Patent Application
20100184779
The present invention relates to a method of treating chronic myeloid leukemia (CML) in a human patient population.
The success of treatment with Imatinib mesylate in the majority of chronic phase CML patients is well documented. Improving treatment outcomes for those patients who perform less well however requires a detailed understanding of the critical determinants of treatment response. It was previously demonstrated that intrinsic sensitivity to Imatinib-induced kinase inhibition is a good predictor of response (White D, Saunders V, Lyons A B, et al. Blood. 2005; 106:2520-2526; Schultheis B, Szydlo R, Mahon F X, Apperley J F, Melo J V. Blood. 2005; 105:4893-4894) and that this is closely related to the intracellular uptake and retention (IUR) of imatinib (White D L, Saunders V A, Dang P, et al. Blood. 2006; 108:697-704). Further it has been demonstrated that the active transport of imatinib is dependent on the organic cation transporter OCT-1 (Thomas J, Wang L, Clark R E, Pirmohamed M. Blood. 2004; 104:3739-3745; White D L, Saunders V A, Dang P, et al, OCT-1 mediated influx is a key determinant of the intracellular uptake of imatinib but not nilotinib. Blood. 2006; 108: 697-704).
The OCT-1 protein is a member of the largest superfamily of transporters, the solute carrier family (Koepsell H, Endou H. Pflugers Arch. 2004; 447:666-676) which transport in an electrogenic fashion a variety of organic cations, including drugs, toxins and other xenobiotics. The transporter is predicted to have 12 transmembrane domains, and binding pockets with partially overlapping interaction domains for different substrates and inhibitors (Koepsell H, Schmitt B M, Gorboulev V. Rev Physiol Biochem Pharmacol. 2003; 150:36-90). Post transcriptional regulation of OCT-1 by phosphorylation status (Ciarimboli G, Schlatter E. Pflugers Arch. 2005; 449:423-441) and such compounds as PKA, Src-like p56 and CaM have also been demonstrated.
It was now found that a significant correlation exists between the IC50imatinib and OCT-1 Activity, and that patients with a low IC50imatinib have significantly greater OCT-1 Activity than patients with high IC50imatinib. That implies that a greater OCT-1 activity is associated with a more efficient inhibition of the drug target(s). By comparing the OCT-1 Activity with molecular response in all patients enrolled to the TIDEL trial (chronic phase newly diagnosed CML patients who received 600 mg Imatinib mesylate up-front), it was demonstrated that patients with high OCT-1 Activity achieve significantly greater molecular responses over 24 months of Imatinib treatment than patients with low OCT-1 Activity. Further it was found that the molecular response of those patients with low OCT-1 Activity is dose dependent, with patients receiving 600 mg of Imatinib mesylate achieving significantly better molecular response by 24 months than those receiving less than 600 mg. Significantly, a group of patients with low OCT-1 Activity was identified who are at higher risk for suboptimal or failed Imatinib response if they do not receive the trial dose of 600 mg or at least 600 mg of Imatinib mesylate per day. Further study results show that a greater proportion of patients with Low OCT-1 Activity randomised to 800 mg imatinib achieve MMR by 12 months, when compared to those randomised to 400 mg. The data disclosed herein demonstrates that dose is an important factor for overcoming a risk of suboptimal response in low OCT-1 Activity patients.
Importantly, using this functional assay to determine OCT-1 Activity at the time of diagnosis may identify CML patients likely to respond well to standard dose Imatinib mesylate, and those who would be most likely to benefit from a higher dose of Imatinib mesylate.
Hence, the present invention pertains to a method of treating CML in a warm-blooded animal comprising the steps of
Preferably, the warm blooded animal is a human.
In a preferred embodiment, the daily dose to be administered is between about 600 and 1000 mg of Imatinib mesylate, e.g. 600 mg/day, 800 mg/day, 1000 mg/day or 1200 mg/day.
In an embodiment, in step (b) a daily dose of at least 400 mg of Imatinib mesylate is administered orally. In an embodiment, in step (b) a daily dose of at least 600 mg of Imatinib mesylate is administered orally. In an embodiment, in step (b) a daily dose of at least 800 mg of Imatinib mesylate is administered orally.
In an embodiment, in step (b) a daily dose of between about 600 and 1000 mg of Imatinib mesylate is administered orally. In an embodiment in step (b) a daily dose of between 600 mg and 1000 mg of Imatinib mesylate is administered orally.
A. Correlation between the IC50imatinib and the IUR in 99 patients Box plot demonstrating the difference in IUR between low and high IC50imatinib groups, and also demonstrating the removal of this difference using Prazosin, a potent inhibitor of OCT-1.
A. Correlation between the IC50imatinib and the OCT-1 Activity in 99 patients
B. Box plot demonstrating the difference in OCT-1 Activity between low and high
IC50imatinib groups.
OCT-1 Activity and Molecular response in A. Between low and high OCT-1 Activity groups. B. The effect of dose in patients with high OCT-1 Activity. C. The effect of dose in patients with low OCT-1 Activity
Kaplan Meier demonstrating the difference in achievement of MMR between low and high OCT-1 Activity groups at various time points.
Demonstrating the effect of dose escalation by dividing patients in to low and high OCT-1 Activity groups and then further subdividing them on the basis of ADD.
The OCT-1 Activity compared to molecular responses (log reduction in BCR-ABL) at 18 months. Response criteria assessed are suboptimal response (failure to achieve MMR by 18 months), and optimal responses (achievement of 3- to 4-log reduction in BCR-ABL, and >4-log reduction by 18 months. Cohorts are divided:
A. All patients irrespective of dose received
B. Patients receiving <600 mg ADD over the first 12 months
C. Patients who failed to dose escalate to 800 mg ADD after 12 months
D. Patients who successfully dose escalated to 800 mg ADD after 12 months of therapy
A. Dot plot showing the correlation between the OCT-1 Activity and OCT-1 mRNA
B. Box plot demonstrating the difference between the levels of OCT-1 mRNA in the low and high OCT-1 Activity groups.
The term “major molecular response (MMR)” as used herein means a 3 log reduction in BCR-ABL transcripts, quantified from peripheral blood using real-time quantitative reverse-transcriptase polymerase chain reaction, preferably after 12 months of therapy, e.g. 12 months Imatinib mesylate therapy.
The term “complete cytogenic response (CCR)” as used herein means 0% Philadelphia-chromosome positive metaphases among at least 20 or 25 cells in metaphase in the bone marrow aspirate (Colombat M, Fort M P, Chollet C, et al. Molecular remission in chronic myeloid leukemia patients with sustained complete cytogenetic remission after imatinib mesylate treatment. Haematologica 2006; 91:162-8.).
Imatinib is generically and specifically disclosed in the patent applications U.S. Pat. No. 5,521,184, in particular in Example 21, the subject-matter of which is hereby incorporated into the present application by reference. Imatinib can also be prepared in accordance with the processes disclosed in WO03/066613.
For the purpose of the present invention, Imatinib is preferably applied in the form of its mono-mesylate salt. Imatinib mono-mesylate can be prepared in accordance with the processes disclosed in U.S. Pat. No. 6,894,051 the subject-matter of which is hereby incorporated into the present application by reference. Comprised are likewise the corresponding polymorphs, e.g. crystal modifications, which are disclosed therein.
Imatinib mono-mesylate can be administered in dosage forms as described in U.S. Pat. No. 5,521,184, U.S. Pat. No. 6,894,051 or US 2005-0267125.
The collecting of a blood sample from CML patients required under step (a) of the methods described herein can be accomplished by standard procedures being state of the art. The blood derived cells from patients could be fresh or frozen to conduct the assay for evaluating OCT-1 activity.
OCT-1 Activity as referred to herein is calculated as the difference in IUR of the drug/imatinib in the absence (total IUR) and presence of prazosin, an inhibitor of OCT-1, to provide a measure of the activity of the OCT-1 protein in the transport of imatinib, as defined by the intracellular level of the drug. For example: [Total IUR 32 ng/200,000 cells)]-[prazosin IUR 23 ng/200,000 cells] gives an OCT-1 Activity of 9 ng/200,000 cells.
As described in the Examples below, OCT-1 Activity was measured in pre-therapy blood from CML patients. The activity of OCT-1 is derived by measuring the difference in intracellular uptake and retention (IUR) of [14C]-Imatinib in cells from CML patients grown in vitro with and without OCT-1 inhibitor, prazocin. 85% of patients with >median (high) OCT-1 Activity achieved major molecular response (MMR) by 24 months, versus 45% with ≦median (low) OCT-1 Activity. Assessing patients receiving 600 mg Imatinib mesylate/day and those averaging <600 mg over 12 months of therapy revealed patients with high OCT-1 Activity achieved excellent molecular response regardless of dose, whereas response of patients with low OCT-1 Activity was highly dose dependent. 45% of patients with low OCT-1 Activity who received <600 mg failed to achieve a 2-log reduction by 12 months, and 82% failed to achieve a MMR by 18 months, compared to 8% and 17% in the cohort with high OCT-1 Activity and dose <600 mg/day (p=0.017 and p=0.022). OCT-1 Activity is an important determinant of molecular response to Imatinib, and has a predictive value closely linked to dose.
The following examples are illustrative, but do not serve to limit the scope of the invention described herein. The examples are meant only to suggest a method of practicing the present invention. The data shown below were collected from samples of CML patients in the TIDEL trial.
Measure Oct-1 Activity by Intracellular Imatinib Concentration
Reagents: Hanks Balanced Salt Solution (HBSS), Ca++ and Mg++ free. SAFC Biosciences Add 0.05 mM Hepes prior to use. Foetal Bovine Serum (FBS). Lymphoprep, Axis Shield. MICROSCINT-20 scintillation fluid, PerkinElmer. RPMI Medium, W/o L-Glutamine, Ca++ and Mg++ free, SAFC Biosciences.
Prazosin—Inhibits OCT-1
(Prazosin Hydrochloride, MW=419.9, Sigma)
Use at 100 μM from 10 mM stock
10 mM stock=4.2 mg/mL methanol
Working Solution of 14C-Imatinib
50% Mixture of Hot and Cold Imatinib, 100 μM—for 1 mL:
Specimen Requirements
Peripheral blood, bone marrow, and other fluids containing haematopoietic cells, collected aseptically in lithium or sodium heparin.
Blood should be ficolled and mononuclear cells (MNC) should be used in this assay. The preparation of the MNC is followed standard lab technique using a density gradient.
Cryopreserved, and subsequently thawed, MNC may be used in place of fresh—ensure to compensate for viability.
Method
1. All assay points must be performed in triplicate.
2. Label 24×2 ml microfuge tubes as detailed below (Appendix 1).
3. Dispense 1 mL RPMI+10% FBS into each tube using stepper pipette.
4. Dispense 200,000 viable MNC into each tube.
5. Add inhibitors of influx/efflux as required, as detailed below (Appendix 1).
6. Add 14C-imatinib to tubes as per Appendix 1.
7. Make up to total volume of 2 mls with RPMI+10% FBS dropwise using a pasteur pipette.
8. Incubate for 2 hours at 37° C./5% CO2 for standard 2 hr assay.
9. Centrifuge tubes 6800 rpm for 5 mins. Pulse spin to 13000 rpm, 30 secs.
10. Take a 20 μl aliquot of supernatant (S/N) from each tube and mix with 100 μl Microscint-20 in 96-well plate as per Appendix 4.
11. Cover plate with adhesive plastic cover-seal and wrap plate in foil.
12. Pulse spin tubes again to 13000 rpm, 30 secs.
13. Aspirate all remaining fluid using a cannula+yellow tip attached to suction line—do not disturb cell pellet!
14. Add 50 μl of Microscint to the pellet using stepper pipette.
15. Resuspend the cells by vortexing. Pulse spin 15 secs at 13000 rpm.
16. Transfer the lysed cells to a secondary 96 well plate as per Appendix 2.
17. Cover plate with adhesive cover-seal and wrap in foil.
18. Count all plates on the TopCount beta counter.
19. Enter count data into IUR-imatinib Assay Template Spreadsheet.
In a smaller series (n=19) a good correlation between the IC50imatinib and the IUR (p=0.014) was demonstrated (White D L, Saunders V A, Dang P, et al. Blood. 2006; 108:697-704). In this current expanded series (n=99) again a strong correlation (r=−0.342; p=0.0005) is demonstrated, confirming the relationship between the two parameters (
The addition of prazosin, a potent inhibitor of OCT-1, to the IUR assay impairs the active transport of Imatinib by OCT-1. Examination of 132 patients enrolled to both trials, reveals a wide variation in OCT-1 Activity (median 8.2: Range 0 to 31.2). In replicate assays of 5 patients the IUR values with prazosin were equal or lower than the values without prazosin. These patients were scored as having negligible (Ong/200,000 cells) OCT-1 Activity.
In 99 patients where both IC50imatinib and OCT-1 Activity were measured, there was a significant correlation between the IC50imatinib and the OCT-1 Activity (r=−0.238; p=0.019) (
The OCT-1 Activity was compared to molecular response over the first 24 months of Imatinib mesylate therapy in 56 patients enrolled to the TIDEL trial. Patients were grouped into low and high OCT-1 Activity based on the median activity for this cohort of 7.2 ng/200,000 cells. As shown in Table 1 and
However, because of tolerability issues, not all patients received 600 mg of Imatinib mesylate consistently over the first 12 months of therapy. To assess the effect of varying dose patients were further sub-grouped into those patients who received an average daily dose (ADD) of 600 mg (n=33) over the first 12 months of Imatinib therapy, and those who received an ADD of less than 600 mg per day (n=23 median ADD 523 mg). Four patients with ADD <400 mg were included in this cohort.
Assessing only those patients with high OCT-1 Activity there is no significant difference in molecular response between those patients who received <600 mg versus those patients who received 600 mg or more over the first 12 months (p=0.449 at 24 months) (
Kaplan Meir Analysis revealed 85% of patients with high OCT-1 Activity achieved a MMR (median time to achieve 9 months) by 24 months versus only 45% of patients with low OCT-1 Activity (median time 24 months) p=0.009 (
In the TIDEL trial, dose increases from the initial 600 mg to 800 mg per day were mandated if a 4 log reduction in BCR-ABL from the standardized baseline was not achieved by 12 months. In the patient cohort of the present Example 46 patients were scheduled to receive dose increases. In this analysis a dose increase was considered to have occurred if the patient received 800 mg ADD for at least one month.
For reasons of toxicity only 29 (63%) patients in this cohort successfully dose increased to 800 mg per day. The primary reason for inability was previous toxicity/tolerance issues which occurred in 13 of those 17 patients who did not dose escalate. Of the remaining 4 patients 2 dose escalated for one month but did not reach 600 mg ADD. It is not known why the remaining 2 patients failed to escalate.
Dose increase occurred by 14 months in all patients who were able to dose increase. To assess the impact of dose escalation patients were grouped into low and high OCT-1 Activity groups as previously, then were further subdivided into those who received increased dose and those who remained on 600 mg or less as shown in
The median OCT-1 Activity in patients who failed to achieve MMR by 18 months is significantly lower than OCT-1 Activity in patients who did achieve MMR (
Suboptimal response has been defined as failure to achieve a major cytogenetic response by 6 months, a complete cytogenetic response by 12 months or a MMR by 18 months (Baccarani M, Saglio G, Goldman J, et al. Blood. 2006; 108:1809-1820). As good correlation between molecular and cytogenetic response has been demonstrated previously (Branford S, Hughes T P, Rudzki Z. Br J Haematol. 1999; 107:587-599) the suboptimal response as failure to achieve 1-log reduction in BCR-ABL by 6 months, a 2-log reduction by 12 months and a S-log reduction (MMR) by 18 months was assessed. Kaplan Meier analysis as reported in Table 2 reveals there is no significant difference in the frequency of suboptimal response at any time-point when patients receive 600 mg ADD, however there is a significant difference at all time-points when patients receive reduced dose, demonstrating patients with low OCT-1 Activity who receive reduced dosing are at substantial risk of suboptimal response to Imatinib mesylate.
Assessing the failure to achieve a 2-log reduction by 18 months (Imatinib failure, Baccarani M, Saglio G, Goldman J, et al. see above) reveals also that a significantly lower proportion of patients with low OCT-1 Activity who receive reduced dose achieve a 2-log reduction by 18 months (high OCT-1 Activity 8% low OCT-1 Activity 36% p-0.04).
The level of OCT-1 mRNA was measured relative to BCR mRNA in 93 patients. The relative mean % mRNA was 0.835 within a range 0.02 to 3.5%. Correlating mRNA with OCT-1 Activity revealed a p-value of 0.002 (r=0.378) (
Limiting analysis to those patients where 24 month molecular follow-up is available (TIDEL patients) a significant difference in the molecular response between the two groups (low and high OCT-1 mRNA) could not be demonstrated (Table 3). Also in contrast to OCT-1 Activity there was no significant difference between the two groups with respect to dose (600 mg or lower), and dose escalation (to 800 mg) (Table 3) Unlike OCT-1 Activity, mRNA analysis did not reveal a group of patients with suboptimal response or at risk for Imatinib failure (data not shown).
This assay was performed in previously untreated, chronic phase CML patients prior to imatinib therapy. Primary End Point: Achievement of MMR (Major Molecular Response:>3 log reduction in BCR-ABL as measured by RQ-PCR) by 12 month.
Statistics Kaplan Meier, Log Rank Survival, t-test and Log Rank Analysis. P value for significance at the <0.05 level as indicated.
Results
A: Achievement of MMR by 12 Months (Percentage of Patients)
B: Median Molecular Response (Log Reduction in BCR-ABL)
This data show that (i) a greater proportion of patients with Low OCT-1 Activity randomised to 800 mg imatinib achieve MMR by 12 months, when compared to those randomised to 400 mg, (ii) There is an outcome benefit observed for patients with low OCT-1 Activity with doses of >400 mg (in this case 800 mg) of imatinib per day; (iii) Patients with Low OCT-1 Activity seem to further benefit from dose increase of higher than 800 mg. This data supports the initial findings of the TIDEL trial and therefore provides confirmatory evidence for the importance of OCT-1 Activity in the achievement of MMR.
| Number | Date | Country | Kind |
|---|---|---|---|
| 07110868.2 | Jun 2007 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AU2008/000911 | 6/20/2008 | WO | 00 | 12/18/2009 |
