The LC-ESI-MS was developed and validated for the analysis of imatinib in plasma and bone marrow samples using deuterated imatinib (D(8)-IM) as an internal standard. The biological samples were extracted using Strata-X-C SPE cartridges and separated on C&lt;sub&gt;8&lt;/sub&gt; column (50 x 3 mm, 3 &#181;m), and methanol: 0.1% formic acid (70:30) was delivered at the rate of 0.7 ml/min as a mobile phase. Imatinib was quantified in samples by monitoring the ions m/z 494.3 for imatinib and 502.3 for D&lt;sub&gt;8&lt;/sub&gt;-imatinib on mass spectrometer. The method was linear in the concentration range of 1-1500 ng/250 &#181;l in spiked human plasma samples and limit of quantification was 5 ng/mL. Inter-day and intra-day variations in spiked human plasma spiked with 50, 250 and 500 ng /mL were less than 3.16%. The repeatability and reproducibility and other parameters of the methods were also validated. The method was employed for the analysis of the imatinib in human plasma and bone marrow samples. The drug levels in bone marrow and plasma samples were correlated to the degree of cytogenetic response. No significant difference of imatinib level between blood and bone marrow in IM-treated patients dosed to steady state was observed

Elliott, M.

Holyoake, T.L.

Iqbal, Z.

Jorgensen, H.G.

Watson, D.G.

English

The LC-ESI-MS was developed and validated for the analysis of imatinib in plasma and bone marrow samples using deuterated imatinib (D(8)-IM) as an internal standard. The biological samples were extracted using Strata-X-C SPE cartridges and separated on C(8) column (50 x 3 mm, 3 μm), and methanol: 0.1% formic acid (70:30) was delivered at the rate of 0.7 ml/min as a mobile phase. Imatinib was quantified in samples by monitoring the ions m/z 494.3 for imatinib and 502.3 for D(8)-imatinib on mass spectrometer. The method was linear in the concentration range of 1-1500 ng/250 μl in spiked human plasma samples and limit of quantification was 5 ng/mL. Inter-day and intra-day variations in spiked human plasma spiked with 50, 250 and 500 ng /mL were less than 3.16%. The repeatability and reproducibility and other parameters of the methods were also validated. The method was employed for the analysis of the imatinib in human plasma and bone marrow samples. The drug levels in bone marrow and plasma samples were correlated to the degree of cytogenetic response. No significant difference of imatinib level between blood and bone marrow in IM-treated patients dosed to steady state was observed

Iqbal, Zafar

Elliott, Moira

Watson, David

Holyoake, Tessa

Jørgensen, Heather

University of Strathclyde Institutional Repository

Activity of a Zafar Iqbal et al.

An elderly case of chronic myeloid leukemia in which BCR/ABL decreased or disappeared, following imatinib therapy after each episode of blast crisis.

Characterization of imatinib metabolites in rat and human liver microsomes: Differentiation of hydroxylation from Noxidation by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. Rapid Commun.

CYP3A activity influences imatinib response in patients with chronic myeloid leukemia: A pilot study on in vivo CYP3A activity.

Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction.

Determination of imatinib (Gleevec) in human plasma by solid-phase extractionliquid chromatography-ultraviolet absorbance detection.

Development and validation of a sensitive assay for the quantification of imatinib using LC/LC-MS/MS in human whole blood and cell culture.

Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia.

Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia.

Functional ABCG2 is overexpressed on primary CML CD34+ cells and is inhibited by imatinib mesylate.

G(2009). New HPLC-MS method for the simultaneous quantification of the antileukemia drugs imatinib, dasatinib, and nilotinib in human plasma,

Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia.

High-throughput quantification of the antileukemia drug ST1571 (Gleevec (TM)) and its main metabolite (CGP 74588) inhuman plasma using liquid chromatography-tandem mass spectrometry.

Hydrophilic interaction liquid chromatography/tandem mass spectrometry for the simultaneous determination of dasatinib, imatinib and nilotinib in mouse plasma.

Imatinib assay by HPLC with photodiode-array UV detection in plasma from patients with chronic myeloid leukemia: Comparison with LC-MS/MS.

Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study.

Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: Results of a phase II study.

Imatinib mesylate (STI571) therapy for Philadelphia chromosome-positive chronic myelogenous leukemia in blast phase.

Liquid chromatographic-mass spectrometric assay for quantitation of imatinib and its main metabolite (CGP 74588) in plasma.

Lonafarnib reduces the resistance of primitive quiescent CML cells to imatinib mesylate in vitro.

Pharmacokinetics and pharmacodynamics of imatinib in a phase I trial with chronic myeloid leukemia patients.

pp.285-291 291 specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.

Punish the parent not the progeny.

Validation of an HPLC method for the determination of imatinib mesylate in pharmaceutical dosage.

Wever I and Oosterom AT Bruijn

Analysis of imatinib in bone marrow and plasma samples of chronic myeloid leukaemia patients using solid phase extraction LC-ESI-MS

Name not available

The LC-ESI-MS was developed and validated for the analysis of imatinib in plasma and bone marrow samples using deuterated imatinib (D(8)-IM) as an internal standard. The biological samples were extracted using Strata-X-C SPE cartridges and separated on C8 column (50 x 3 mm, 3 &#181;m), and methanol: 0.1% formic acid (70:30) was delivered at the rate of 0.7 ml/min as a mobile phase. Imatinib was quantified in samples by monitoring the ions m/z 494.3 for imatinib and 502.3 for D8-imatinib on mass spectrometer. The method was linear in the concentration range of 1-1500 ng/250 &#181;l in spiked human plasma samples and limit of quantification was 5 ng/mL. Inter-day and intra-day variations in spiked human plasma spiked with 50, 250 and 500 ng /mL were less than 3.16%. The repeatability and reproducibility and other parameters of the methods were also validated. The method was employed for the analysis of the imatinib in human plasma and bone marrow samples. The drug levels in bone marrow and plasma samples were correlated to the degree of cytogenetic response. No significant difference of imatinib level between blood and bone marrow in IM-treated patients dosed to steady state was observed

Enlighten: Publications

 
 
 
 
 
 
 
Iqbal, Z., Elliott, M., Watson, D.G., Holyoake, T.L. and Jorgensen, 
H.G. (2011) Analysis of imatinib in bone marrow and plasma samples of 
chronic myeloid leukaemia patients using solid phase extraction LC-ESI-
MS. Pakistan Journal of Pharmaceutical Sciences, 24 (3). pp. 285-291. 
ISSN 1011-601X 
 
http://eprints.gla.ac.uk/54114/ 
 
Deposited on: 24 November 2011 
 
 
Enlighten – Research publications by members of the University of Glasgow 
http://eprints.gla.ac.uk 
1 
ZAFAR IQBAL1, MOIRA ELLIOTT1, DAVID G WATSON1, TESSA HOLYOAKE2 
AND HEATHER JØRGENSEN2 
 
1Division of Pharmaceutical Sciences, Strathclyde Institute for Pharmacy and Biomedical Sciences, 
University of Strathclyde, Glasgow, United Kingdom 
2Section of Experimental Haematology, Division of Cancer Sciences & Molecular Pathology, 
University of Glasgow, Royal Infirmary, Glasgow, United Kingdom 
 
 
ABSTRACT 
 
The LC-ESI-MS was developed and validated for the analysis of imatinib in plasma 
and bone marrow samples using deuterated imatinib (D8-IM) as an internal standard. 
The biological samples were extracted using Strata-X-C SPE cartridges and separated 
on C8 column (50 x 3 mm, 3 µm), and methanol: 0.1% formic acid (70:30) was 
delivered at the rate of 0.7 ml/min as a mobile phase. Imatinib was quantified in 
samples by monitoring the ions m/z 494.3 for imatinib and 502.3 for D8-imatinib on 
mass spectrometer. The method was linear in the concentration range of 1-1500 
ng/250 µL in spiked human plasma samples and limit of quantification was 5 ng/mL. 
Inter-day and intra-day variations in spiked human plasma spiked with 50, 250 and 
500 ng /mL were less than 3.16%. The repeatability and reproducibility and other 
parameters of the methods were also validated. The method was employed for the 
analysis of the imatinib in human plasma and bone marrow samples. The drug levels 
in bone marrow and plasma samples were correlated to the degree of cytogenetic 
response. No significant difference of imatinib level between blood and bone marrow 
in IM-treated patients dosed to steady state was observed. 
 
Keywords: Bone marrow, Imatinib, Leukaemic, LC- ESI-MS, Human plasma.  
 
 
2 
INTRODUCTION  
 
The selective Abl tyrosine kinase inhibitor, imatinib mesylate (IM; Gleevec or 
Glivec) has become the gold standard treatment of Chronic Myeloid Leukaemia 
(CML)1,2.  Discovered from a rational drug design program, IM blocks the activity of 
BCR-ABL, the oncogenic tyrosine kinase created by the Philadelphia (Ph) 
chromosome translocation diagnostic of CML.  Although clinical studies in the three 
sequential stages of the disease (chronic phase3, accelerated phase4, and terminal blast 
crisis5,6) confirm the substantial activity of the drug in vivo, responses are not 
sustained in advanced phases.  Further, the majority of patients in the early chronic 
phase do not achieve the deepest level of response, i.e. molecular remission that is 
polymerase chain reaction (PCR) negativity for BCR-ABL7. 
 
Knowing that CML is a clonal disease of stem cell origin, it has been postulated that 
low stem cell turnover in vivo may facilitate survival of ‘protected’ quiescent stem 
cells able to maintain and repopulate the disease8,9.  Physiologically, bone marrow is 
the major compartment for white cell production, and this is the compartment in 
which quiescent CML stem cells are presumed to reside.  In the face of emerging 
resistance it would be of interest to know the level of IM achieved in patient marrow.  
We sought to assess whether inadequate patient response was in part related to failure 
to achieve a sufficiently high IM level in bone marrow as compared to peripheral 
blood.  
 
3 
The aim of the present work was to relate drug levels to the clinical status of patients 
at the time of sampling using a suitably sensitive, precise and accurate method for the 
analysis of IM in peripheral blood and matched bone marrow plasma samples. 
 
4 
DESIGN AND METHODS  
 
Reagents and solutions 
Methanol, acetonitrile, high pressure liquid chromatography (HPLC) water, 98% v/v 
formic acid and 35% v/v ammonia solution (analytical reagent grade) were all 
obtained from BDH Laboratory Supplies (Poole, UK).  IM and the internal standard 
D8-IM were kind gifts from Novartis Pharma, Basle, Switzerland.  Stock IM and 
internal standard D8-IM solutions at 1mg/mL were prepared in 50:50 methanol:1% 
w/v formic acid and stored at +4ºC until use.  The stock solution was diluted for 
HPLC analysis with 1% w/v formic acid.   
 
Clinical samples 
Peripheral blood was drawn into ethylene diamine tetra-acetic acid (EDTA) tubes 
with informed consent from CML patients or healthy volunteers according to the 
Declaration of Helsinki.  Bone marrow aspirate, excess to clinical diagnostic 
requirements, was also harvested from the CML patients.  CML marrow was sampled 
at 3 or 4 months follow-up post-initiation of IM. The plasma supernatant was 
separated by centrifugation of whole blood at 1200rpm for 10 minutes and stored 
frozen at -20°C until required.  Glasgow Royal Infirmary Local Research Ethics 
Committee approved the use of human tissue in this study. 
 
Solid phase extraction (SPE) method for plasma and bone marrow samples 
Frozen peripheral blood or bone marrow plasma samples were thawed at room 
temperature.  0.25mL was transferred to a 1mL plastic vial to which was added 
0.25mL 0.1M phosphate buffer pH3.0 with an appropriate volume of internal standard 
solution.  The solution was vortex mixed for 30 seconds then transferred to a Strata-
5 
X-C column (Phenomenex, Macclesfield, UK) previously washed with 1mL of 
phosphate buffer.  The column was washed with 1mL water, 1mL methanol, and then 
twice eluted with 1mL of 2M ammonium hydroxide in methanol.  The eluent was 
collected, evaporated under a stream of nitrogen and dissolved in 0.1% w/v formic 
acid. 
 
High pressure liquid chromatography mass spectrometry (LC-MS) for analysis of 
IM in biological fluids  
IM analysis of plasma or bone marrow samples was performed using a Spectra 
Systems HPLC equipped with a P2000 pump, AS1000 autosampler with 20µL fixed 
injection loop and UV2000 detector (ThermoSeparations, Hemel Hempstead, Herts, 
UK).  Chromatographic separation was at ambient temperature using an ACE 3µm 
C8 column (50 x 3mm internal diameter; HiChrom, Reading, UK).  Mobile phase 
(methanol:formic acid 0.1% w/v) in the ratio of 70:30 was delivered in isocratic mode 
at a flow rate of 0.7mL / min over 10 minutes.   
 
Analytes were detected using an LCQ Finnigan Automass LC-MS system 
(ThermoSeparations).  Samples at 20µL were injected via the autosampler into the 
mass spectrometer without splitting at a flow rate of 0.7mL / min.  The interface was 
operated in positive ion electrospray mode at 4.5 kV.  The capillary temperature was 
adjusted to 270°C.  The MS data were acquired under selected ion monitoring mode 
by monitoring for the IM ion at m/z 494.3 and at 502.3 for D8-IM.  
 
 
6 
RESULTS AND DISCUSSION  
 
 
Based on our group’s research interest in persistent quiescent CML stem cells within 
the bone marrow compartment, we postulated that truly successful chemotherapeutic 
disease eradication may only be achieved if local drug delivery is optimal.  An 
investigation into the correlation of peripheral blood and bone marrow plasma drug 
levels was therefore planned for which a sensitive, precise and accurate analytical 
methodology had to be developed.   
 
Extraction of IM from biological samples using an SPE method proved to be superior 
to the solvent based approach of existing methods.11-13  Using the latter methodology, 
the poorest result was achieved with acetonitrile as the extraction solvent with only 
68% drug recovery in comparison to 89% ± 5% IM recovery by SPE (IM at 200ng in 
250µL, n = 5).  
 
The LC-MS analytical methodology proved to be both specific without interference 
from other peripheral blood or bone marrow plasma components, and sensitive with a 
limit of detection (LOD) of 5ng / 250µL of IM.  Method linearity was proven by 
direct injection of standards in the range 1ng to 500ng / 250µL mobile phase (7 data 
points) described by the regression equation y = -0.0245 + 0.00203x (r2 = 0.998). 
Method precision (expressed as co-efficient of variation, % CV) and accuracy were 
studied at IM concentrations of 50, 250 and 500ng / 250µL.  Results were entirely 
satisfactory with intra-day accuracy in the range 89% to 98%, and intra-day precision 
(% CV) ranging from 0.6% to 3.5%.  These results were reproducible in inter-day 
studies.  
 
7 
From a cohort of 10 CML patients enrolled in a clinical trial studying IM in chronic 
phase (Novartis studies 0106 and 0113), matched peripheral blood and bone marrow 
aspirate samples were processed to determine drug levels.  The average IM level in 
peripheral blood samples taken at 3 or 4 months post-initiation of IM therapy (Table 
1) was in keeping with published human pharmacokinetic data dosed to steady state10.  
At this time, all patients had achieved an haematological remission (to < 10 x 106 / 
mL). However, only half had achieved the next level of response that is a complete 
cytogenetic response (CCR).  None of the patients were PCR-negative for BCR-ABL 
at this stage.  
 
On analysis of total data from all patients, there was no significant difference in the 
average drug concentration between peripheral blood and bone marrow plasma. This 
suggests that 400mg IM daily is sufficient to sustain equilibrium between peripheral 
blood and bone marrow plasma at a therapeutic level (approximately 2 µM). When 
the levels measured in the cytogenetically responding patients were compared with 
the non-responders, there were still no significant differences between sub-groups in 
peripheral blood or bone marrow.  
 
In summary, using a sensitive assay for measurement of IM in biological samples, we 
have found that the IM concentration in peripheral blood and bone marrow plasma 
from CML patients dosed to steady state is in equilibrium. Despite the relatively 
limited patient numbers studied, these data were nonetheless consistent demonstrating 
good distribution of the drug into this previously un-assayed tissue. However, as we 
believe that higher intracellular levels of IM are required to kill stem cells than mature 
leucocytes in vitro, due to over-expression of BCR-ABL protein and / or multi-drug 
8 
resistance pumps14,15, we now hypothesise that more effective intracellular stem cell 
drug loading may be required within the bone marrow compartment.  
 
9 
REFERENCES  
 
1.  Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, et al.  Efficacy 
and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid 
leukemia. N Engl J Med 2001; 344: 1031–37. 
  
2. Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Fernandes-Reese S, Ford JM, et 
al.  Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis 
of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia 
chromosome.  N Engl J Med 2001; 344: 1038–42. 
 
3. Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-
Passerini C, et al.  Hematologic and cytogenetic responses to imatinib mesylate in 
chronic myelogenous leukemia.   N Engl J Med 2002; 346: 645–52.  
 
4. Talpaz M, Silver RT, Druker BJ, Goldman JM, Gambacorti-Passerini C, Guilhot F, 
et al.  Imatinib induces durable hematologic and cytogenetic responses in patients 
with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood 
2002; 99:1928–37.  
 
5. Kantarjian HM, Cortes J, O’Brien S, Giles FJ, Albitar M, Rios MB, et al.  Imatinib 
mesylate (STI571) therapy for Philadelphia chromosome-positive chronic 
myelogenous leukemia in blast phase. Blood  2002; 99:3547–53. 
  
10 
6. Sawyers CL, Hochhaus A, Feldman E, Goldman JM, Miller CB, Ottmann OG, et 
al.  Imatinib induces hematologic and cytogenetic responses in patients with chronic 
myelogenous leukemia in myeloid blast crisis: results of a Phase II study. Blood 2002; 
99:3530–39.  
 
7.  Hughes TP, Kaeda J, Branford S, Rudzki Z, Hochhaus A, Hensley ML et al.  
Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine 
in newly diagnosed chronic myeloid leukemia.  N Engl J Med 2003; 349:1423–32. 
 
8. Elrick LJ, Jørgensen HG, Mountford JC, and Holyoake TL. Punish the parent not 
the progeny. Blood 2005; 105: 1862–66. 
 
9. Jørgensen HG, Allan EK, Graham SM, Godden J, Richmond L, Elliott MA, et al. 
Lonafarnib reduces the resistance of primitive quiescent CML cells to imatinib 
mesylate in vitro.  Leukemia 2005; 19: 1184-91. 
 
10.  Peng B, Hayes M, Resta D, Racine-Poon A, Druker BJ, Talpaz M, et al. 
Pharmacokinetics and pharmacodynamics of imatinib in a Phase I trial with chronic 
myeloid leukemia patients.  J Clin Oncol 2004; 22(5):935-42. 
 
11. Parise RA, Ramanathan RK, Hayes MJ and Egorin MJ. Liquid chromatographic–
mass spectrometric assay for quantitation of imatinib and its main metabolite (CGP 
74588) in plasma.  J Chromatogr B 2003; 791(1-2):39-44. 
 
11 
12. Bakhtiar R, Lohne J, Ramos L, Khemani L, Hayes M and Tse F. High-throughput 
quantification of the anti-leukemia drug STI571 (Gleevec™) and its main metabolite 
(CGP 74588) in human plasma using liquid chromatography–tandem mass 
spectrometry.  J Chromatogr B 2002; 768(2):325-40.  
 
13. Widmer N, Béguin A, Rochat B, Buclin T, Kovacsovics T, Duchosal MA, et al.  
Determination of imatinib (Gleevec®) in human plasma by solid-phase extraction–
liquid chromatography–ultraviolet absorbance detection.  J Chromatogr B 2004; 
805(2):285-92. 
 
14. Copland M, Hamilton A, Elrick LJ, Baird JW, Allan EK, Jordanides N, et al. 
Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in 
primary CML, but does not eliminate the quiescent fraction. Blood 2006; 107:4532-
39. 
 
15. Jordanides NE, Jørgensen HG, Holyoake TL and Mountford JC.  Functional 
ABCG2 is over-expressed on primary CML CD34+ cells and is inhibited by imatinib 
mesylate. Blood 2006; 108:1370-73. 
 
 
12 
TITLES AND LEGENDS  
 
Table 1.  Concentration of IM in matched bone marrow and plasma samples from 
treated CML patients at haematological remission (3 or 4 months). 
 
 
 
 
 
 
 
 
 
 
 
  
 
          
 
 
 
 
 
 
 
13 
 
 
Table 1.  Concentration of IM in matched bone marrow and plasma samples from 
treated CML patients at haematological remission (3 or 4 months) 
 
   IM Concentration (µmolar)   
Sample  n   Peripheral blood  Bone marrow   
All patients 10      1.64 ± 0.07      2.14 ± 0.08    
CCR    5      1.78 ± 0.21      2.15 ± 0.12   
No CCR  5      1.49 ± 0.12      2.13 ± 0.24  
 


Enlighten

       Iqbal, Z., Elliott, M., Watson, D.G., Holyoake, T.L. and Jorgensen, H.G. (2011) Analysis of imatinib in bone marrow and plasma samples of chronic myeloid leukaemia patients using solid phase extraction LC-ESI-MS. Pakistan Journal of Pharmaceutical Sciences, 24 (3). pp. 285-291. ISSN 1011-601X  http://eprints.gla.ac.uk/54114/  Deposited on: 24 November 2011   Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk 1 ZAFAR IQBAL1, MOIRA ELLIOTT1, DAVID G WATSON1, TESSA HOLYOAKE2 AND HEATHER JØRGENSEN2  1Division of Pharmaceutical Sciences, Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom 2Section of Experimental Haematology, Division of Cancer Sciences & Molecular Pathology, University of Glasgow, Royal Infirmary, Glasgow, United Kingdom   ABSTRACT  The LC-ESI-MS was developed and validated for the analysis of imatinib in plasma and bone marrow samples using deuterated imatinib (D8-IM) as an internal standard. The biological samples were extracted using Strata-X-C SPE cartridges and separated on C8 column (50 x 3 mm, 3 µm), and methanol: 0.1% formic acid (70:30) was delivered at the rate of 0.7 ml/min as a mobile phase. Imatinib was quantified in samples by monitoring the ions m/z 494.3 for imatinib and 502.3 for D8-imatinib on mass spectrometer. The method was linear in the concentration range of 1-1500 ng/250 µL in spiked human plasma samples and limit of quantification was 5 ng/mL. Inter-day and intra-day variations in spiked human plasma spiked with 50, 250 and 500 ng /mL were less than 3.16%. The repeatability and reproducibility and other parameters of the methods were also validated. The method was employed for the analysis of the imatinib in human plasma and bone marrow samples. The drug levels in bone marrow and plasma samples were correlated to the degree of cytogenetic response. No significant difference of imatinib level between blood and bone marrow in IM-treated patients dosed to steady state was observed.  Keywords: Bone marrow, Imatinib, Leukaemic, LC- ESI-MS, Human plasma.    2 INTRODUCTION   The selective Abl tyrosine kinase inhibitor, imatinib mesylate (IM; Gleevec or Glivec) has become the gold standard treatment of Chronic Myeloid Leukaemia (CML)1,2.  Discovered from a rational drug design program, IM blocks the activity of BCR-ABL, the oncogenic tyrosine kinase created by the Philadelphia (Ph) chromosome translocation diagnostic of CML.  Although clinical studies in the three sequential stages of the disease (chronic phase3, accelerated phase4, and terminal blast crisis5,6) confirm the substantial activity of the drug in vivo, responses are not sustained in advanced phases.  Further, the majority of patients in the early chronic phase do not achieve the deepest level of response, i.e. molecular remission that is polymerase chain reaction (PCR) negativity for BCR-ABL7.  Knowing that CML is a clonal disease of stem cell origin, it has been postulated that low stem cell turnover in vivo may facilitate survival of ‘protected’ quiescent stem cells able to maintain and repopulate the disease8,9.  Physiologically, bone marrow is the major compartment for white cell production, and this is the compartment in which quiescent CML stem cells are presumed to reside.  In the face of emerging resistance it would be of interest to know the level of IM achieved in patient marrow.  We sought to assess whether inadequate patient response was in part related to failure to achieve a sufficiently high IM level in bone marrow as compared to peripheral blood.   3 The aim of the present work was to relate drug levels to the clinical status of patients at the time of sampling using a suitably sensitive, precise and accurate method for the analysis of IM in peripheral blood and matched bone marrow plasma samples.  4 DESIGN AND METHODS   Reagents and solutions Methanol, acetonitrile, high pressure liquid chromatography (HPLC) water, 98% v/v formic acid and 35% v/v ammonia solution (analytical reagent grade) were all obtained from BDH Laboratory Supplies (Poole, UK).  IM and the internal standard D8-IM were kind gifts from Novartis Pharma, Basle, Switzerland.  Stock IM and internal standard D8-IM solutions at 1mg/mL were prepared in 50:50 methanol:1% w/v formic acid and stored at +4ºC until use.  The stock solution was diluted for HPLC analysis with 1% w/v formic acid.    Clinical samples Peripheral blood was drawn into ethylene diamine tetra-acetic acid (EDTA) tubes with informed consent from CML patients or healthy volunteers according to the Declaration of Helsinki.  Bone marrow aspirate, excess to clinical diagnostic requirements, was also harvested from the CML patients.  CML marrow was sampled at 3 or 4 months follow-up post-initiation of IM. The plasma supernatant was separated by centrifugation of whole blood at 1200rpm for 10 minutes and stored frozen at -20°C until required.  Glasgow Royal Infirmary Local Research Ethics Committee approved the use of human tissue in this study.  Solid phase extraction (SPE) method for plasma and bone marrow samples Frozen peripheral blood or bone marrow plasma samples were thawed at room temperature.  0.25mL was transferred to a 1mL plastic vial to which was added 0.25mL 0.1M phosphate buffer pH3.0 with an appropriate volume of internal standard solution.  The solution was vortex mixed for 30 seconds then transferred to a Strata-5 X-C column (Phenomenex, Macclesfield, UK) previously washed with 1mL of phosphate buffer.  The column was washed with 1mL water, 1mL methanol, and then twice eluted with 1mL of 2M ammonium hydroxide in methanol.  The eluent was collected, evaporated under a stream of nitrogen and dissolved in 0.1% w/v formic acid.  High pressure liquid chromatography mass spectrometry (LC-MS) for analysis of IM in biological fluids  IM analysis of plasma or bone marrow samples was performed using a Spectra Systems HPLC equipped with a P2000 pump, AS1000 autosampler with 20µL fixed injection loop and UV2000 detector (ThermoSeparations, Hemel Hempstead, Herts, UK).  Chromatographic separation was at ambient temperature using an ACE 3µm C8 column (50 x 3mm internal diameter; HiChrom, Reading, UK).  Mobile phase (methanol:formic acid 0.1% w/v) in the ratio of 70:30 was delivered in isocratic mode at a flow rate of 0.7mL / min over 10 minutes.    Analytes were detected using an LCQ Finnigan Automass LC-MS system (ThermoSeparations).  Samples at 20µL were injected via the autosampler into the mass spectrometer without splitting at a flow rate of 0.7mL / min.  The interface was operated in positive ion electrospray mode at 4.5 kV.  The capillary temperature was adjusted to 270°C.  The MS data were acquired under selected ion monitoring mode by monitoring for the IM ion at m/z 494.3 and at 502.3 for D8-IM.    6 RESULTS AND DISCUSSION    Based on our group’s research interest in persistent quiescent CML stem cells within the bone marrow compartment, we postulated that truly successful chemotherapeutic disease eradication may only be achieved if local drug delivery is optimal.  An investigation into the correlation of peripheral blood and bone marrow plasma drug levels was therefore planned for which a sensitive, precise and accurate analytical methodology had to be developed.    Extraction of IM from biological samples using an SPE method proved to be superior to the solvent based approach of existing methods.11-13  Using the latter methodology, the poorest result was achieved with acetonitrile as the extraction solvent with only 68% drug recovery in comparison to 89% ± 5% IM recovery by SPE (IM at 200ng in 250µL, n = 5).   The LC-MS analytical methodology proved to be both specific without interference from other peripheral blood or bone marrow plasma components, and sensitive with a limit of detection (LOD) of 5ng / 250µL of IM.  Method linearity was proven by direct injection of standards in the range 1ng to 500ng / 250µL mobile phase (7 data points) described by the regression equation y = -0.0245 + 0.00203x (r2 = 0.998). Method precision (expressed as co-efficient of variation, % CV) and accuracy were studied at IM concentrations of 50, 250 and 500ng / 250µL.  Results were entirely satisfactory with intra-day accuracy in the range 89% to 98%, and intra-day precision (% CV) ranging from 0.6% to 3.5%.  These results were reproducible in inter-day studies.   7 From a cohort of 10 CML patients enrolled in a clinical trial studying IM in chronic phase (Novartis studies 0106 and 0113), matched peripheral blood and bone marrow aspirate samples were processed to determine drug levels.  The average IM level in peripheral blood samples taken at 3 or 4 months post-initiation of IM therapy (Table 1) was in keeping with published human pharmacokinetic data dosed to steady state10.  At this time, all patients had achieved an haematological remission (to < 10 x 106 / mL). However, only half had achieved the next level of response that is a complete cytogenetic response (CCR).  None of the patients were PCR-negative for BCR-ABL at this stage.   On analysis of total data from all patients, there was no significant difference in the average drug concentration between peripheral blood and bone marrow plasma. This suggests that 400mg IM daily is sufficient to sustain equilibrium between peripheral blood and bone marrow plasma at a therapeutic level (approximately 2 µM). When the levels measured in the cytogenetically responding patients were compared with the non-responders, there were still no significant differences between sub-groups in peripheral blood or bone marrow.   In summary, using a sensitive assay for measurement of IM in biological samples, we have found that the IM concentration in peripheral blood and bone marrow plasma from CML patients dosed to steady state is in equilibrium. Despite the relatively limited patient numbers studied, these data were nonetheless consistent demonstrating good distribution of the drug into this previously un-assayed tissue. However, as we believe that higher intracellular levels of IM are required to kill stem cells than mature leucocytes in vitro, due to over-expression of BCR-ABL protein and / or multi-drug 8 resistance pumps14,15, we now hypothesise that more effective intracellular stem cell drug loading may be required within the bone marrow compartment.   9 REFERENCES   1.  Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, et al.  Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344: 1031–37.   2. Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Fernandes-Reese S, Ford JM, et al.  Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.  N Engl J Med 2001; 344: 1038–42.  3. Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-Passerini C, et al.  Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia.   N Engl J Med 2002; 346: 645–52.   4. Talpaz M, Silver RT, Druker BJ, Goldman JM, Gambacorti-Passerini C, Guilhot F, et al.  Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood 2002; 99:1928–37.   5. Kantarjian HM, Cortes J, O’Brien S, Giles FJ, Albitar M, Rios MB, et al.  Imatinib mesylate (STI571) therapy for Philadelphia chromosome-positive chronic myelogenous leukemia in blast phase. Blood  2002; 99:3547–53.   10 6. Sawyers CL, Hochhaus A, Feldman E, Goldman JM, Miller CB, Ottmann OG, et al.  Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a Phase II study. Blood 2002; 99:3530–39.   7.  Hughes TP, Kaeda J, Branford S, Rudzki Z, Hochhaus A, Hensley ML et al.  Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia.  N Engl J Med 2003; 349:1423–32.  8. Elrick LJ, Jørgensen HG, Mountford JC, and Holyoake TL. Punish the parent not the progeny. Blood 2005; 105: 1862–66.  9. Jørgensen HG, Allan EK, Graham SM, Godden J, Richmond L, Elliott MA, et al. Lonafarnib reduces the resistance of primitive quiescent CML cells to imatinib mesylate in vitro.  Leukemia 2005; 19: 1184-91.  10.  Peng B, Hayes M, Resta D, Racine-Poon A, Druker BJ, Talpaz M, et al. Pharmacokinetics and pharmacodynamics of imatinib in a Phase I trial with chronic myeloid leukemia patients.  J Clin Oncol 2004; 22(5):935-42.  11. Parise RA, Ramanathan RK, Hayes MJ and Egorin MJ. Liquid chromatographic–mass spectrometric assay for quantitation of imatinib and its main metabolite (CGP 74588) in plasma.  J Chromatogr B 2003; 791(1-2):39-44.  11 12. Bakhtiar R, Lohne J, Ramos L, Khemani L, Hayes M and Tse F. High-throughput quantification of the anti-leukemia drug STI571 (Gleevec™) and its main metabolite (CGP 74588) in human plasma using liquid chromatography–tandem mass spectrometry.  J Chromatogr B 2002; 768(2):325-40.   13. Widmer N, Béguin A, Rochat B, Buclin T, Kovacsovics T, Duchosal MA, et al.  Determination of imatinib (Gleevec®) in human plasma by solid-phase extraction–liquid chromatography–ultraviolet absorbance detection.  J Chromatogr B 2004; 805(2):285-92.  14. Copland M, Hamilton A, Elrick LJ, Baird JW, Allan EK, Jordanides N, et al. Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML, but does not eliminate the quiescent fraction. Blood 2006; 107:4532-39.  15. Jordanides NE, Jørgensen HG, Holyoake TL and Mountford JC.  Functional ABCG2 is over-expressed on primary CML CD34+ cells and is inhibited by imatinib mesylate. Blood 2006; 108:1370-73.   12 TITLES AND LEGENDS   Table 1.  Concentration of IM in matched bone marrow and plasma samples from treated CML patients at haematological remission (3 or 4 months).                                13   Table 1.  Concentration of IM in matched bone marrow and plasma samples from treated CML patients at haematological remission (3 or 4 months)     IM Concentration (µmolar)   Sample  n   Peripheral blood  Bone marrow   All patients 10      1.64 ± 0.07      2.14 ± 0.08    CCR    5      1.78 ± 0.21      2.15 ± 0.12   No CCR  5      1.49 ± 0.12      2.13 ± 0.24   

Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.

Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML, but does not eliminate the quiescent fraction. Blood

Determination of imatinib (Gleevec ®) in human plasma by solid-phase extraction– liquid chromatography–ultraviolet absorbance detection.

Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia.

Functional ABCG2 is over-expressed on primary CML CD34+ cells and is inhibited by imatinib mesylate. Blood

High-throughput quantification of the anti-leukemia drug STI571 (Gleevec™) and its main metabolite (CGP 74588) in human plasma using liquid chromatography–tandem mass spectrometry.

Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood

Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a Phase II study. Blood

Imatinib mesylate (STI571) therapy for Philadelphia chromosome-positive chronic myelogenous leukemia in blast phase. Blood

Liquid chromatographic– mass spectrometric assay for quantitation of imatinib and its main metabolite (CGP 74588) in plasma.

Lonafarnib reduces the resistance of primitive quiescent CML cells to imatinib mesylate in vitro. Leukemia

Pharmacokinetics and pharmacodynamics of imatinib in a Phase I trial with chronic myeloid leukemia patients.

Punish the parent not the progeny. Blood

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Analysis of imatinib in bone marrow and plasma samples of chronic myeloid leukaemia patients using solid phase extraction LC-ESI-MS

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