Leukemia & Lymphoma, November 2014; 55(11): 2614–2619 © 2014 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2014.885515
ORIGINAL ARTICLE: RESEARCH
Correlation of plasma trough levels of imatinib with molecular response in patients with chronic myeloid leukemia Hemant Malhotra1, Pratibha Sharma1,2, Shipra Bhargava2,3, Om Singh Rathore1, Bharti Malhotra4 & Madhu Kumar2 1Division of Medical Oncology, Department of Medicine and 4Department of Microbiology, SMS Medical College, Jaipur, India
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and 2Department of Zoology and 3Department of Botany, University of Rajasthan, Jaipur, India
and progress to accelerated or blast phase [7]. Various mechanisms of resistance to imatinib have been identified, such as gene mutations in the kinase domain of BCR–ABL, BCR–ABL gene amplification, overexpression of Src related kinases, low expression and activity of drug influx transporters, drug efflux mediated by the P-glycoprotein which is encoded by the MDR1 gene and suboptimal plasma levels of imatinib. It has been suggested in recent studies that trough levels of imatinib in plasma may correlate with clinical responses in patients with CML [8–10] and that maintaining the plasma concentration above a threshold of about 1000 ng/mL (1.695 μM) may be important for improving response rates [9,10]. In this study, we assessed the correlation between mean trough imatinib plasma level and molecular response (as determined by BCR–ABL/ABL ratio by real time polymerase chain reaction [RQ-PCR]) in patients with chronic phase CML at our center. The imatinib threshold level that correlated with a better molecular response was determined using receiver operating characteristic (ROC) curve analysis. Furthermore, patients were divided into two categories: those who were taking Glivec and those who were taking generic/biosimilar imatinib. By statistical analysis, we analyzed whether there was a significant difference in mean trough levels of the drug between these two groups of patients who received different formulations of imatinib.
Abstract The present study looked at the correlation between mean trough Imatinib plasma levels and molecular response in 131 CML patients on imatinib. Patients receiving Glivec versus generic Imatinib were also compared. A ROC curve was constructed to estimate a threshold level that correlates with a favourable response. Patients were grouped into Responders (BCR/ABL ration by RQ-PCR less than 1) and Non Responders (ration 1). The mean trough imatinib plasma level in the responders was significantly higher than in the non responders (p 0.001). The area under ROC curve was 0.733, with best sensitivity (51.85%) and specificity (89.42%) at a plasma threshold of 0.988 μg/ml [1.675 μM]. Levels in the patients on Glivec versus generic drug (p 0.05) were comparable. Trough Imatinib plasma levels may be a marker for suboptimal response and may identify patients in whom increase of drug dose or change in therapy may be indicated. Keywords: Chronic myeloid leukemia (CML), imatinib, major molecular response (MMR), real time polymerase chain reaction (RQ-PCR), receiver operating characteristic (ROC) curve
Introduction Chronic myeloid leukemia (CML) is a malignant chronic myeloproliferative disorder (MPD) characterized by a marked increase in granulocytes, marked bone marrow hyperplasia and splenomegaly [1]. CML primarily affects adults between 25 and 60 years of age and accounts for 15–20% of all cases of leukemia [2]. It represents the most common type of adult leukemia in India [3,4]. Imatinib mesylate (Glivec) is currently regarded as the most specific and effective drug for patients with CML [5]. This drug works by directly inhibiting the cancer-causing protein BCR–ABL that is found inside leukemia cells, and therefore is the first successful targeted treatment for cancer [6]. Although most patients with chronic phase CML treated with imatinib mesylate have well-controlled disease, a considerable number of patients relapse
Materials and methods Patients and methods One hundred and thirty-one patients with CML in chronic phase who were on imatinib therapy for more than 24 months and in complete hematological remission (CHR) and attending the outpatient department of the leukemia/lymphoma clinic of the Birla Cancer Center, SMS Medical College Hospital, Jaipur, India were included in this study. The SMS Hospital (SMSH) at Jaipur, Rajasthan is a 2000-bed, tertiary care, super-specialty referral hospital, one of the largest in North India. The RK Birla Cancer Center, situated on the
Correspondence: Prof. Dr. Hemant Malhotra, Division of Medical Oncology, Department of Medicine, SMS Medical College & Hospital, Jawahar Lal Nehru Marg, Jaipur-302004, India. Tel: 91-141-2620600, 2620400. Fax: 91-141-2622899, 5105589. E-mail: [email protected] Received 12 October 2013; revised 26 November 2013; accepted 14 January 2014
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Plasma imatinib levels and molecular response 2615 SMSH campus, caters for more than 10 000 cases of cancer per year, and has a well-established leukemia/lymphoma clinic (since 1991). Out of 131 patients, 47 were taking generic imatinib/biosimilars while 84 patients were taking Glivec. Glivec was provided to patients through the Glivec International Patient Assistance Program (GIPAP). Glivec was provided to patients who were socioeconomically compromised, did not have medical insurance and did not have any source of reimbursement for the drug (as per the standard operating procedure [SOP] of the Max Foundation, the non-governmental organization [NGO] appointed by Novartis for conduct of the GIPAP program globally). All patients potentially eligible for Glivec through the GIPAP program were started on generic imatinib, but were shifted to Glivec as soon as Max Foundation approval for Glivec was obtained (usually within 2–3 weeks of the application). Informed consent was given by all patients for participation in this study. The study was approved by the Ethics Committee of the institution. Diagnosis of CML was established at presentation in all patients with demonstration of BCR–ABL gene quantification by RQ-PCR. As we do not have an in-house cytogenetics laboratory, or availability of samples, cytogenetics was not performed at baseline in any patient. All patients were started on 400 mg/day with monitoring of hematological responses every 4 weeks and molecular responses (by RQPCR on peripheral blood) every 6 months. Response criteria were: complete hematological remission (CHR), defined as a white blood cell (WBC) count of less than 10 109/L, a platelet count of less than 450 109/L, differential without immature granulocytes and with less than 5% basophils and non-palpable spleen; partial hematological remission (PHR), defined as above except for the presence of immature cells, platelet count 50% of pretreatment value but 450 109/L and persistent splenomegaly but 50% of pretreatment size; complete molecular response (CMR), defined as BCR–ABL mRNA transcript levels non-quantifiable and non-detectable by RQ-PCR; major molecular response (MMR), defined by the presence of a BCR–ABL/ABL ratio 1% as quantified by RQ-PCR. For BCR–ABL quantification, 10 mL of fresh peripheral blood was collected from each patient in ethylenediaminetetraacetic acid (EDTA) and the sample was kept at 4°C until further processing, within 2 h of collection. The sample was treated with red blood cell (RBC) lysis buffer (containing 155 mM NH4Cl and 0.1 mM NaHCO3) to lyse the red cells and the mononuclear cells were separated. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The yield, concentration and purity of the RNA were assessed using an Infinite 200 PRO NanoQuant (Tecan, Durham, NC), and only those samples with absorbance (A) A260/A280 ratios between 1.9 and 2.1 were further considered. Two micrograms of total RNA were reverse transcribed with a High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA) in a final volume of 20 μL according to the manufacturer’s instructions. cDNA was then used for quantification of BCR–ABL relative to ABL. Plasmid DNA standards having copy numbers 5 103, 5 104, 5 105, 5 106 and 5 107 were used as calibrators for the generation of standard curves for both BCR–ABL and
ABL. Reactions were performed on an ABI PRISM 7700 SDS (Applied Biosystems) instrument. The reaction conditions and the primer and probe sequences for BCR–ABL and ABL were the same as described previously [11]. A no-template control (NTC) containing nuclease-free water was included in each run. RQ-PCR results were analyzed using the standard curves, which were used to calculate the quantity of BCR–ABL and ABL transcripts (copy numbers) in the samples. A change in the BCR–ABL transcript level was expressed as the ratio of BCR–ABL/ABL. The patients were divided into two groups: responders, patients who achieved a CMR or a MMR (BCR-ABL/ABL ratio 1% as assessed by RQ-PCR); and non-responders, those without CMR or MMR (BCR–ABL/ABL ratio 1% as assessed by RQ-PCR). Imatinib plasma quantification was done using a high performance liquid chromatography ultraviolet (HPLC-UV) detection method as developed by Velpandian et al. [12], which included single-step extraction by methanol. As patients were followed up every 4 weeks, they were counseled about blood collection and compliance on the visit before the sample collection. One day before sample collection, the dose of imatinib was given under supervision and the sample was collected 22–24 h after this dose. Plasma was separated from the blood samples of patients by centrifugation at 2500 rpm for 15 min. These plasma samples were then stored frozen at 80°C until laboratory analysis. Basic measures of location (i.e. mean, median, range, etc.) were calculated to provide a detailed description of group characteristics. Student’s t-test was done to test the significance of means between the two groups. Fisher’s exact and χ2 tests were carried out for comparison of proportions. Receiver operating curve (ROC) curve analysis was done to determine the plasma threshold of imatinib that correlated with MMR.
Results One hundred and thirty-one patients with CML were included in this study. The patients’ baseline characteristics are shown in Table I. Patients were divided into two groups according to their molecular response to imatinib therapy: those with MMR (BCR–ABL/ABL ratio 1% as assessed by RQ-PCR) or better (responders, n 104) and those without MMR (BCR–ABL/ ABL ratio 1% as assessed by RQ-PCR) (non-responders, n 27). Characteristics of responders and non-responders are given in Table II. Plasma imatinib trough level was measured once for every patient. The imatinib trough levels in our subjects ranged from 0.288 to 7.11 μg/mL (0.488 μM to 12.05 μM). The mean plasma imatinib level in responders was 2.10 μg/mL (3.561 μM), range 0.37–7.11 μg/mL (0.63– 12.06 μM); 92 (88.46%) patients in the imatinib responder group had plasma imatinib levels 1 μg/mL ( 1.695 μM). The mean plasma imatinib level in non-responders was 1.31 μg/mL (2.22 μM), range 0.288–2.89 μg/mL (0.49– 4.9 μM). The mean trough imatinib plasma level in responders was significantly higher (2.10 1.18 μg/mL/3.56 2.00 μM) than in non-responders (1.31 0.72 μg/mL/2.22 1.22 μM) (p 0.001). ROC curve analysis was done to determine the plasma threshold of imatinib that correlated with a favorable
2616 H. Malhotra et al. Table I. Baseline characteristics of patients.
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Characteristic
Value
Age (years) Median Range Sex (n) Male Female Splenomegaly 10 cm below costal margin, n (%) Hemoglobin level (g/dL) Median Range Basophils (%) Median Range WBC count (/mm3) Median Range Platelet count (/mm3) Median Range Peripheral blasts 3% Sokal score, n (%) Low Intermediate High
32 18–63 81 50 20 (15) 10.4 6.7–16.3 1 0–4 52 000 3000–210 000 295 000 105 000–910 000 42 (32%) 41 (31.30) 50 (38.17) 40 (30.53)
WBC, white blood cell.
response.TheareaundertheROCcurvewas0.733,withbestsensitivity (51.85%) and specificity (89.42%) at a plasma threshold of 0.988 μg/mL (1.675 μM). Thus a plasma threshold of 0.988 μg/mL (1.675 μM) was significantly associated with the presence of a MMR or CMR, i.e. the two groups could be best separated at this concentration of imatinib in plasma. Patients were also classified into those who were on Glivec (n 84) and those who were on generic/biosimilar imatinib (n 47). Characteristics of patients in these two groups are shown in Table III. The mean imatinib plasma trough level in patients who were on Glivec was 2.07 1.15 μg/mL (3.51 1.95 μM) and in those who were on generic/ biosimilar imatinib was 1.70 1.11 μg/mL (2.88 1.88 μM), and the difference was found to be insignificant (p 0.079). The majority of the treated patients experienced adverse reactions some time during therapy. The toxicity profile of imatinib is given in Table IV.
Discussion In this study, we determined the minimum blood plasma concentration (“trough” level) of imatinib occurring just prior to taking the next dose by HPLC assay, which is a rapid
and sensitive technique and requires small blood samples for analysis. There are several published methods to determine levels of imatinib and its main metabolite norimatinib in human plasma. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) is recommended for most quantitative determinations of imatinib in the plasma of patients with CML, and has been described by several authors [13–16]. However, LC–MS/MS facilities are not always available in standard hospital laboratories as the equipment is quite expensive, as is its use [13,17,18]. Several authors have reported the use of HPLC methods with UV detection for the quantitation of imatinib in human plasma. Some recent studies have compared HPLC with UV/diode array detection to LC-MS/MS and found no significant differences between the two methods. In our study, we used the HPLC UV detection method developed by Velpandian et al. [12], which includes single-step extraction, and is rapid and simple to perform. The imatinib trough levels in our subjects ranged from 0.288 to 7.11 μg/mL (0.488–12.06 μM). This suggests that people differ markedly in how fast their bodies process and excrete imatinib. This difference might be due to variations in the expression and activity of metabolizing enzymes in the liver such as cytochrome P450. CYP3A4 is the major enzyme responsible for the metabolism of imatinib. Some polymorphisms affecting the expression and inducibility of CYP3A4 result in a 10-fold variability in CYP3A4 activity among different subjects, and this is one of the most important factors responsible for the large intra- and interindividual pharmacokinetics variability observed during drug treatments [19], which may further be influenced by a number of inhibitory or inducing compounds present in the environment. A decrease in the plasma concentration of imatinib can occur in patients treated with CYP3A4 inducers such as rifampicin [20] and St John’s wort, i.e. Hypericum perforatum L. extracts [21,22]. In contrast, an increase in plasma imatinib concentration may occur by co-administration of a CYP3A4 inhibitor such as ketoconazole [23]. Since imatinib is also an inhibitor of CYP3A4, co-interactions may occur between imatinib and inhibitors or inducers of these enzymes, leading to changes in the plasma concentration of imatinib. In addition, the binding of imatinib with plasma proteins such as alpha glycoprotein (AGP) may result in its altered plasma concentration, and may have potential consequences on the clinical response [24]. The bioavailability of
Table II. Characteristics of patients classified according to their molecular response to imatinib therapy. Characteristic Age* Plasma level, μg/mL (μM)* Sex category† Male Female Sokal risk group† Low risk ( 0.8) Intermediate risk (0.8–1.2) High risk ( 1.2)
Responders
Non-responders
p-Value
34.25 13.36 2.10 1.18 (3.56 2.00)
35.96 15.55 1.31 0.72 (2.22 1.22)
0.567 0.001
60.58% (63) 39.42% (41)
66.67% (18) 33.33% (9)
0.659
31.73% (33) 37.50% (39) 30.77% (32)
29.63% (8) 40.74% (11) 29.63% (8)
0.951
*Statistical analysis and p-value calculation by Students t-test. †Statistical analysis and p-value calculation by Fisher ’s exact test or χ2 test.
Plasma imatinib levels and molecular response 2617 Table III. Characteristics of patients classified according to formulation of imatinib. Characteristic Plasma level, μg/mL (μM)* Sokal score* Response category† Responder Non-responder Sokal risk group† Low risk Intermediate risk High risk Sex category† Male Female
Glivec
Generic imatinib
p-value
2.07 1.15 (3.51 1.95)
1.70 1.11 (2.88 1.88)
0.079
1.03 0.44
1.23 0.73
0.049
75.00% (63) 25.00% (21)
87.23% (41) 12.77% (6)
0.117
35.71% (30) 39.29% (33) 25.00% (21)
23.40% (11) 36.17% (17) 40.43% (19)
65.48% (55) 34.52% (29)
55.32% (26) 44.68% (21)
0.143 0.266
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*Statistical analysis and p-value calculation by Students t-test. †Statistical analysis and p-value calculation by Fisher ’s exact test or χ2 test.
imatinib can be substantially modified by the presence of high AGP levels that could increase the half-life of imatinib and could alter its tissue distribution and cellular uptake, which would interfere with the access of imatinib to its intracellular target protein, BCR–ABL [25]. However, the therapeutic concentrations of imatinib generally exceed the total binding capacity of AGP, thereby counterbalancing the effect of this plasma protein. Moreover, interindividual differences in the expression and activity of the imatinib influx (OCT-1) [26–29] and efflux transporters (ABC transporters) [30], which are the key determinants of drug disposition and response, lead to a highly variable drug elimination rate, and are therefore responsible for interindividual differences in circulating drug concentrations and therapeutic response. Individual patient characteristics, including age, sex, race and weight and inherited differences in drug absorption, metabolism, transport and disposition of drugs and interactions between prescribed medications, may also affect drug levels. Sampling time error could be another source of variability, although all samples were collected within 2 h, within acceptable sampling time period. Some patients had very low imatinib levels, and this might be due to poor compliance to drug therapy. There are no published data from India regarding compliance to imatinib, but data from
the West [31] indicate that this may be a major issue in suboptimal responders. In our study, the measure of a patient’s adherence to drug therapy was based on self-reporting by the patient. Although patients’ compliance was assured by repeated counseling, some patients might not have taken the medication and may not even have reported this behavior to the clinician. The reasons for poor compliance could be either side effects or cost issues. In our study, many of the side effects of imatinib were observed in the study population (Table IV), and these may account for the low compliance in some of the patients. In our study, we categorized the patients into two groups: those who were taking Glivec and those who were taking generic/biosimilar imatinib. By statistical analysis, we analyzed whether there was a significant difference in the mean trough levels of the drug between these two groups of patients. Our analysis data showed that there was no significant difference between the plasma levels of these two categories of patients (p 0.05). Based on these results, it is concluded that both products can be considered equally effective and interchangeable in medical practice. We also correlated the mean imatinib plasma levels with molecular responses to imatinib therapy in our patients. Patients were classified into responders and non-responders on the basis of molecular response to imatinib therapy as
Table IV. Toxicity profile of imatinib in patients with chronic myeloid leukemia. Glivec (n 84) Toxicity Superficial edema Nausea Muscle cramps Musculoskeletal pain Rashes and related events Diarrhea Headache Joint pain Abdominal pain Myalgia Vomiting Fatigue Dyspepsia Increased weight Insomnia Discontinuations due to AEs AE, adverse event.
Generic/biosimilar imatinib (n 47)
Grade 1/2, n (%)
Grade 3/4, n (%)
Grade 1/2, n (%)
Grade 3/4, n (%)
52 (61.9) 32 (38.1) 47 (55.95) 38 (45.23) 34 (40.47) 39 (46.42) 33 (39.28) 27 (32.14) 23 (27.38) 20 (23.80) 18 (21.42) 32 (38.09) 15 (17.85) 11 (13.09) 13 (15.47)
3 (3.5) 2 (2.38) 0 (0) 4 (4.76) 3 (3.5) 2 (2.38) 0 (0) 2 (2.38) 4 (4.76) 1 (1.19) 1 (1.19) 0 (0) 0 (0) 1 (1.19) 0 (0)
30 (63.8) 19 (40.42) 27 (57.44) 21 (44.68) 18 (38.29) 21 (44.68) 20 (42.55) 14 (29.78) 13 (27.65) 11 (23.40) 11 (23.40) 19 (40.42) 9 (19.14) 7 (14.89) 8 (17.02)
2 (4.25) 1 (2.12) 0 (0) 2 (4.25) 2 (4.25) 1 (2.12) 0 (0) 1 (2.12) 2 (4.25) 1 (2.12) 0 (0) 1 (2.12) 0 (0) 1 (2.12) 0 (0)
0 (0)
0 (0)
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2618 H. Malhotra et al. determined by the BCR–ABL/ABL ratio by RQ-PCR. Out of 131 patients, 104 who achieved either CMR or MMR were included in the responders while 27 patients who did not achieve either CMR or MMR were included in the nonresponders. Student’s t-test results showed that the mean plasma levels in responders (2.10 1.18 μg/mL/3.56 2.00 μM) were significantly higher than in non-responders (1.31 0.72 μg/mL/2.22 1.22 μM) (p 0.001). Several earlier studies also correlated the plasma imatinib trough levels with clinical responses in patients with CML and compared plasma imatinib levels in patients classified into responders and non-responders based upon molecular or cytogenetic responses. It was reported previously that patients having plasma imatinib levels 1 μg/mL (1.695 μM) achieved a CHR and that low plasma levels of imatinib were associated with inadequate hematological response [32,33]. In the study by Picard et al. [10], trough imatinib levels were found to correlate with the achievement of MMR, with patients achieving trough imatinib levels of 1002 ng/mL (1.699 μM) being significantly more likely to achieve a MMR (p 0.001). Larson et al. also studied the correlation of imatinib trough plasma concentrations with clinical responses, event-free survival (EFS) and adverse events (AEs) [9]. Imatinib trough plasma concentrations were found to be significantly higher in patients who achieved CCR versus those who did not achieve it (1009 544 ng/mL/1.711 0.922 μM vs. 812 409 ng/mL/1.377 0.694 μM, p 0.01). Also, patients with high imatinib exposure were found to have better rates of CCR, MMR and EFS. Singh et al. also performed a similar study in which the correlation of plasma level of imatinib with response to therapy was investigated, and it was demonstrated that the mean plasma imatinib level in responders were significantly higher than that in non-responders (2.34 vs. 0.70 μM, respectively, p 0.002) [34]. Awidi et al. carried out a similar study in Jordanian patients with CML who were on imatinib therapy for at least 12 months and found that the mean trough levels in patients with MMR or better were significantly higher than in those without MMR (2766 811 ng/ mL/4.69 1.38 μM vs. 1777 624 ng/mL/3.013 1.058 μM, respectively) [8]. Ishikawa et al. also obtained similar results in their study, and found that the plasma trough levels of imatinib among patients who achieved MMR during treatment (n 52) were higher than those for patients who did not achieved MMR (n 8) [35]. Marin et al. found that patients with plasma imatinib levels less than 1 μg/mL (1.695 μM) had a lower probability of being in MMR, and these results were in concordance with previous studies [31]. Based on the results of our study and similar previous studies, it can be concluded that trough imatinib levels provide a good indicator of response outcome in imatinib-treated patients. Receiver operating characteristic (ROC) curve analysis demonstrated plasma threshold imatinib levels and their discrimination potential for MMR with the best sensitivity (51.85%) and specificity (89.42%) at a threshold of 0.988 μg/mL (1.675 μM) (Figure 1). This threshold value is consistent with the results of earlier studies in which it was reported that maintaining plasma trough levels at or above the mean population concentration of approximately
Figure 1. ROC curve for determining imatinib trough level threshold value beyond which imatinib level correlates with a favorable response (MMR or CMR). Imatinib trough level at best sensitivity and specificity corresponds to threshold level, which was found to be 0.988 μg/mL (1.675 μM).
1 μg/mL (1.695 μM) may be important for achieving improved CCR and MMR rates [9,10,18]. The area under curve was 0.733 at this threshold level, and was significantly associated with the presence of a MMR. A box plot was drawn to show the dispersion around the median for responders (n 104, median 1.83 μg/mL/3.10 μM) and non-responders (n 27, median 0.976 μg/mL/1.66 μM) (Figure 2). Achieving maximum benefit with imatinib therapy requires optimal dosing as well as compliance to drug therapy. Monitoring of trough imatinib plasma levels may help to determine a patient’s adherence to therapy. Poor compliance to drug may cause a relapse, and therefore reinforcing compliance would help to achieve optimal responses in these patients. Excessive side effects of imatinib in some patients may indicate the need to minimize the drug dose while still maintaining the therapeutic plasma levels of the drug. Thus, the determination of trough plasma levels would help in drug dose adjustments on a patient-by-patient basis, thereby preventing relapse when used along with standard quantitative BCR–ABL transcript monitoring by RQ-PCR.
Figure 2. Box plot graph of trough imatinib plasma levels. Graph shows dispersion around the median for responders (n 104, median 1.83) and non-responders (n 27, median 0.976).
Plasma imatinib levels and molecular response 2619
Acknowledgements The authors gratefully acknowledge the Indian Council of Medical Research (ICMR), Government of India, for a research fellowship awarded to to Pratibha Sharma (Grant No. 3/2/2/7/2010/NCD-III) and the Indian Cooperative Oncology network (ICON) for a research grant for this project. We thank Dr. Vikram Gotta, Advanced Center for Treatment, Research & Education in Cancer, Mumbai and Dr. Ashok Kumar and the Radiation and Cancer Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur for help in HPLC analysis.
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Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.
References [1] Steensma DP. The chronic myeloproliferative disorders: an historical perspective. Curr Hematol Rep 2003;2:221–230. [2] Cortes J, Talpaz M, O’Brien S, et al. Effects of age on prognosis with imatinib mesylate therapy for patients with Philadelphia chromosomepositive chronic myelogenous leukemia. Cancer 2003;98:1105–1113. [3] Bhutani M, Vora A , Kumar L, et al. Lympho-hemopoietic malignancies in India. Med Oncol 2002;19:141–150. [4] Aziz Z, Iqbal J, Akram M, et al. Treatment of chronic myeloid leukemia in the imatinib era: perspective from a developing country. Cancer 2007;109:1138–1145. [5] Deininger MW, Druker BJ. Specific targeted therapy of chronic myelogenous leukemia with imatinib. Pharmacol Rev 2003;55:401–423. [6] Maekawa T, Ashihara E, Kimura S. The Bcr-Abl tyrosine kinase inhibitor and promising new agents against Philadelphia chromosome-positive leukemias. Int J Clin Oncol 2007;12:327–340. [7] Hochhaus A . Cytogenetic and molecular mechanisms of resistance to imatinib. Semin Hematol 2003;40:69–79. [8] Awidi A , Ayeda AO, Bsoul N, et al. Relationship of serum imatinib trough level and response in CML patients: long term follow-up. Leuk Res 2010;34:1573–1575. [9] Larson RA , Druker BJ, Guilhot F, et al. Imatinib pharmacokinetics and its correlation with response and safety in chronic-phase chronic myeloid leukemia:a subanalysis of the IRIS study. Blood 2008;111: 4022–4028. [10] Picard S, Titier K , Etienne G, et al. Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia. Blood 2007;109:3496–3499. [11] Guo JQ, Lin H, Kantarjian H, et al. Comparison of competitivenested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients. Leukemia 2002;16:2447–2453. [12] Velpandian T, Mathur R, Agarwal NK , et al. Development and validation of a simple liquid chromatographic method with ultraviolet detection for the determination of imatinib in biological samples. J Chromatogr B Analyt Technol Biomed Life Sci 2004;804:431–434. [13] Titier K, Picard S, Ducint D, et al. Quantification of imatinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry. Ther Drug Monit 2005;27:634–640. [14] Bakhtiar R, Khemani L, Hayes M, et al. Quantification of the anti-leukemia drug STI571 (Gleevec) and its metabolite (CGP74588) in monkey plasma using a semi-automated solid phase extraction procedure and liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal 2002;28:1183–1194. [15] Bakhtiar R, Lohne J, Ramos L, et al. High-throughput quantification of the anti-leukemia drug STI571 (Gleevec) and its main
metabolite (CGP74588) in human plasma using liquid chromatography tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 2002;768:325–340. [16] Parise RA , Ramanathan RK, Hayes MJ, et al. Liquid chromatographic-mass spectrometric assay for quantitation of imatinib and its main metabolite (CGP74588) in plasma. J Chromatogr B Anal Technol Biomed Life Sci 2003;791:39–44. [17] Awidi A , Salemb II, Najibb N, et al. Determination of imatinib plasma levels in patients with chronic myeloid leukemia by high performance liquid chromatography-ultraviolet detection and liquid chromatography-tandem mass spectrometry: methods’ comparison. Leuk Res 2010;34:714–717. [18] Pasqualotto AC, Xavier MO, Andreolla HF, et al. Voriconazole therapeutic drug monitoring: focus on safety. Expert Opin Drug Saf 2010;9:125–137. [19] Wojnowski L. Genetics of the variable expression of CYP3A in humans. Ther Drug Monit 2004;26:192–199. [20] Bolton AE, Peng B, Hubert M, et al. Effect of rifampicin on the pharmacokinetics of imatinib mesylate (Gleevec, STI571) in healthy subjects. Cancer Chemother Pharmacol 2004;53:102–106. [21] Smith P, Bullock JM, Booker BM et al. The influence of St. John’s wort on the pharmacokinetics and protein binding of imatinib mesylate. Pharmacotherapy. 2004;24:1508–1514. [22] Frye RF, Fitzgerald SM, Lagattuta TF et al. Effect of St John’s wort on imatinib mesylate pharmacokinetics. Clin Pharmacol Ther. 2004;76:323–332. [23] Dutreix C, Peng B, Mehring G et al. Pharmacokinetic interaction between ketoconazole and Imatinib mesylate (Glivec) in healthy subjects. Cancer Chemother Pharmacol. 2004;54:290–294. [24] Widmer N, Decosterd LA , Csajka C et al. Population pharmacokinetics of Imatinib and the role of alpha-acid glycoprotein. Br J Clin Pharmacol. 2006;62:97–112. [25] Gambacorti-Passerini C, Barni R, le Coutre P et al. Role of alpha1 acid glycoprotein in the in vivo resistance of human BCRABL( ) leukemic cells to the abl inhibitor STI571. J Natl Cancer Inst 2000;92:1641–1650. [26] Crossman LC, Druker BJ, Deininger MW et al. hOCT 1 and resistance to imatinib. Blood 2005;106:1133–1134. [27] Wang L, Giannoudis A , Lane S et al. Expression of the uptake drug transporter hOCT1 is an important clinical determinant of the response to imatinib in chronic myeloid leukemia. Clin Pharmacol Ther 2008;83:258–264. [28] White DL, Dang P, Engler J et al. Functional activity of the OCT-1 protein is predictive of Long-Term outcome in patients with chronicphase chronic myeloid leukemia treated with imatinib. J Clin Oncol 2010;28:2761–2767. [29] White DL, Saunders VA , Dang P, et al. Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: higher doses of imatinib may overcome the negative impact of low OCT-1 activity. Blood 2007;110:4064–4072. [30] Widmer N, Colombo S, Buclin T, et al. Functional consequence of MDR1 expression on imatinib intracellular concentrations. Blood 2003;102:1142. [31] Marin D, Bazeos A , Mahon FX, et al. Adherence is the critical factor for achieving molecular responses in patients with chronic myeloid leukemia who achieve complete cytogenetic responses on imatinib. J Clin Oncol 2010;28:2381–2388. [32] Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996;2:561–566. [33] Peng B, Lloyd P, Schran H. Clinical pharmacokinetics of imatinib. Clin Pharmacokinet 2005;44:879–894. [34] Singh N, Kumar L, Meena R, et al. Drug monitoring of imatinib levels in patients undergoing therapy for chronic myeloid leukemia: comparing plasma levels of responders and non-responders. Eur J Clin Pharmacol 2009;65:545–549. [35] Ishikawa Y, Kiyoi H, Watanabe K , et al. Trough plasma concentration of imatinib reflects BCR–ABL kinase inhibitory activity and clinical response in chronic-phase chronic myeloid leukemia: a report from the BINGO study. Cancer Sci 2010;101: 2186–2192.
ORIGINAL ARTICLE: RESEARCH
Correlation of plasma trough levels of imatinib with molecular response in patients with chronic myeloid leukemia Hemant Malhotra1, Pratibha Sharma1,2, Shipra Bhargava2,3, Om Singh Rathore1, Bharti Malhotra4 & Madhu Kumar2 1Division of Medical Oncology, Department of Medicine and 4Department of Microbiology, SMS Medical College, Jaipur, India
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and 2Department of Zoology and 3Department of Botany, University of Rajasthan, Jaipur, India
and progress to accelerated or blast phase [7]. Various mechanisms of resistance to imatinib have been identified, such as gene mutations in the kinase domain of BCR–ABL, BCR–ABL gene amplification, overexpression of Src related kinases, low expression and activity of drug influx transporters, drug efflux mediated by the P-glycoprotein which is encoded by the MDR1 gene and suboptimal plasma levels of imatinib. It has been suggested in recent studies that trough levels of imatinib in plasma may correlate with clinical responses in patients with CML [8–10] and that maintaining the plasma concentration above a threshold of about 1000 ng/mL (1.695 μM) may be important for improving response rates [9,10]. In this study, we assessed the correlation between mean trough imatinib plasma level and molecular response (as determined by BCR–ABL/ABL ratio by real time polymerase chain reaction [RQ-PCR]) in patients with chronic phase CML at our center. The imatinib threshold level that correlated with a better molecular response was determined using receiver operating characteristic (ROC) curve analysis. Furthermore, patients were divided into two categories: those who were taking Glivec and those who were taking generic/biosimilar imatinib. By statistical analysis, we analyzed whether there was a significant difference in mean trough levels of the drug between these two groups of patients who received different formulations of imatinib.
Abstract The present study looked at the correlation between mean trough Imatinib plasma levels and molecular response in 131 CML patients on imatinib. Patients receiving Glivec versus generic Imatinib were also compared. A ROC curve was constructed to estimate a threshold level that correlates with a favourable response. Patients were grouped into Responders (BCR/ABL ration by RQ-PCR less than 1) and Non Responders (ration 1). The mean trough imatinib plasma level in the responders was significantly higher than in the non responders (p 0.001). The area under ROC curve was 0.733, with best sensitivity (51.85%) and specificity (89.42%) at a plasma threshold of 0.988 μg/ml [1.675 μM]. Levels in the patients on Glivec versus generic drug (p 0.05) were comparable. Trough Imatinib plasma levels may be a marker for suboptimal response and may identify patients in whom increase of drug dose or change in therapy may be indicated. Keywords: Chronic myeloid leukemia (CML), imatinib, major molecular response (MMR), real time polymerase chain reaction (RQ-PCR), receiver operating characteristic (ROC) curve
Introduction Chronic myeloid leukemia (CML) is a malignant chronic myeloproliferative disorder (MPD) characterized by a marked increase in granulocytes, marked bone marrow hyperplasia and splenomegaly [1]. CML primarily affects adults between 25 and 60 years of age and accounts for 15–20% of all cases of leukemia [2]. It represents the most common type of adult leukemia in India [3,4]. Imatinib mesylate (Glivec) is currently regarded as the most specific and effective drug for patients with CML [5]. This drug works by directly inhibiting the cancer-causing protein BCR–ABL that is found inside leukemia cells, and therefore is the first successful targeted treatment for cancer [6]. Although most patients with chronic phase CML treated with imatinib mesylate have well-controlled disease, a considerable number of patients relapse
Materials and methods Patients and methods One hundred and thirty-one patients with CML in chronic phase who were on imatinib therapy for more than 24 months and in complete hematological remission (CHR) and attending the outpatient department of the leukemia/lymphoma clinic of the Birla Cancer Center, SMS Medical College Hospital, Jaipur, India were included in this study. The SMS Hospital (SMSH) at Jaipur, Rajasthan is a 2000-bed, tertiary care, super-specialty referral hospital, one of the largest in North India. The RK Birla Cancer Center, situated on the
Correspondence: Prof. Dr. Hemant Malhotra, Division of Medical Oncology, Department of Medicine, SMS Medical College & Hospital, Jawahar Lal Nehru Marg, Jaipur-302004, India. Tel: 91-141-2620600, 2620400. Fax: 91-141-2622899, 5105589. E-mail: [email protected] Received 12 October 2013; revised 26 November 2013; accepted 14 January 2014
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Plasma imatinib levels and molecular response 2615 SMSH campus, caters for more than 10 000 cases of cancer per year, and has a well-established leukemia/lymphoma clinic (since 1991). Out of 131 patients, 47 were taking generic imatinib/biosimilars while 84 patients were taking Glivec. Glivec was provided to patients through the Glivec International Patient Assistance Program (GIPAP). Glivec was provided to patients who were socioeconomically compromised, did not have medical insurance and did not have any source of reimbursement for the drug (as per the standard operating procedure [SOP] of the Max Foundation, the non-governmental organization [NGO] appointed by Novartis for conduct of the GIPAP program globally). All patients potentially eligible for Glivec through the GIPAP program were started on generic imatinib, but were shifted to Glivec as soon as Max Foundation approval for Glivec was obtained (usually within 2–3 weeks of the application). Informed consent was given by all patients for participation in this study. The study was approved by the Ethics Committee of the institution. Diagnosis of CML was established at presentation in all patients with demonstration of BCR–ABL gene quantification by RQ-PCR. As we do not have an in-house cytogenetics laboratory, or availability of samples, cytogenetics was not performed at baseline in any patient. All patients were started on 400 mg/day with monitoring of hematological responses every 4 weeks and molecular responses (by RQPCR on peripheral blood) every 6 months. Response criteria were: complete hematological remission (CHR), defined as a white blood cell (WBC) count of less than 10 109/L, a platelet count of less than 450 109/L, differential without immature granulocytes and with less than 5% basophils and non-palpable spleen; partial hematological remission (PHR), defined as above except for the presence of immature cells, platelet count 50% of pretreatment value but 450 109/L and persistent splenomegaly but 50% of pretreatment size; complete molecular response (CMR), defined as BCR–ABL mRNA transcript levels non-quantifiable and non-detectable by RQ-PCR; major molecular response (MMR), defined by the presence of a BCR–ABL/ABL ratio 1% as quantified by RQ-PCR. For BCR–ABL quantification, 10 mL of fresh peripheral blood was collected from each patient in ethylenediaminetetraacetic acid (EDTA) and the sample was kept at 4°C until further processing, within 2 h of collection. The sample was treated with red blood cell (RBC) lysis buffer (containing 155 mM NH4Cl and 0.1 mM NaHCO3) to lyse the red cells and the mononuclear cells were separated. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The yield, concentration and purity of the RNA were assessed using an Infinite 200 PRO NanoQuant (Tecan, Durham, NC), and only those samples with absorbance (A) A260/A280 ratios between 1.9 and 2.1 were further considered. Two micrograms of total RNA were reverse transcribed with a High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA) in a final volume of 20 μL according to the manufacturer’s instructions. cDNA was then used for quantification of BCR–ABL relative to ABL. Plasmid DNA standards having copy numbers 5 103, 5 104, 5 105, 5 106 and 5 107 were used as calibrators for the generation of standard curves for both BCR–ABL and
ABL. Reactions were performed on an ABI PRISM 7700 SDS (Applied Biosystems) instrument. The reaction conditions and the primer and probe sequences for BCR–ABL and ABL were the same as described previously [11]. A no-template control (NTC) containing nuclease-free water was included in each run. RQ-PCR results were analyzed using the standard curves, which were used to calculate the quantity of BCR–ABL and ABL transcripts (copy numbers) in the samples. A change in the BCR–ABL transcript level was expressed as the ratio of BCR–ABL/ABL. The patients were divided into two groups: responders, patients who achieved a CMR or a MMR (BCR-ABL/ABL ratio 1% as assessed by RQ-PCR); and non-responders, those without CMR or MMR (BCR–ABL/ABL ratio 1% as assessed by RQ-PCR). Imatinib plasma quantification was done using a high performance liquid chromatography ultraviolet (HPLC-UV) detection method as developed by Velpandian et al. [12], which included single-step extraction by methanol. As patients were followed up every 4 weeks, they were counseled about blood collection and compliance on the visit before the sample collection. One day before sample collection, the dose of imatinib was given under supervision and the sample was collected 22–24 h after this dose. Plasma was separated from the blood samples of patients by centrifugation at 2500 rpm for 15 min. These plasma samples were then stored frozen at 80°C until laboratory analysis. Basic measures of location (i.e. mean, median, range, etc.) were calculated to provide a detailed description of group characteristics. Student’s t-test was done to test the significance of means between the two groups. Fisher’s exact and χ2 tests were carried out for comparison of proportions. Receiver operating curve (ROC) curve analysis was done to determine the plasma threshold of imatinib that correlated with MMR.
Results One hundred and thirty-one patients with CML were included in this study. The patients’ baseline characteristics are shown in Table I. Patients were divided into two groups according to their molecular response to imatinib therapy: those with MMR (BCR–ABL/ABL ratio 1% as assessed by RQ-PCR) or better (responders, n 104) and those without MMR (BCR–ABL/ ABL ratio 1% as assessed by RQ-PCR) (non-responders, n 27). Characteristics of responders and non-responders are given in Table II. Plasma imatinib trough level was measured once for every patient. The imatinib trough levels in our subjects ranged from 0.288 to 7.11 μg/mL (0.488 μM to 12.05 μM). The mean plasma imatinib level in responders was 2.10 μg/mL (3.561 μM), range 0.37–7.11 μg/mL (0.63– 12.06 μM); 92 (88.46%) patients in the imatinib responder group had plasma imatinib levels 1 μg/mL ( 1.695 μM). The mean plasma imatinib level in non-responders was 1.31 μg/mL (2.22 μM), range 0.288–2.89 μg/mL (0.49– 4.9 μM). The mean trough imatinib plasma level in responders was significantly higher (2.10 1.18 μg/mL/3.56 2.00 μM) than in non-responders (1.31 0.72 μg/mL/2.22 1.22 μM) (p 0.001). ROC curve analysis was done to determine the plasma threshold of imatinib that correlated with a favorable
2616 H. Malhotra et al. Table I. Baseline characteristics of patients.
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Characteristic
Value
Age (years) Median Range Sex (n) Male Female Splenomegaly 10 cm below costal margin, n (%) Hemoglobin level (g/dL) Median Range Basophils (%) Median Range WBC count (/mm3) Median Range Platelet count (/mm3) Median Range Peripheral blasts 3% Sokal score, n (%) Low Intermediate High
32 18–63 81 50 20 (15) 10.4 6.7–16.3 1 0–4 52 000 3000–210 000 295 000 105 000–910 000 42 (32%) 41 (31.30) 50 (38.17) 40 (30.53)
WBC, white blood cell.
response.TheareaundertheROCcurvewas0.733,withbestsensitivity (51.85%) and specificity (89.42%) at a plasma threshold of 0.988 μg/mL (1.675 μM). Thus a plasma threshold of 0.988 μg/mL (1.675 μM) was significantly associated with the presence of a MMR or CMR, i.e. the two groups could be best separated at this concentration of imatinib in plasma. Patients were also classified into those who were on Glivec (n 84) and those who were on generic/biosimilar imatinib (n 47). Characteristics of patients in these two groups are shown in Table III. The mean imatinib plasma trough level in patients who were on Glivec was 2.07 1.15 μg/mL (3.51 1.95 μM) and in those who were on generic/ biosimilar imatinib was 1.70 1.11 μg/mL (2.88 1.88 μM), and the difference was found to be insignificant (p 0.079). The majority of the treated patients experienced adverse reactions some time during therapy. The toxicity profile of imatinib is given in Table IV.
Discussion In this study, we determined the minimum blood plasma concentration (“trough” level) of imatinib occurring just prior to taking the next dose by HPLC assay, which is a rapid
and sensitive technique and requires small blood samples for analysis. There are several published methods to determine levels of imatinib and its main metabolite norimatinib in human plasma. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) is recommended for most quantitative determinations of imatinib in the plasma of patients with CML, and has been described by several authors [13–16]. However, LC–MS/MS facilities are not always available in standard hospital laboratories as the equipment is quite expensive, as is its use [13,17,18]. Several authors have reported the use of HPLC methods with UV detection for the quantitation of imatinib in human plasma. Some recent studies have compared HPLC with UV/diode array detection to LC-MS/MS and found no significant differences between the two methods. In our study, we used the HPLC UV detection method developed by Velpandian et al. [12], which includes single-step extraction, and is rapid and simple to perform. The imatinib trough levels in our subjects ranged from 0.288 to 7.11 μg/mL (0.488–12.06 μM). This suggests that people differ markedly in how fast their bodies process and excrete imatinib. This difference might be due to variations in the expression and activity of metabolizing enzymes in the liver such as cytochrome P450. CYP3A4 is the major enzyme responsible for the metabolism of imatinib. Some polymorphisms affecting the expression and inducibility of CYP3A4 result in a 10-fold variability in CYP3A4 activity among different subjects, and this is one of the most important factors responsible for the large intra- and interindividual pharmacokinetics variability observed during drug treatments [19], which may further be influenced by a number of inhibitory or inducing compounds present in the environment. A decrease in the plasma concentration of imatinib can occur in patients treated with CYP3A4 inducers such as rifampicin [20] and St John’s wort, i.e. Hypericum perforatum L. extracts [21,22]. In contrast, an increase in plasma imatinib concentration may occur by co-administration of a CYP3A4 inhibitor such as ketoconazole [23]. Since imatinib is also an inhibitor of CYP3A4, co-interactions may occur between imatinib and inhibitors or inducers of these enzymes, leading to changes in the plasma concentration of imatinib. In addition, the binding of imatinib with plasma proteins such as alpha glycoprotein (AGP) may result in its altered plasma concentration, and may have potential consequences on the clinical response [24]. The bioavailability of
Table II. Characteristics of patients classified according to their molecular response to imatinib therapy. Characteristic Age* Plasma level, μg/mL (μM)* Sex category† Male Female Sokal risk group† Low risk ( 0.8) Intermediate risk (0.8–1.2) High risk ( 1.2)
Responders
Non-responders
p-Value
34.25 13.36 2.10 1.18 (3.56 2.00)
35.96 15.55 1.31 0.72 (2.22 1.22)
0.567 0.001
60.58% (63) 39.42% (41)
66.67% (18) 33.33% (9)
0.659
31.73% (33) 37.50% (39) 30.77% (32)
29.63% (8) 40.74% (11) 29.63% (8)
0.951
*Statistical analysis and p-value calculation by Students t-test. †Statistical analysis and p-value calculation by Fisher ’s exact test or χ2 test.
Plasma imatinib levels and molecular response 2617 Table III. Characteristics of patients classified according to formulation of imatinib. Characteristic Plasma level, μg/mL (μM)* Sokal score* Response category† Responder Non-responder Sokal risk group† Low risk Intermediate risk High risk Sex category† Male Female
Glivec
Generic imatinib
p-value
2.07 1.15 (3.51 1.95)
1.70 1.11 (2.88 1.88)
0.079
1.03 0.44
1.23 0.73
0.049
75.00% (63) 25.00% (21)
87.23% (41) 12.77% (6)
0.117
35.71% (30) 39.29% (33) 25.00% (21)
23.40% (11) 36.17% (17) 40.43% (19)
65.48% (55) 34.52% (29)
55.32% (26) 44.68% (21)
0.143 0.266
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*Statistical analysis and p-value calculation by Students t-test. †Statistical analysis and p-value calculation by Fisher ’s exact test or χ2 test.
imatinib can be substantially modified by the presence of high AGP levels that could increase the half-life of imatinib and could alter its tissue distribution and cellular uptake, which would interfere with the access of imatinib to its intracellular target protein, BCR–ABL [25]. However, the therapeutic concentrations of imatinib generally exceed the total binding capacity of AGP, thereby counterbalancing the effect of this plasma protein. Moreover, interindividual differences in the expression and activity of the imatinib influx (OCT-1) [26–29] and efflux transporters (ABC transporters) [30], which are the key determinants of drug disposition and response, lead to a highly variable drug elimination rate, and are therefore responsible for interindividual differences in circulating drug concentrations and therapeutic response. Individual patient characteristics, including age, sex, race and weight and inherited differences in drug absorption, metabolism, transport and disposition of drugs and interactions between prescribed medications, may also affect drug levels. Sampling time error could be another source of variability, although all samples were collected within 2 h, within acceptable sampling time period. Some patients had very low imatinib levels, and this might be due to poor compliance to drug therapy. There are no published data from India regarding compliance to imatinib, but data from
the West [31] indicate that this may be a major issue in suboptimal responders. In our study, the measure of a patient’s adherence to drug therapy was based on self-reporting by the patient. Although patients’ compliance was assured by repeated counseling, some patients might not have taken the medication and may not even have reported this behavior to the clinician. The reasons for poor compliance could be either side effects or cost issues. In our study, many of the side effects of imatinib were observed in the study population (Table IV), and these may account for the low compliance in some of the patients. In our study, we categorized the patients into two groups: those who were taking Glivec and those who were taking generic/biosimilar imatinib. By statistical analysis, we analyzed whether there was a significant difference in the mean trough levels of the drug between these two groups of patients. Our analysis data showed that there was no significant difference between the plasma levels of these two categories of patients (p 0.05). Based on these results, it is concluded that both products can be considered equally effective and interchangeable in medical practice. We also correlated the mean imatinib plasma levels with molecular responses to imatinib therapy in our patients. Patients were classified into responders and non-responders on the basis of molecular response to imatinib therapy as
Table IV. Toxicity profile of imatinib in patients with chronic myeloid leukemia. Glivec (n 84) Toxicity Superficial edema Nausea Muscle cramps Musculoskeletal pain Rashes and related events Diarrhea Headache Joint pain Abdominal pain Myalgia Vomiting Fatigue Dyspepsia Increased weight Insomnia Discontinuations due to AEs AE, adverse event.
Generic/biosimilar imatinib (n 47)
Grade 1/2, n (%)
Grade 3/4, n (%)
Grade 1/2, n (%)
Grade 3/4, n (%)
52 (61.9) 32 (38.1) 47 (55.95) 38 (45.23) 34 (40.47) 39 (46.42) 33 (39.28) 27 (32.14) 23 (27.38) 20 (23.80) 18 (21.42) 32 (38.09) 15 (17.85) 11 (13.09) 13 (15.47)
3 (3.5) 2 (2.38) 0 (0) 4 (4.76) 3 (3.5) 2 (2.38) 0 (0) 2 (2.38) 4 (4.76) 1 (1.19) 1 (1.19) 0 (0) 0 (0) 1 (1.19) 0 (0)
30 (63.8) 19 (40.42) 27 (57.44) 21 (44.68) 18 (38.29) 21 (44.68) 20 (42.55) 14 (29.78) 13 (27.65) 11 (23.40) 11 (23.40) 19 (40.42) 9 (19.14) 7 (14.89) 8 (17.02)
2 (4.25) 1 (2.12) 0 (0) 2 (4.25) 2 (4.25) 1 (2.12) 0 (0) 1 (2.12) 2 (4.25) 1 (2.12) 0 (0) 1 (2.12) 0 (0) 1 (2.12) 0 (0)
0 (0)
0 (0)
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2618 H. Malhotra et al. determined by the BCR–ABL/ABL ratio by RQ-PCR. Out of 131 patients, 104 who achieved either CMR or MMR were included in the responders while 27 patients who did not achieve either CMR or MMR were included in the nonresponders. Student’s t-test results showed that the mean plasma levels in responders (2.10 1.18 μg/mL/3.56 2.00 μM) were significantly higher than in non-responders (1.31 0.72 μg/mL/2.22 1.22 μM) (p 0.001). Several earlier studies also correlated the plasma imatinib trough levels with clinical responses in patients with CML and compared plasma imatinib levels in patients classified into responders and non-responders based upon molecular or cytogenetic responses. It was reported previously that patients having plasma imatinib levels 1 μg/mL (1.695 μM) achieved a CHR and that low plasma levels of imatinib were associated with inadequate hematological response [32,33]. In the study by Picard et al. [10], trough imatinib levels were found to correlate with the achievement of MMR, with patients achieving trough imatinib levels of 1002 ng/mL (1.699 μM) being significantly more likely to achieve a MMR (p 0.001). Larson et al. also studied the correlation of imatinib trough plasma concentrations with clinical responses, event-free survival (EFS) and adverse events (AEs) [9]. Imatinib trough plasma concentrations were found to be significantly higher in patients who achieved CCR versus those who did not achieve it (1009 544 ng/mL/1.711 0.922 μM vs. 812 409 ng/mL/1.377 0.694 μM, p 0.01). Also, patients with high imatinib exposure were found to have better rates of CCR, MMR and EFS. Singh et al. also performed a similar study in which the correlation of plasma level of imatinib with response to therapy was investigated, and it was demonstrated that the mean plasma imatinib level in responders were significantly higher than that in non-responders (2.34 vs. 0.70 μM, respectively, p 0.002) [34]. Awidi et al. carried out a similar study in Jordanian patients with CML who were on imatinib therapy for at least 12 months and found that the mean trough levels in patients with MMR or better were significantly higher than in those without MMR (2766 811 ng/ mL/4.69 1.38 μM vs. 1777 624 ng/mL/3.013 1.058 μM, respectively) [8]. Ishikawa et al. also obtained similar results in their study, and found that the plasma trough levels of imatinib among patients who achieved MMR during treatment (n 52) were higher than those for patients who did not achieved MMR (n 8) [35]. Marin et al. found that patients with plasma imatinib levels less than 1 μg/mL (1.695 μM) had a lower probability of being in MMR, and these results were in concordance with previous studies [31]. Based on the results of our study and similar previous studies, it can be concluded that trough imatinib levels provide a good indicator of response outcome in imatinib-treated patients. Receiver operating characteristic (ROC) curve analysis demonstrated plasma threshold imatinib levels and their discrimination potential for MMR with the best sensitivity (51.85%) and specificity (89.42%) at a threshold of 0.988 μg/mL (1.675 μM) (Figure 1). This threshold value is consistent with the results of earlier studies in which it was reported that maintaining plasma trough levels at or above the mean population concentration of approximately
Figure 1. ROC curve for determining imatinib trough level threshold value beyond which imatinib level correlates with a favorable response (MMR or CMR). Imatinib trough level at best sensitivity and specificity corresponds to threshold level, which was found to be 0.988 μg/mL (1.675 μM).
1 μg/mL (1.695 μM) may be important for achieving improved CCR and MMR rates [9,10,18]. The area under curve was 0.733 at this threshold level, and was significantly associated with the presence of a MMR. A box plot was drawn to show the dispersion around the median for responders (n 104, median 1.83 μg/mL/3.10 μM) and non-responders (n 27, median 0.976 μg/mL/1.66 μM) (Figure 2). Achieving maximum benefit with imatinib therapy requires optimal dosing as well as compliance to drug therapy. Monitoring of trough imatinib plasma levels may help to determine a patient’s adherence to therapy. Poor compliance to drug may cause a relapse, and therefore reinforcing compliance would help to achieve optimal responses in these patients. Excessive side effects of imatinib in some patients may indicate the need to minimize the drug dose while still maintaining the therapeutic plasma levels of the drug. Thus, the determination of trough plasma levels would help in drug dose adjustments on a patient-by-patient basis, thereby preventing relapse when used along with standard quantitative BCR–ABL transcript monitoring by RQ-PCR.
Figure 2. Box plot graph of trough imatinib plasma levels. Graph shows dispersion around the median for responders (n 104, median 1.83) and non-responders (n 27, median 0.976).
Plasma imatinib levels and molecular response 2619
Acknowledgements The authors gratefully acknowledge the Indian Council of Medical Research (ICMR), Government of India, for a research fellowship awarded to to Pratibha Sharma (Grant No. 3/2/2/7/2010/NCD-III) and the Indian Cooperative Oncology network (ICON) for a research grant for this project. We thank Dr. Vikram Gotta, Advanced Center for Treatment, Research & Education in Cancer, Mumbai and Dr. Ashok Kumar and the Radiation and Cancer Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur for help in HPLC analysis.
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References [1] Steensma DP. The chronic myeloproliferative disorders: an historical perspective. Curr Hematol Rep 2003;2:221–230. [2] Cortes J, Talpaz M, O’Brien S, et al. Effects of age on prognosis with imatinib mesylate therapy for patients with Philadelphia chromosomepositive chronic myelogenous leukemia. Cancer 2003;98:1105–1113. [3] Bhutani M, Vora A , Kumar L, et al. Lympho-hemopoietic malignancies in India. Med Oncol 2002;19:141–150. [4] Aziz Z, Iqbal J, Akram M, et al. Treatment of chronic myeloid leukemia in the imatinib era: perspective from a developing country. Cancer 2007;109:1138–1145. [5] Deininger MW, Druker BJ. Specific targeted therapy of chronic myelogenous leukemia with imatinib. Pharmacol Rev 2003;55:401–423. [6] Maekawa T, Ashihara E, Kimura S. The Bcr-Abl tyrosine kinase inhibitor and promising new agents against Philadelphia chromosome-positive leukemias. Int J Clin Oncol 2007;12:327–340. [7] Hochhaus A . Cytogenetic and molecular mechanisms of resistance to imatinib. Semin Hematol 2003;40:69–79. [8] Awidi A , Ayeda AO, Bsoul N, et al. Relationship of serum imatinib trough level and response in CML patients: long term follow-up. Leuk Res 2010;34:1573–1575. [9] Larson RA , Druker BJ, Guilhot F, et al. Imatinib pharmacokinetics and its correlation with response and safety in chronic-phase chronic myeloid leukemia:a subanalysis of the IRIS study. Blood 2008;111: 4022–4028. [10] Picard S, Titier K , Etienne G, et al. Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia. Blood 2007;109:3496–3499. [11] Guo JQ, Lin H, Kantarjian H, et al. Comparison of competitivenested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients. Leukemia 2002;16:2447–2453. [12] Velpandian T, Mathur R, Agarwal NK , et al. Development and validation of a simple liquid chromatographic method with ultraviolet detection for the determination of imatinib in biological samples. J Chromatogr B Analyt Technol Biomed Life Sci 2004;804:431–434. [13] Titier K, Picard S, Ducint D, et al. Quantification of imatinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry. Ther Drug Monit 2005;27:634–640. [14] Bakhtiar R, Khemani L, Hayes M, et al. Quantification of the anti-leukemia drug STI571 (Gleevec) and its metabolite (CGP74588) in monkey plasma using a semi-automated solid phase extraction procedure and liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal 2002;28:1183–1194. [15] Bakhtiar R, Lohne J, Ramos L, et al. High-throughput quantification of the anti-leukemia drug STI571 (Gleevec) and its main
metabolite (CGP74588) in human plasma using liquid chromatography tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 2002;768:325–340. [16] Parise RA , Ramanathan RK, Hayes MJ, et al. Liquid chromatographic-mass spectrometric assay for quantitation of imatinib and its main metabolite (CGP74588) in plasma. J Chromatogr B Anal Technol Biomed Life Sci 2003;791:39–44. [17] Awidi A , Salemb II, Najibb N, et al. Determination of imatinib plasma levels in patients with chronic myeloid leukemia by high performance liquid chromatography-ultraviolet detection and liquid chromatography-tandem mass spectrometry: methods’ comparison. Leuk Res 2010;34:714–717. [18] Pasqualotto AC, Xavier MO, Andreolla HF, et al. Voriconazole therapeutic drug monitoring: focus on safety. Expert Opin Drug Saf 2010;9:125–137. [19] Wojnowski L. Genetics of the variable expression of CYP3A in humans. Ther Drug Monit 2004;26:192–199. [20] Bolton AE, Peng B, Hubert M, et al. Effect of rifampicin on the pharmacokinetics of imatinib mesylate (Gleevec, STI571) in healthy subjects. Cancer Chemother Pharmacol 2004;53:102–106. [21] Smith P, Bullock JM, Booker BM et al. The influence of St. John’s wort on the pharmacokinetics and protein binding of imatinib mesylate. Pharmacotherapy. 2004;24:1508–1514. [22] Frye RF, Fitzgerald SM, Lagattuta TF et al. Effect of St John’s wort on imatinib mesylate pharmacokinetics. Clin Pharmacol Ther. 2004;76:323–332. [23] Dutreix C, Peng B, Mehring G et al. Pharmacokinetic interaction between ketoconazole and Imatinib mesylate (Glivec) in healthy subjects. Cancer Chemother Pharmacol. 2004;54:290–294. [24] Widmer N, Decosterd LA , Csajka C et al. Population pharmacokinetics of Imatinib and the role of alpha-acid glycoprotein. Br J Clin Pharmacol. 2006;62:97–112. [25] Gambacorti-Passerini C, Barni R, le Coutre P et al. Role of alpha1 acid glycoprotein in the in vivo resistance of human BCRABL( ) leukemic cells to the abl inhibitor STI571. J Natl Cancer Inst 2000;92:1641–1650. [26] Crossman LC, Druker BJ, Deininger MW et al. hOCT 1 and resistance to imatinib. Blood 2005;106:1133–1134. [27] Wang L, Giannoudis A , Lane S et al. Expression of the uptake drug transporter hOCT1 is an important clinical determinant of the response to imatinib in chronic myeloid leukemia. Clin Pharmacol Ther 2008;83:258–264. [28] White DL, Dang P, Engler J et al. Functional activity of the OCT-1 protein is predictive of Long-Term outcome in patients with chronicphase chronic myeloid leukemia treated with imatinib. J Clin Oncol 2010;28:2761–2767. [29] White DL, Saunders VA , Dang P, et al. Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: higher doses of imatinib may overcome the negative impact of low OCT-1 activity. Blood 2007;110:4064–4072. [30] Widmer N, Colombo S, Buclin T, et al. Functional consequence of MDR1 expression on imatinib intracellular concentrations. Blood 2003;102:1142. [31] Marin D, Bazeos A , Mahon FX, et al. Adherence is the critical factor for achieving molecular responses in patients with chronic myeloid leukemia who achieve complete cytogenetic responses on imatinib. J Clin Oncol 2010;28:2381–2388. [32] Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996;2:561–566. [33] Peng B, Lloyd P, Schran H. Clinical pharmacokinetics of imatinib. Clin Pharmacokinet 2005;44:879–894. [34] Singh N, Kumar L, Meena R, et al. Drug monitoring of imatinib levels in patients undergoing therapy for chronic myeloid leukemia: comparing plasma levels of responders and non-responders. Eur J Clin Pharmacol 2009;65:545–549. [35] Ishikawa Y, Kiyoi H, Watanabe K , et al. Trough plasma concentration of imatinib reflects BCR–ABL kinase inhibitory activity and clinical response in chronic-phase chronic myeloid leukemia: a report from the BINGO study. Cancer Sci 2010;101: 2186–2192.
