当前位置: 首页 > 期刊 > 《临床肿瘤学》 > 2005年第6期 > 正文
编号:11329450
Pharmacokinetics of 5-Azacitidine Administered With Phenylbutyrate in Patients With Refractory Solid Tumors or Hematologic Malignancies
http://www.100md.com 《临床肿瘤学》
     the Divisions of Medical Oncology, Experimental Therapeutics, and Hematologic Malignancy, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD

    ABSTRACT

    PURPOSE: To characterize the pharmacokinetic behavior of 5-azacitidine (5-AC), a cytidine nucleoside analog, when given with phenylbutyrate, a histone deaceytlase inhibitor.

    PATIENTS AND METHODS: Pharmacokinetic data were obtained from two trials involving patients with solid tumor and hematologic malignancies. 5-AC at doses ranging from 10 to 75 mg/m2/d was administered once daily as a subcutaneous injection for 5 to 21 days in combination with phenylbutyrate administered as a continuous intravenous infusion for varying dose and duration every 28 or 35 days. Serial plasma samples were collected up to 24 hours after 5-AC administration. 5-AC was quantitated using a validated liquid chromatograph/tandem mass spectrometry method.

    RESULTS: 5-AC was rapidly absorbed with the mean Tmax occurring at 0.47 hour. Average maximum concentration (Cmax) and area under the curve (AUC0-) values increased in a dose-proportionate manner with increasing dose from 10 to 75 mg/m2/d

    the mean ± SD Cmax and AUC0- at 10 mg/m2/d were 776 ± 459 nM and 1,355 ± 1,125 h*nM, respectively, and at 75 mg/m2/d were 4,871 ± 1,398 nM and 6,582 ± 2,560 h*nM, respectively. Despite a short terminal half-life of 1.5 ± 2.3 hours, inhibition of DNA methyl transferase activity in tumors of patients receiving 5-AC has been documented.

    CONCLUSION: 5-AC is rapidly absorbed and eliminated when administered subcutaneously. Sufficient 5-AC exposure is achieved to produce pharmacodynamic effects in tumors.

    INTRODUCTION

    5-azacitidine (5-AC

    Vidaza

    Pharmion Corp, Boulder, CO), a cytidine nucleoside analog, inhibits DNA methyltransferase, which leads to hypomethylation in the promoter region of transcriptionally silenced genes. The hypomethylation, which results in the re-expression of the silenced genes (ie, tumor suppressor or other genes), is one treatment strategy in anticancer therapy. 5-AC has anticancer properties and has been utilized in the treatment of various malignancies.1-3 5-AC was recently approved by the US Food and Drug Administration (US FDA) in the treatment of all myelodysplastic syndromes (MDS) subtypes (http://www.vidaza.com/corporateweb/vidazaus/home.nsf/Content/Home).4 Despite US FDA approval, the pharmacokinetics of 5-AC when given on varying dose and administration schedules has not been described, most likely as a result of the lack of sufficiently selective and sensitive methods of quantitation and inherent chemical instability of 5-AC. 5-AC instability has been noted in aqueous solutions and in biologic fluids, which is due to rapid hydrolysis to byproducts including 5-azacytosine and 5-azauracil.5-7

    Sequential addition of histone deacetylase inhibitors to cells preincubated with DNA methyltransferase inhibitors synergistically reactivates the expression of genes silenced through promoter methylation.8,9 The combination of 5-AC and phenylbutyrate was taken into clinical trials for both refractory solid tumors and hematologic malignancies (MDS and acute myeloid leukemia [AML]). These trials were designed to determine the pharmacologic dose where DNA methyltransferase inhibition, gene re-expression, or clinical response occurs. 5-AC at varying doses were administered subcutaneously for 5 to 21 days in combination with phenylbutyrate administered as a continuous intravenous infusion for varying dose and duration every 28 or 35 days. Pharmacologic studies were incorporated in both clinical trials to characterize the pharmacokinetics of 5-AC using a selective and sensitive liquid chromatograph/tandem mass spectrometry (LC/MS/MS) assay that was recently developed, validated, and confirmed to be reproducible and reliable over time.10

    PATIENTS AND METHODS

    Drug Administration

    5-AC was supplied as 100 mg of white, lyophilized powder with 100 mg of mannitol in 30-mL flint vials by the Cancer Therapy Evaluation Program, National Cancer Institute (NCI

    Bethesda, MD). Each vial was reconstituted with 4 mL of sterile water or 0.9% sodium chloride to provide a concentration of 25 mg/mL. Doses were divided so that no single injection constituted greater than 2 mL of slurry. 5-AC does not go into solution but forms a loose slurry when reconstituted in this fashion and must be used within 30 minutes of preparation. The dose was administered subcutaneously by the patient, with daily rotation of the injection site, for 7 to 21 days every 28 or 35 days. A dosing diary was maintained by the patient, who recorded the administration times. When pharmacokinetic studies were performed, the 5-AC dose was administered by trained research staff at the Sidney Kimmel Comprehensive Cancer Center (SKCCC) at Johns Hopkins University (Baltimore, MD).

    Clinical Trial Design

    Patients with advanced AML and MDS or refractory solid tumors were treated on one of two phase I trials at the SKCCC at Johns Hopkins University.1,11 The trials involved administration of 5-AC given subcutaneously in combination with phenylbutyrate, administered as a continuous intravenous infusion, to determine the safety, toxicity, and pharmacokinetics of 5-AC.

    In both studies, patients were required to meet the following eligibility criteria: > 18 years of age, Eastern Cooperative Oncology Group performance status 2, no active infections at the time of study entry, serum creatinine < 2 mg/dL, total serum bilirubin < 2.0 mg/dL, and negative pregnancy test (female patients of reproductive age). Previously treated or untreated patients with hematologic malignancies also were required to have no clinical evidence of CNS or pulmonary leukostasis, disseminated intravascular coagulation, CNS leukemia, and administration of hematopoietic growth factors must have been discontinued 3 weeks before protocol entry and was prohibited while on study. For the solid tumor trial, all patients were required to have a documented malignancy that was a solid tumor or lymphoma and required to have failed therapy of proven efficacy for their disease or for which no conventional therapy was available. Patients with solid tumors were also eligible if they had received chemotherapy if it was completed more than 4 weeks before treatment and with recovery from toxicity. All of the patients gave written informed consent approved by the institutional review board under guidelines of the Department of Health and Human Services. All toxicities were graded by the National Cancer Institute Common Toxicity Criteria (version 2.0) of the Cancer Therapy Evaluation Program.12

    The AML and MDS trial involved 5-AC doses of 25, 50, or 75 mg/m2/d administered for 5, 10, or 14 days with constant doses of phenylbutyrate (375 mg/kg/d administered as a continuous infusion beginning on the final day of 5-AC for 7 days). The solid tumor trial involved multiple combinations of 5-AC with phenylbutyrate utilizing three separate dosing regimens: (1) low doses of 5-AC, 10 or 15 mg/m2/d, for 14 days with phenylbutyrate administered as a 24-hour continuous infusion of 400 mg/kg/d on days 6 and 13

    (2) the approved dose of 5-AC, 75 mg/m2/d, for 7 days with phenylbutyrate administered as a 7-day continuous infusion of 200 or 400 mg/kg/d following 5-AC administration

    (3) low doses of 5-AC, 10 or 12.5 mg/m2/d, for 21 days with phenylbutyrate administered as a 24-hour continuous infusion of 400 mg/kg/d on days 6, 13, and 20. Accrual to the solid tumor trial is still ongoing at the schedule evaluating low-dose 5-AC for 21 days.

    Pharmacokinetic Sampling and Assay

    Blood samples were collected before 5-AC administration, then at 0.25, 0.5, 1, 2, 4, 8, and 24 hours after 5-AC. For the solid tumor study, an additional 6-hour sample was included. For the low-dose regimens in the solid tumor trial (see dosing regimen 3 in Clinical Trial Design section), pharmacokinetic sampling was repeated after daily dosing on day 7, which was the day after the completion of the phenylbutyrate 24-hour continuous intravenous infusion. Samples were processed and frozen within 30 minutes by centrifugation at 3,000 g for 5 minutes in a refrigerated centrifuge. Plasma (300 μL) was then aliquoted into two tubes with the remaining plasma in a third tube. Plasma was to be placed in the freezer within 30 minutes and stored at –80°C until analysis. Initially, samples were collected without tetrahydrouridine (THU). Due to presumed breakdown of 5-AC by cytidine deaminase, THU was added to the plasma supernatant at a final concentration of 100 μM to increase the stability of drug in plasma during storage in the freezer. THU increased freezer stability from 7 days to approximately 21 days when frozen at –80°C.10,13

    5-AC was quantitated in plasma using a previously described and validated LC/MS/MS method.10 Briefly, plasma samples (200 μL) were extracted using acetonitrile then cleaned up by Oasis MCX ion exchange solid-phase extraction cartridges (Waters Corp, Milford, MA). 5-AC was separated on an YMC Jsphr M80TM C18 column (YMC Co, Ltd, Kyoto, Japan) with gradient elution of ammonium acetate (2 mM) with 0.1% formic acid and methanol mobile phase. Due to the instability of 5-AC in plasma, all processing and handling of 5-AC samples was performed on ice until the samples were dried and reconstituted. Identification was through positive-ion mode and multiple reaction monitoring mode at m/z+ 244.9113.0 and 242.0126.0 for 5-AC and the internal standard, 5-methyl-2'-deoxycytidine, respectively. 5-AC and the internal standard eluted at approximately 5.4 and 4.6 minutes, respectively, and with a total run time of 30 minutes. The lower limit of quantitation of 5-AC was 5 ng/mL (20.4 nM) in human plasma, and linearity was observed from 5 to 500 ng/mL (20.4 to 2,041.6 nM) fitted by linear regression with 1/x weight.

    Pharmacokinetic Analysis and Statistics

    Individual concentration-time data were analyzed using noncompartmental methods using WinNonlin Professional version 3.1 (Pharsight, Mountain View, CA).14 Maximum plasma concentration (Cmax) was the observed value, as was the time to Cmax (Tmax). The terminal rate constant (z) was determined from the slope of the terminal phase of the plasma concentration-time curve using uniform weight. The terminal half-life (T1/2) was calculated as 0.693 divided by z. Area under the concentration-time curve (AUC) was calculated using the log/linear trapezoidal rule. The AUC was extrapolated to infinity (AUC0–) by using the equation, AUC0– = AUC0–t + Clast/z, where Clast was the final quantifiable concentration. The percent extrapolated was determined using the equation = (AUC0– – AUC0–t)/AUC0– x 100%. Dose-normalized AUC0– was calculated by dividing AUC0– by the nominal dose administered. Apparent systemic clearance (Cl/F) was calculated by dividing the dose by AUC0–. Apparent volume of distribution (Vd/F) was calculated by dividing apparent systemic clearance by the terminal rate constant.

    Pharmacokinetic parameters were summarized using descriptive statistics. Graphical presentation of concentration-time profiles consisted of the average and standard deviation of the 5-AC concentration determined at each time point. Dose-independent pharmacokinetic parameters (Tmax, T1/2, AUC percent extrapolated, Cl/F, and Vd/F) were compared using a Student's t test between the six pharmacokinetic periods, which were analyzed within 21 days of obtaining the samples and the remaining 22 pharmacokinetic periods that were not. The freezer stability in plasma spiked with known amounts of 5-AC was determined to be 21 days.10 One-way analysis of variance (ANOVA) was used to compare the differences in clearance and dose-normalized AUC0– as a function of dose level. ANOVA and Student's t test were performed using JMP Statistical Discovery software (version 4.0.4

    SAS Institute, Cary, NC). The a priori level of significance was set at P < .05.

    RESULTS

    5-AC was administered to 32 patients with hematologic malignancies and 25 patients with solid tumors. THU was added to the plasma starting with the 18th patient (total of 15 of 32) for the hematologic malignancies trial and the 17th patient (total of 9 of 25) for the solid tumor trial. Since 5-AC has been determined to be unstable in plasma that is not supplemented with THU, the data from the first 33 patients (17 hematologic malignancies and 16 solid tumor patients) was not included in this analysis.10,13 In addition, due to the presumed instability, we did not include any samples that were not initially processed from whole blood to plasma within 30 minutes or samples that would have been thawed more than once. Pharmacokinetic data from 24 patients (Table 1), involving 30 pharmacokinetic study periods, were included in this analysis. There were six patients from the solid tumor study that had 5-AC pharmacokinetics performed twice during treatment as per protocol. Of the 30 pharmacokinetic study periods, data for two were not assessable for pharmacokinetic analysis since the majority of samples were below the assay detection limit and therefore not reportable. Pharmacokinetic parameters were compared in all assessable pharmacokinetic periods between those patients whose 5-AC samples were analyzed within the documented freezer stability of 21 days (n = 6) and those who were not (n = 22). There was no statistically significant difference in Tmax (P = .84), T1/2 (P = .54), AUC percent extrapolated (P = .25), Cl (P = .14), and Vd (P = .29). Therefore, for all analyses, all 28 assessable pharmacokinetic periods were treated the same.

    Mean concentration-time profiles at the different 5-AC dose levels are illustrated in Figure 1. 5-AC was rapidly absorbed with the median Tmax occurring at 0.50 hours, the second pharmacokinetic observation. However, in 11 of 30 concentration-time profiles, in 11 different patients, Tmax occurred at the first pharmacokinetic observation (0.25 hours) while the maximal Tmax observed was at 1.07 hours. Mean ± standard deviation Cmax values increased with increasing dose, and was 190.0 ± 112.5 ng/mL (775.8 ± 459.2 nM) at 10 mg/m2/d and 1,192.8 ± 342.4 ng/mL (4,870.6 ± 1,398.1 nM) at 75 mg/m2/d, respectively (Table 1 and Fig 2). Consistent with the increase in Cmax with increasing dose level, AUC0- increased from 331.8 ± 275.4 h*ng/mL (1,354.8 ± 1,124.5 h*nM) at 10 mg/m2/d to 1,611.8 ± 626.9 h*ng/mL (6,581.5 ± 2,559.7 h*nM) at 75 mg/m2/d (Table 1 and Fig 3). Interpatient variation in AUC0-, which was assessed as the difference between the minimum and maximum value at a given dose level, was 2.1- to 4.7-fold. It is noteworthy that some of the variability may stem from the two separate sampling schemas used in the trials. Dose-normalized AUC0- was 10.8 ± 6.1 h*ng/mL/mg (44.1 ± 24.9 h*nM/mg) and was similar among the different dose levels (P = .14).

    The overall T1/2 was short and determined to be 1.50 ± 2.30 hours, which is consistent with 5-AC being undetectable at 8 hours post-treatment in most samples. The apparent volume of distribution was 192.5 ± 201.8 L (mean ± SD). Apparent clearance was 114.4 ± 51.2 L/h (mean ± SD) with a coefficient of variation of 44.8% and was similar among the different dose levels (P = .37). In the four solid tumor patients with assessable pharmacokinetics with repeated 5-AC administration of 10 mg/m2/d, 5-AC pharmacokinetic parameters did not change significantly (P > .05). In addition, no accumulation was observed in the pretreatment samples after repetitive dosing as evidenced by no detectable 5-AC in the pretreatment samples.

    DISCUSSION

    Due to the instability of 5-AC in plasma, including short-term storage at room-temperature, and long-term storage in the freezer, and the difficulties in developing and validating a specific, sensitive, and reproducible assay, minimal studies have been performed to characterize the pharmacokinetics of 5-AC.5-7,10 Quantitation in plasma was initially described using a microbiologic assay, but this assay was not specific for 5-AC due to the degradation products having residual cytostatic properties.15 A more recent attempt to quantitate 5-AC in plasma involved an ion pairing high-performance liquid chromatography method with the lower limit of quantitation of 250 ng/mL.7 Before this study, only three trials examined the pharmacokinetics of 5-AC: two quantitated 5-AC using radio-labeled 5-AC and one used the microbiologic assay.16-18 Lomen et al16 explored the pharmacokinetics of continuous infusion 5-AC at doses of 150 to 200 mg/m2/d for 5 days using the microbiologic assay. No quantifiable 5-AC was found in plasma and only 0.9% of 5-AC was recovered in the urine over a 96-hour collection period. In the two separate studies utilizing radio-labeled 5-AC administered either intravenously or subcutaneously, quantifiable levels of radioactivity were determined in both urine and plasma. The half-life of total radioactivity in plasma ranged between 3.2 and 6.4 hours when 5-AC was administered subcutaneously, as an intravenous bolus, or as a continuous intravenous infusion.17,18 Between 73% and 98% of the radioactivity was recovered in the urine within 72 hours after intravenous administration, suggesting that renal elimination of 5-AC and the metabolites is the main elimination route.17 Of note, minimal amounts of radioactivity were also present in erythrocytes, leukocytes, feces, acsites, cerebral spinal fluid, and tumor tissues, suggesting limited distribution of 5-AC and the metabolites.17,18 Despite minimal data being available regarding the pharmacokinetics and clinical pharmacology, 5-AC was approved by the US FDA because of the clinical effects in MDS.

    In order to characterize the clinical pharmacokinetics of 5-AC utilizing low-dose, daily administration schedules, a more sensitive and specific method of quantitation using LC/MS/MS was developed and validated to quantitate 5-AC as low as 5 ng/mL.10 With this method, a plasma concentration-time profile could be measured and described. Tmax varied at all dosage levels and may be due, in part, to 5-AC being administered subcutaneously as a slurry which is not fully dissolved upon initial administration. However, 5-AC being administered as a slurry did not prevent 5-AC from being absorbed, as evidenced by linearity observed in the dose-normalized AUC0- and the apparent clearance within the dose range studied. The interpatient variability in exposure could be attributed to several factors, including: an artifact of different pharmacokinetic sampling schemas being used between the two trials, instability of 5-AC in plasma, and inherent pharmacokinetic variability. To account for the variability as a result of potential degradation of 5-AC, the cytidine deaminase inhibitor, THU, was added to plasma at 100 μM which increased freezer stability from 7 days to approximately 21 days.10,13

    5-AC was eliminated rapidly from patients with a t1/2 of 1.5 hours, suggesting that daily dosing of 5-AC would be appropriate since there is no accumulation noted with 5-AC in the four patients with two pharmacokinetic observation periods. In addition, despite 5-AC concentrations being below the assay limit of quantitation by 6 to 8 hours, there has been significant inhibition of methyltransferase observed in bone marrow mononuclear-cells (42% to 85%) in multiple patients with hematologic malignancies and clinical benefit was observed in nine of 17 patients (53%) with hematologic malignancies.11,19 Methyltransferase inhibition was also observed in one patient with hepatocellular carcinoma at the 15 mg/m2/d dose. Only the higher doses of 5-AC (75 mg/m2/d) achieved plasma concentrations of 5 μM that were shown in vitro to cause methyltransferase inhibition.2 Given the measurable inhibition of methytransferase at lower concentrations than laboratory assays potentially suggests that pharmacodynamic effects can occur even at low concentrations. The disconnect between 5-AC plasma concentrations achieved at lower 5-AC doses and concentrations required in vitro to produce pharmacodynamic effects needs to be examined further. Several aspects for further study will involve characterizing the plasma protein binding of 5-AC and measurement of drug concentrations intracellularly and in tumor.

    The pharmacokinetics of 5-AC administered subcutaneously appear to be linear with respect to Cmax and AUC0- at the dose levels studied. Despite the short half-life of 5-AC, pharmacodynamic effects have been observed in the hematologic malignancy and solid tumor trials. Additional pharmacokinetic studies should further address characterization of the pharmacokinetics of 5-AC following repeated administration to aid in determining the optimal duration of 5-AC administration in combination with a deaceytlase inhibitor.

    Authors' Disclosures of Potential Conflicts of Interest

    The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Consultant: Steven D. Gore, Pharmion. Stock Ownership: Steven D. Gore, Pharmion. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section of Information for Contributors found in the front of every issue.

    Acknowledgment

    We thank the following people from Johns Hopkins University for their support during both clinical trials: Rana Adkamar, Kathleen Burks, Tianna Dauses, and Suzanne Dolan for nursing support

    Bettye Carr and Jill Stewart for data management

    Jatandra Birney, Susan Davidson, and Yelena Zabelina for their assistance in the pharmacokinetic quantitation.

    NOTES

    Supported by National Institutes of Health grants P30CA069773, U01CA70095, R01CA75525, and R01CA87760. Steven D. Gore was the recipient of a Scholar Award in Clinical Research from the Leukemia and Lymphoma Society of America.

    Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org.

    Authors' disclosures of potential conflicts of interest are found at the end of this article.

    REFERENCES

    Carducci MA, Gilbert J, Herman J, et al: Clinical trials with HDAC and DNA methylation inhibitors. Clin Cancer Res 7:3829s-3830s, 2001 (suppl)

    Claus R, Lubbert M: Epigenetic targets in hematopoietic malignancies. Oncogene 22:6489-6496, 2003

    Gilbert J, Gore SD, Herman JG, et al: The clinical application of targeting cancer through histone acetylation and hypomethylation. Clin Cancer Res 10:4589-4596, 2004

    Pharmion's Vidaza approved in US to treat MDS: Retracts EU thalidomide application. Cancer Drug News 122:8, 2004

    Notari RE, DeYoung JL: Kinetics and mechanisms of degradation of the antileukemic agent 5-azacytidine in aqueous solutions. J Pharm Sci 64:1148-1157, 1975

    Kissinger LD, Stemm NL: Determination of the antileukemia agents cytarabine and azacitidine and their respective degradation products by high-performance liquid chromatography. J Chromatogr 353:309-318, 1986

    Rustum AM, Hoffman NE: High-performance liquid chromatographic determination of 5-azacytidine in plasma. J Chromatogr 421:387-391, 1987

    Zhu WG, Lakshmanan RR, Beal MD, et al: DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors. Cancer Res 61:1327-1333, 2001

    Cameron EE, Bachman KE, Myohanen S, et al: Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 21:103-107, 1999

    Zhao M, Rudek MA, He P, et al: Quantification of 5-azacytidine in plasma by electrospray tandem mass spectrometry coupled with high-performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 813:81-88, 2004

    Gore SD, Baylin SB, Carducci MA, et al: Sequential DNA methyltransferase and histone deacetylase inhibition to re-express silenced genes: Pre-clinical and early clinical modeling. Proc Am Assoc Cancer Res 42:681, 2001

    Wittes R: Manual of Oncologic Therapeutics. Philadelphia, PA, Lippincott, 1991

    Zhao M, Newman EM, Doroshow JH, et al: Sensitive and specific method for the determination of 5-aza-2'-deoxycyticine in human plasma by LC-MS-MS. Proc Am Assoc Cancer Res 43:1067, 2002

    Gibaldi M, Perrier D: Noncompartmental Analysis Based on Statistical Moment Theory, Pharmacokinetics (ed 2). New York, NY, Marcel Dekker, 1982, pp 409-417

    Pittillo RF, Woolley C: 5-azacytidine: Microbiological assay in mouse blood. Appl Microbiol 18:284-286, 1969

    Lomen PL, Baker LH, Neil GL, et al: Phase I study of 5-azacytidine (NSC-102816) using 24-hour continuous infusion for 5 days. Cancer Chemother Rep 59:1123-1126, 1975

    Israili ZH, Vogler WR, Mingioli ES, et al: The disposition and pharmacokinetics in humans of 5-azacytidine administered intravenously as a bolus or by continuous infusion. Cancer Res 36:1453-1461, 1976

    Troetel WM, Weiss AJ, Stambaugh JE, et al: Absorption, distribution, and excretion of 5-azacytidine (NSC-102816) in man. Cancer Chemother Rep 56:405-411, 1972

    Gore SD, Baylin SB, Dauses T, et al: Changes in promoter methylation and gene expression in patients with MDS and MDS-AML treated with 5-azacitidine and sodium phenylbutyrate. Blood 104:469a, 2004(Michelle A. Rudek, Ming Z)