine), and dFdCTP AUC0-24 of 40 h*ng/mg protein or 13.5 h*pmol per 106 PBMCs (~100- to 200-fold lower compared with i.v. gemcitabine). After single i.v. gemcitabine, dFdCTP AUC0-24 values of ~1,400 h*pmol per 106 PBMCs (29–31) and a cumulative dFdCTP AUC >3 weeks of 9,700 h*pmol per 106 PBMCs have been reported (11). These data illustrate the significant difference in pharmacokinetics of gemcitabine after p.o. and i.v. administration.

The main pattern of toxicity observed in this study is gastrointestinal toxicity, whereas hardly any hematologic toxicity was observed, probably consistent with the low systemic exposure to dFdC. The incidence of gastrointestinal-related toxicity was similar between part A and part B, although the severity seemed to be less in part B (every other day) compared with part A (once daily). In the single patient in part A with lethal liver and kidney toxicity, exposure levels to dFdC, dFdU, and dFdCTP were comparable with those of other patients. However, the exposure to dFdUTP in this patient was high, with an AUC of 694 h*pmol/mg protein on day 14, compared with the other patients, in which AUC levels ranged from 20 to 270 h*pmol/mg protein after multiple daily dosing.

The study was terminated before the MTD was achieved because of the unfavorable pharmacokinetics of oral gemcitabine (low exposure to dFdC and dFdCTP and long t1/2 of dFdU) and the lethal liver toxicity observed in one patient. We hypothesized that there was a relationship between the pharmacokinetics findings and toxicity of oral gemcitabine. Therefore, we carried out preclinical studies to investigate cytotoxicity, uptake, metabolism, and biodistribution of dFdC, dFdU, and phosphorylated metabolites. In brief, in HepG2 cells, the IC50 values of dFdC and dFdU were ~1 nmol/L (~0.3 ng/mL) and 2.5 µmol/L (~660 ng/mL; ref. 32), respectively, after 14 days of drug exposure, comparable with the concentrations found in patients in this study. Furthermore, we discovered that dFdUTP is formed (via deamination of gemcitabine monophosphate to dFdUMP and via phosphorylation of dFdU) and incorporated into DNA and RNA (33). The long t1/2 of dFdU and its accumulation in patients after continuous oral gemcitabine administration might result in pharmacologically relevant concentrations for prolonged periods of time, and dFdU might contribute to the activity/toxicity of gemcitabine (e.g., by incorporation of dFdUTP into nucleic acids). Of note, accumulation of arabinofuranosyladenine-uridine triphosphate in leukemic blasts has been reported after treatment with cytarabine (1-β-D-arabinofuranosylcytosine) 0.5 g/m2/h administered as 2- or 4-hour infusion (34).

The best response was stable disease in seven patients with a median duration of three cycles in six of seven patients. Remarkably, the patient with advanced leiomyosarcoma had stable disease during 39 cycles of therapy after low dose oral gemcitabine. Second-line treatment with single agent i.v. gemcitabine was shown to be active in patients with leiomyosarcoma (35, 36).

In conclusion, this is the first study that tested oral administration of gemcitabine in patients. It shows that the systemic exposure to dFdC was low due to extensive first-pass metabolism to dFdU, which must be overcome to successfully deliver gemcitabine p.o. We discovered for the first time the formation of dFdUTP in PBMCs of patients. Recently, different approaches have been attempted to decrease deamination of dFdC to dFdU, such as coupling a long chain fatty acid or an isoprenoid chain of squalene to the terminal amino group of dFdC (37, 38), thereby protecting it from deamination by cytidine deaminase. Whether these strategies might ultimately lead to higher intracellular concentrations of dFdC and its active phosphorylated metabolites, possibly leading to an increased cytotoxicity, has to be further elucidated.


   Disclosure of Potential Conflicts of Interest
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 Abstract
 Patients and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
S. Callies, C.M. Visseren-Grul, S. Kloeker-Rhoades, V. Andre, and C.A. Slapak are employed by Eli Lilly and Company.


   Acknowledgments
 
We thank Annemarie Nol for her logistic assistance; Enaksha Wickremsinhe, Irina Kapsh, Vanaja Raguvaran, Ernest Wong, and Henrianna Pang for their work on the analysis of the dFdC and dFdU samples; and Philip Marder, Lisa Green, and Joanne Hom for their work on the T-cell proliferation assay.


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

7 Unpublished. Back

8 Unpublished. Back

Received 10/ 3/07; revised 2/ 6/08; accepted 2/15/08.


   References
Top
 Abstract
 Patients and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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