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© 2002 American Society for Clinical Oncology Prolonged Infusion of Gemcitabine: Clinical and Pharmacodynamic Studies During a Phase I Trial in Relapsed Acute Myelogenous LeukemiaByFrom the Departments of Experimental Therapeutics and Leukemia, University of Texas M.D. Anderson Cancer Center, Houston, TX. Address reprint requests to Varsha Gandhi, PhD, Department of Experimental Therapeutics, Box 71, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; email: vgandhi{at}mdanderson.org
PURPOSE: To determine the maximum tolerated duration of infusions at the fixed gemcitabine dose rate of 10 mg/m2/min and to analyze the pharmacodynamic actions in leukemia blasts during gemcitabine therapy. PATIENTS AND METHODS: The study was conducted in a phase I trial by escalating the duration of gemcitabine infusion at a fixed-dose rate of 10 mg/m2/min. Patients with relapsed or refractory acute myelogenous leukemia (AML) received gemcitabine for 8.0 (n = 3), 10.0 (n = 3), 12.5 (n = 8), 15.5 (n = 3), or 18.0 hours (n = 2). Pharmacokinetic and pharmacodynamic investigations were undertaken in circulating AML blasts. RESULTS: Gemcitabine was infused for up to 18 hours at the fixed-dose rate. Four patients had grade 3 toxicities at longer infusion schedules. One patient had a partial remission; two others had a reduction in blasts and concomitant rise in neutrophils. Gemcitabine triphosphate was detectable in AML cells even at 1 hour after the start of infusion in eight patients. The concentration ranged from 130 to 900 µmol/L at the end of the infusion. Consistently, there was a rapid decline in DNA synthesis, which remained suppressed at 85% to 95% during and for at least 10 hours after the end of the infusion. Compared with levels in cells measured before therapy, at 8 hours after the start of the infusion, there was a decline in the cellular purine deoxynucleotide pools. CONCLUSION: At the fixed-dose rate of 10 mg/m2/min, gemcitabine could be administered for longer than 12 hours without untoward toxicity. The favorable toxicity profile and pharmacokinetic and pharmacodynamic features warrant combination with DNA-damaging agents.
GEMCITABINE HAS been proven to be an effective agent in the treatment of a wide variety of solid tumors1-5 and hematologic malignancies,6,7 either as a single agent or in combination with other chemotherapeutic agents and modalities, such as irradiation. However, the dosing and scheduling vary greatly in these clinical investigations. Importantly, the maximum tolerated dose (MTD) is heavily dependent on the schedule and frequency of infusion.8,9 The effectiveness of gemcitabine depends on its metabolism to its monophosphate, diphosphate, and triphosphate, with the rate-limiting factor being the phosphorylation of the drug to its monophosphate, which is catalyzed by the deoxycytidine (dCyd) kinase.10,11 The more active monophosphokinases and diphosphokinases convert gemcitabine monophosphate efficiently to its diphosphate and triphosphate, respectively.12 Hence, maximizing the efficiency of triphosphate accumulation is dependent on achieving plasma gemcitabine concentrations that use the full capacity of dCyd kinase without greatly exceeding such concentrations. Previous studies using purified enzyme demonstrated that gemcitabine is an effective substrate for phosphorylation by dCyd kinase, having a Km value of 5 to 10 µmol/L.11,13 Furthermore, inhibition of the reaction has been shown at high concentrations of the substrate.11 Therefore, the rate of phosphorylation is expected to become saturated at gemcitabine concentrations greater than 20 µmol/L, with a negative effect occurring at higher levels of this agent. This contrasts with other nucleoside analogs that are phosphorylated by the dCyd kinase.14 Consistent with this observation using cells in culture, the rate of gemcitabine triphosphate accumulation is maximal at 10 to 20 µmol/L gemcitabine.10,15,16 Similar results were obtained when primary tumor cells such as acute or chronic leukemia blasts or lymphocytes were used in an in vitro system.17,18 Additionally, because of the efficient phosphorylation of gemcitabine (relative Vmax of 44 compared with natural substrate dCyd),14 it is expected that the rate of phosphorylation would be linear.11,14 When tested clinically, pharmacokinetic investigations have strongly suggested that the rate of gemcitabine triphosphate accumulation is saturated at 10 to 20 µmol/L gemcitabine in plasma.6,19 This concentration is achieved in plasma when gemcitabine is infused at a dose rate of 8 to 10 mg/m2/min.6 Clinically, doses of gemcitabine ranging from 800 to 2,600 mg/m2 are generally administered via intravenous infusion over 30 minutes,1-5 which generates gemcitabine concentrations in plasma that greatly exceed the level that saturates the rate of triphosphate accumulation18,19 and may in fact inhibit this process.11 Thus, under such conditions, it is not possible for the target cells to use a substantial portion of the infused drug, which is metabolically cleared through deamination.19 Although these studies strongly support the use of pharmacologically directed infusions of gemcitabine, there are no data regarding the tolerable duration of such administration. Hence, it is not known how long gemcitabine can be infused safely at the fixed-dose rate of 10 mg/m2/min if given as a single infusion per course. With regard to cellular pharmacology, the length of time that the accumulation of gemcitabine triphosphate remains linear throughout the infusion duration is not known, although linear rates have been observed for 8 hours.6 Similarly, previous studies have not considered the pharmacodynamic effect of gemcitabine on cellular deoxynucleotide pools in leukemia blasts. The present investigation was initiated to answer these questions with a focus on identifying the duration of infusion that would provide maximum pharmacologic and pharmacodynamic benefit.
Patients Nineteen patients with relapsed or primary refractory acute myelogenous leukemia (AML) received treatment at the University of Texas M.D. Anderson Cancer Center (Table 1). These patients were typical of those entered onto phase I trials. In particular, although they had relatively normal renal and hepatic functions (serum creatinine and bilirubin level < 2), they were felt to have a disease that was highly unlikely to respond to treatment (< 5% probability) given the number of unsuccessful regimens they had received previously and the brevity of their previous initial complete remission (CRs).
Early death was defined as death occurring within 2 weeks of the start of gemcitabine and, thus, in most cases, before evaluation of antileukemia effect can be made. The criteria for partial remission (PR) were a marrow with 6% to 25% blasts, a neutrophil count greater than 1,000/µL, and a platelet count greater than 100,000/µL. Hematologic improvement required either that the neutrophil count at least doubled and was greater than 1,000/µL or that the platelet count at least doubled and was greater than 100,000/µL. The treatment plan was based on our previous experience with gemcitabine6,17,18 using the pharmacologically guided fixed-dose rate of 10 mg/m2/min; the infusion duration was increased in a phase I setting. The starting infusion duration was 8 hours and was increased in increments of 20% to 25%; the final duration was 18 hours. This course was repeated every 21 to 28 days if there was evidence of a response to the first course. The protocol, along with its pharmacologic investigations, was approved by the M.D. Anderson Cancer Center institutional review board. All patients were informed about the investigational nature of this program in accord with institutional policies, and they signed consent forms to enter the study.
Drug and Other Chemicals
Blood Samples for Clinical Pharmacology Blood samples (10 to 20 mL) were obtained by laboratory phlebotomists and transferred to green-stopper Vacutainer tubes containing heparin and 1 µmol/L tetrahydrouridine (obtained from the National Cancer Institute, Bethesda, MD) to inhibit the conversion of gemcitabine to difluorodeoxyuridine by cytidine deaminase.19 The tubes were then immediately placed in an ice-water bath and transported to the laboratory. Control studies demonstrated that leukemic blasts are stable under these conditions with respect to their size and membrane integrity.20 The cellular nucleotide content was stable for at least 15 hours under these conditions.
Cellular Pharmacology
Determination of Intracellular Deoxynucleotide Triphosphate Pool
Inhibition of DNA Synthesis
Calculations and Statistical Analysis
Patient Characteristics and Clinical Responses Nineteen patients with relapsed (n = 14) or primary refractory (n = 5) AML were entered onto this phase I trial (Table 1). The median duration of first CR in all 19 patients was 12 weeks; in patients with relapsed AML, it was 19 weeks. All patients had received cytarabine-containing regimens as initial therapy. Three of the 19 patients received gemcitabine as first therapy after relapse (n = 2) or after failing initial induction therapy (n = 1). The remaining 16 patients had received one (n = 8) or two (n = 8) salvage regimens before receiving gemcitabine. These generally were other investigational agents, such as topotecan, pyrazine diazohydroxide, and tallimustine. The WBC counts listed in Table 1 were obtained before beginning gemcitabine. Similar to WBC, there were differences in the percent blast in peripheral blood and bone marrow (Table 1). Hydroxyurea was not used to control patients’ disease; rather, patients started treatment with gemcitabine. The dose rate of gemcitabine was the same in all 19 patients (10 mg/m2/min). The starting infusion duration was 8 hours and was escalated to 18 hours. As expected given the characteristics illustrated in Table 1, none of the 19 patients had a CR. However, one patient (no. 13) achieved partial remission, and two others (nos. 6 and 10) had an increase in neutrophils meeting the criteria for hematologic improvement described above. Patient no. 13 (first CR duration, 12 weeks; gemcitabine as first salvage therapy) began treatment with a neutrophil count of 200/µL, a platelet count of 43,000/µL, and a marrow that showed 40% blasts. After 2 courses of gemcitabine, the neutrophil count had risen to 1,500/µL, the platelet count had risen to 175,000/µL, and the blasts in the marrow had been reduced to 12%. Four subsequent courses of gemcitabine were given at monthly intervals, and the response lasted for 6 months. The neutrophil count of patient no. 6 (primary refractory; gemcitabine as second salvage therapy) rose from less than 100/µL before treatment to 1,300/µL after one course. Five additional courses of gemcitabine were given, and the neutrophil count remained above 800/µL for 5 months. Similarly, patient no. 10 (first CR duration, 25 weeks; gemcitabine as first salvage therapy) began gemcitabine with less than 100/µL neutrophils, with the neutrophil count increasing to 2,400/µL after one course of therapy. A second course was administered, and the response lasted for 1 month. None of the six patients who received gemcitabine at 10 mg/m2/min for less than 12 hours had severe toxicity compared with one of eight, one of two, and two of three patients in whom this infusion rate was extended to 12, 15.4, and 18 hours, respectively. Although it is thus difficult to ascertain the exact MTD, it seems likely that the MTD is between 12 and 15.4 hours. For example, the incidences of toxicity at infusion durations of less than 15.4 and more than 12 hours are significantly different (one of 14 v three of five, Fisher’s exact test; P = .04), (Table 1). Taken together, these data suggested that the maximum tolerated duration of infusion at this fixed-dose rate is between 12 and 15.4 hours. Only the two patients described as responders received more than one course of gemcitabine, but there was nothing to suggest cumulative toxicity.
Cellular Pharmacokinetics of Gemcitabine Triphosphate
To determine if cells had variations regarding retention of gemcitabine triphosphate, cells were collected after the end of the gemcitabine infusions and the level of triphosphate was quantitated. As shown in Fig 2, the elimination profile was monophasic, with a half-life of 7 hours. Although such a detailed investigation of gemcitabine triphosphate retention was not possible in all patients, less complete data for each patient suggested similar half-life in the circulating AML blasts. Taken together, these observations indicate that the differences in the intracellular concentration of gemcitabine triphosphate were attributable to variations in the phosphorylation of gemcitabine and the formation of the triphosphate, further stressing the importance of dCyd kinase in this equation.
Inhibition of DNA Synthesis During Therapy To determine the effect of gemcitabine infusion on DNA synthesis in circulating AML blasts, cells were isolated before therapy (control), at 1 hour during infusion, at the end of infusion, and 24 hours after infusion. These cells were incubated ex vivo with [3H]dThd, and incorporation into DNA was measured (Figs 1A and 1B). Compared with the control values, there was an abrupt decrease in DNA synthesis after 1 hour of the infusion that reached 5% of pretreatment value in one patient (Fig 1A) and less than 20% in another (Fig 1B). The same level of inhibition was observed at the end of the gemcitabine infusion in both patients. Interestingly, the decline in DNA synthesis was maintained up to 15 hours after the end of the infusion (24-hour value). The gemcitabine triphosphate concentration at 24 hours in these patients’ blasts was 260 and 160 µmol/L, respectively, indicating that DNA synthesis recovery did not occur at these levels (Table 2). Similar data were obtained in six patients (Table 2) with a median value of 90% inhibition at the end of the gemcitabine infusion (range, 79% to 99%). The gemcitabine triphosphate concentration at these times varied from 130 to almost 1 mmol/L. The inhibition of DNA synthesis was maintained in all of these patients, including those whose blasts retained only 33 µmol/L gemcitabine triphosphate at this time point. This may have been attributable to a sustained inhibition of DNA synthesis because of incorporated gemcitabine molecules or lowered deoxynucleotide pools, as shown below.
Cellular dNTP Pools During Therapy
Although the absolute concentrations of dATP in circulating AML blasts varied (median, 8.1 µmol/L; range, 1 to 63 µmol/L), the effects of gemcitabine infusions on this pool were similar (Table 3). In two cases, there was a slight increase initially in the concentration of dATP. However, there was a decline by the end of the infusion. Interestingly, the suppressed cellular dATP concentrations were maintained after the end of the infusion. At 24 hours, the decrease ranged from 22% to 60% (median, 40%). This decrease was not dependent on the infusion duration, because there was up to a 60% decrease in the dATP pool even at the shortest duration (hours). Similar to the dATP pool, there was a decline in the other purine deoxynucleotide, dGTP, in all but one patient (data not shown). At 24 hours, from 25% to 75% of the initial dGTP concentration remained in the cells.
Pyrimidine deoxynucleotides were similarly quantitated and compared for perturbation after gemcitabine treatment. Specifically, the median deoxycytidine triphosphate (dCTP) concentration in circulating AML blasts before therapy was 15 µmol/L (range, 1 to 19 µmol/L). By 24 hours, the value either was similar to the control level or had increased. The dTTP pool data were available only in three patient samples. In each case, by 24 hours, there was a 25% to 50% decline in the concentration of this deoxynucleotide.
Gemcitabine is the epitome of chemotherapeutic agents that have a strong association between the MTD and the infusion schedule.8 When administered frequently (ie, daily for 5 days), the MTD was found to be only 9 mg/m2/d when infused over 30 minutes.23 This is in sharp contrast to the MTD of more than 4,000 mg/m2/d when the drug is administered using a biweekly schedule.24 Additionally, doses ranging from 700 to 2,600 mg/m2/d have been successfully administered to patients as a 30-minute infusion on a weekly schedule (for 3 weeks).1-5 The commonality among these investigations is the duration of infusion, which by convention has been 30 minutes. This has become a standard infusion duration,25 even though it produces plasma concentrations of gemcitabine that are unlikely to be used completely for intracellular conversion to gemcitabine triphosphate.6,17-19 In contrast, the fixed infusion rate of 10 mg/m2/min serves as a pharmacologically guided dose rate to provide plasma concentrations (approximately 20 µmol/L)18,19 that are sufficient to maximize the rate of gemcitabine triphosphate accumulation. Such concentrations are achieved and maintained even when infusion duration is up to 21 hours.7 Also, when this fixed dose of gemcitabine was administered in phase I trials to patients with previously treated solid tumors, the MTD values were shown to be 1,500 mg/m2 over 150 minutes26 and 1,800 mg/m2 over 180 minutes weekly for 3 weeks.27 In contrast, patients with relapsed acute leukemia are able to withstand considerably greater doses of gemcitabine on this schedule. In a previous investigation, gemcitabine has been infused in patients with leukemia for up to 8 hours, ie, 4,800 mg/m2/wk for 3 consecutive weeks. This is consistent with most drugs that induce marrow suppression as the dose-limiting toxicity. The present investigation in patients with AML suggests that gemcitabine may be infused at this dose rate for up to 15 hours without untoward toxicity. This provides a pharmacokinetically optimized, pharmacodynamically characterized administration schedule that can serve as the basis for strategies that add a second drug or modality to capitalize on both the high accumulation of gemcitabine triphosphate and a decline in the dNTP concentrations to treat patients with AML. Several investigations have used gemcitabine at the fixed-dose rate of 10 mg/m2/min in patients with a variety of malignancies.26-31 The plasma pharmacology studies demonstrated that the gemcitabine concentrations of 10 to 20 µmol/L required to maximize the rate of triphosphate accumulation were achieved in plasma.6 The cellular accumulation of gemcitabine triphosphate was linear during this infusion for as long as 8 hours, an infusion duration that resulted in accumulation of substantially greater triphosphate concentrations than if gemcitabine were administered for a lesser duration.6 The fact that the peak level of gemcitabine triphosphate is frequently achieved at the end of the infusion (Table 2) further strengthens the rationale for using a prolonged infusion at a pharmacologically guided dose rate. Furthermore, a randomized comparative study of equitoxic regimens of gemcitabine infused over 30 minutes in a cohort of patients versus a fixed-dose rate infusion over 150 minutes in the second treatment arm demonstrated a linear rate of triphosphate accumulation in peripheral-blood mononuclear cells receiving a dose of 10 mg/m2/min.32 This contrasted with the hyperbolic kinetics of triphosphate accumulation over the same period after a conventional infusion of gemcitabine. In addition, direct comparison of a 1,000-mg/m2 dose of gemcitabine infused at two different dose rates in individual patients demonstrated a pharmacologic advantage of using a 150-minute infusion of gemcitabine (dose rate, 7 mg/m2/min) over using a standard 30-minute infusion.33 Clinical benefits in patients having pancreatic carcinomas after the 150-minute infusion compared with the 30-minute infusion provide additional support for the use of a pharmacologically optimized dose rate in gemcitabine infusion.32 The principal cytotoxic action of gemcitabine is inhibition of DNA synthesis,34 which results from decreased deoxynucleotide pools after inhibition of ribonucleotide reductase10,16,35 by gemcitabine diphosphate and incorporation of gemcitabine triphosphate into DNA.36-38 The immediate inhibition (1 hour after the start of the gemcitabine infusion) of DNA synthesis in AML blasts (Fig 1) suggests that, at least initially, this action may be attributable to incorporation of gemcitabine triphosphate into replicating DNA rather than an effect on deoxynucleotide pools. This is consistent with results obtained in vitro in whole cells, demonstrating that gemcitabine triphosphate incorporation into DNA is necessary for inhibition of DNA synthesis and induction of apoptosis.34 Such observations emphasize the need for maintaining the gemcitabine triphosphate pool intracellularly for a long period of time using prolonged infusion of the parent drug. The fact that the accumulation of gemcitabine triphosphate by AML blasts in the present study continued throughout infusions lasting 18 hours further strengthens the rationale for using prolonged infusion of the drug. In contrast to the inhibition of DNA synthesis, which occurred abruptly after 1 hour of the gemcitabine infusion, the effect on deoxynucleotide pools took a long time. This may have been attributable to low gemcitabine diphosphate levels in the cells during the early part of the infusion or repletion of deoxynucleotide pools through salvage pathways. Furthermore, by definition, the inhibition of DNA synthesis after incorporation of gemcitabine nucleotide decreased the use of deoxynucleotides. These postulates and findings are consistent with the need for a prolonged infusion of gemcitabine to derive maximum cytotoxic advantage.
Previous detailed investigations using the purified ribonucleotide reductase elucidated the mechanism-based inhibition of this protein by gemcitabine diphosphate.39-41 In this in vitro system, the effect of gemcitabine diphosphate was observed on both subunits.42 However, the reversible action of gemcitabine on the R2 subunit in the presence of reductant, which is likely to be present in circulating leukemia cells, suggests irreversible inactivation of the R1 subunit as the reasonable target for enzyme inactivation. Such inhibition of the R1 subunit depends on the availability of gemcitabine diphosphate and on the synthesis of new proteins to restore enzyme activity. In circulating leukemia cells, the concentration of gemcitabine diphosphate during prolonged infusion of gemcitabine is not known. Nonetheless, gemcitabine triphosphate is present at high concentrations in these cells throughout and up to 24 hours after the start of the infusion therapy (Fig 1 and Table 2). In cultured cells, there is a proportional amount of gemcitabine diphosphate ( The fluctuation for the protein level of the R2 subunit, although a maintained presence of the R1 subunit, also has been demonstrated in vitro in response to DNA damage in a p53-dependent manner47 in yeast,48 mouse and human cell lines,49,50 and human primary leukemia cells.51 The evolutionary measure that apparently evoked this response pathway is the need to provide deoxynucleotides for DNA repair-patch synthesis. Hence, the combination of gemcitabine with DNA repair-initiating agents, such as irradiation,52,53 cisplatin54,55 ifosfamide,56 and mitoxantrone,7 is a mechanistically rationalized strategy. Gemcitabine infusion has a dual effect on inhibition of DNA repair synthesis, causing direct incorporation of gemcitabine triphosphate into the repair patch and inhibition of ribonucleotide reductase by gemcitabine diphosphate. These findings additionally suggest the use of prolonged infusion of gemcitabine at a pharmacologically guided dose rate for maximal pharmacokinetic and pharmacodynamic advantage.
Supported in part by grant nos. PA30, CA16672, CA32839, CA55164, and CA57629 from the National Cancer Institute, Department of Health and Human Services, Bethesda, MD. We thank Deborah Cox for help in the preparation of the manuscript and Don Norwood for critically editing the manuscript.
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
5' exonuclease activity (United States Biochemical Corp, Cleveland, OH) was used to start a reaction in a mixture that contained 100 mmol/L HEPES buffer (pH 7.3), 10 mmol/L MgCl2, 7.5 µg bovine serum albumin, and synthetic oligonucleotides of defined sequences as templates annealed to a primer, [3H]deoxyadenosine triphosphate (dATP) or [3H]deoxythymidine triphosphate (dTTP), and either standard dNTP or the extract from 1 or 2 x 106 leukemia cells before and after therapy. Reactants were incubated for 1 hour and applied to filter discs. After they were washed to remove unincorporated [3H]dATP or [3H]dTTP, the radioactivity within the oligodeoxynucleotides retained on the discs was determined using liquid scintillation counting and compared with that with in the standard deoxyguanosine triphosphate (dGTP) samples. 
). Black bar below the abscissa indicates the duration of gemcitabine infusion.
), and dGTP (
) pools were measured. Black bar below the abscissa indicates the duration of gemcitabine infusion.
5% to 10% of the amount of intracellular gemcitabine triphosphate).
