Abstract
The safety, pharmacokinetics and biological effect of plerixafor in children as part of a conditioning regimen for chemo-sensitization in allogeneic hematopoietic stem cell transplantation (HSCT) have not been studied. This is a phase I study of plerixafor designed to evaluate its tolerability at dose of 0.24 mg/kg given intravenously on day -4 (level 1), day -4, and day -3 (level 2), or day -4, -3, and day -2 (level 3) in combination with fludarabine, thiotepa, melphalan, and rabbit anti-thymocytic globulin for a second allogeneic HSCT in children with refractory or relapsed leukemia. Immunophenotype analysis was performed on blood and bone marrow prior to and after plerixafor administration. Twelve patients were enrolled. Plerixafor at all 3 levels was well tolerated without dose-limiting toxicity. Transient gastrointestinal side effects of National Cancer Institute grade 1 or 2 in severity were the most common adverse events. The area under the concentration-time curve increased proportionally to the dose level. Plerixafor clearance was higher in males, and increased linearly with body weight, and glomerular filtration rate. The clearance decreased and the elimination half-life increased significantly from dose level 1 to 3 (P < 0.001). Biologically, the proportion of CXCR4-positive blasts and lymphocytes both in the bone marrow and peripheral blood, increased after plerixafor administration.
Keywords: plerixafor, children, allogeneic, stem cell transplantation
Introduction
The outcome in children with relapsed leukemia undergoing a second allogeneic hematopoietic stem cell transplantation (HSCT) is poor, with a 5-year overall survival ranging from 30% [1, 2, 3] to 48% [4]. Children may experience lower regimen-related toxicity from a second transplant compared to adults [2, 4], however, recurrent disease remains a predominant cause of death. Thus novel therapeutic strategies are needed to overcome leukemia resistance to improve the outcome after second HSCT. Interaction between leukemia cells and the bone marrow stromal environment is postulated to be an important mediator of this resistance [5, 6]. The chemokine receptor CXCR4 is expressed in acute myeloid leukemia [AML; 7] and acute lymphoblastic leukemia ALL; 8], binds to CXCL12 expressed by the marrow stroma, and promotes survival of the leukemic cells. Increased expression of CXCR4 has been associated with an increased risk of relapse and poor outcome in ALL [9], and AML [10, 11].
Plerixafor (Mozobil™, Sanofi USA, Bridgewater, NJ) is a reversible inhibitor of the binding of CXCL12 to CXCR4. It is FDA approved for use in combination with granulocyte colony stimulating factor (G-CSF) to mobilize hematopoietic stem cells in patients with non-Hodgkin's lymphoma and multiple myeloma undergoing autologous transplantation [12, 13]. In a murine model of AML, mice treated with chemotherapy plus plerixafor had lower tumor burdens and improved overall survival compared with mice treated with chemotherapy alone [14]. A phase 1/2 study of plerixafor in adults with relapsed or refractory acute myeloid leukemia showed that the drug was well tolerated, and disrupted the CXCR4/CXCL12 axis [15].
The toxicity, pharmacokinetics, and biological effect of plerixafor when used in children as part of conditioning for an allogeneic HSCT is not known. We conducted a phase I trial to investigate the maximum tolerated dose of plerixafor, pharmacokinetics, and cell surface expression of CXCR4.
Methods
Study Population
The study was conducted at St. Jude Children's Research Hospital in Memphis, Tennessee, and was approved by the hospital's Institutional Review Board. Consent was obtained from all parents / guardians, and assent was obtained from all children more than 7 years of age.
Eligibility criteria included age ≤ 21 years, a hematologic malignancy that had relapsed after prior allogeneic HSCT, and scheduled to receive a bone marrow stem cell graft from a 7/8 or 8/8 HLA allele-matched related or unrelated donor. Patients had to have adequate renal, hepatic, cardiac and pulmonary function as determined by institutional guidelines. Exclusion criteria included active central nervous system malignancy, neuromuscular dysfunction, or ongoing treatment for acute or chronic graft-versus-host disease (GVHD). All patients had a performance score of 100.
Conditioning regimen included fludarabine 30 mg/m2 on day -9 through day -5, thiotepa 5 mg/kg/dose for 2 doses on day -4, melphalan 70 mg/m2 on day -3 and -2, and rabbit anti-thymocytic globulin (rATG) 3 mg/kg/day on days -3 through -1 after a test dose of 1mg/kg on day -4. GVHD prophylaxis included tacrolimus starting day -2, sirolimus starting day 0 and methotrexate 5 mg/m2 on days +1, +3 and +6. Patients at risk for cytomegalovirus or herpes simplex reactivation received prophylaxis with acyclovir, and all patients received prophylaxis with metronidazole, co-trimoxazole and antifungals in accordance with institutional guidelines.
Plerixafor dose schedule
Plerixafor 0.24 mg/kg/day was given intravenously at 3 dose levels (1 dose; day -4; level 1), (2 doses; days -4 and -3; level 2), (3 doses; days -4, -3 and -2; level 3). Plerixafor was administered 5 hours prior to chemotherapy at each dose level.
Pharmacokinetic Testing
Blood samples for pharmacokinetic testing were obtained prior to plerixafor and at 30 minutes, 1, 2, 6, 12, and 24 hours after infusion of each dose. Samples were spun, and sera were cryopreserved, batched, and run at a later date. Samples prepared by a protein precipitation extraction procedure in sodium heparin plasma were analyzed by liquid chromatography / tandem mass spectrometry over the concentration range of 5 to1000 ng/mL. The API 5000 was operated in the Multiple Reaction Monitoring (MRM) mode under optimized conditions for detection of plerixafor and AMD16617 positive ions formed by electrospray ionization. Calibration standards were placed at the beginning and end of each bio-analytical run.
Pharmacokinetic Analysis
The population pharmacokinetic and individual post-hoc estimates were determined using non-linear mixed effects modeling performed with Monolix (version 4.2.2, www.monolix.org). A two-compartment pharmacokinetic model with first-order elimination was fit to the data. Parameters estimated included systemic clearance (CL, L/hr or L/hr/kg), volume of distribution (V, L or L/kg), inter-compartmental clearance (Q, L/hr or L/hr/kg), and volume of peripheral compartment (V2, mL or L/kg). The inter- individual variability of the parameters was assumed to be log normally distributed. A proportional residual error model was used with assumed normal distribution of the residuals. Estimates of area under the concentration-time curve from 0 to 72 hrs (AUC, ng*hr/mL), maximum concentration (Cmax, ng/mL), and minimum concentration (Cmin, ng/mL) were determined using the individual post-hoc estimates.
Covariates, including demographics, glomerular filtration rate (GFR) as assessed by Tc99m renal clearance, serum creatinine, AST, ALT, bilirubin, and absolute neutrophil count were evaluated to determine their significance in explaining pharmacokinetic variability. These covariates were considered significant in a univariate analysis if their addition to the model reduced the objective function value (OFV) at least 3.84 units (P < 0.05, based on the χ2 test for the difference in the -2 log-likelihood between 2 hierarchical models that differ by 1 degree of freedom), and the covariate term was significantly different than zero (P < 0.05, t-test).
Immunophenotype analysis
Blood samples and bone marrow aspirate for immunophenotype analysis was obtained prior to plerixafor administration on day -5 and after its administration on day -4 (prior to thiotepa administration). The cell content was phenotyped by flow cytometry using BD FACSCanto™ II flow cytometer, BD FACSDiva 6.0 software, and a red cell lysis/multi-color antibody protocol. The following monoclonal antibodies against cell surface or intracellular markers were used: Anti-CD45 APC-H7 (Clone 2D1), Anti-CD33 PE-Cy7 (Clone P67.6), Anti-sCD3 V450 (Clone UCHT1), Anti-CD7 FITC (Clone M-T701), Anti-CD5 PE-Cy7 (Clone L17F12), Anti-CD19 APC (Clone SJ25C1), Anti-CD33 APC (Clone P67.6), Anti-HLADR APC-H7 (Clone L243), Anti-CD184 (CXCR4) PE (Clone ID9), Anti-IgG 2a PE (Clone X-39; all 6from BD Biosciences, San Jose, CA); Anti-CD34 PerCP (Clone 581), Anti-cCD3 PerCP (Clone SK7; all from Biolegend, San Diego, CA); Anti-CD38 FITC (Clone T16; Beckman Coulter, Pasadena, CA) and Anti-CD133 APC (Clone AC133, Miltenyi Biotec, Cambridge, MA). Patient-specific combinations of 6 or 8 antibodies and Boolean gating scheme were used to identify the blasts for each patient and determine their CXCR4 expression. Matched isotype control was used to determine the upper limit of fluorescent background.
Toxicity
Dose limiting toxicity (DLT) was defined as any grade IV organ toxicity not due to conditioning or underlying malignancy, attributable to plerixafor from the first dose on day -4 through day +7 post-HSCT. Adverse events and toxicities due to plerixafor were assessed using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events, version 4.0.
Routine evaluation
GVHD was assessed in accordance with published criteria [16]. Daily physical examination and blood testing, including complete blood count and serum chemistries were obtained. The day of engraftment was defined as the first of 3 measurements on consecutive days of achieving an absolute neutrophil count > 500 cells/μL. Primary graft failure was defined as an ANC never meeting or exceeding 500 cells/μL for 3 measurements on consecutive days by day +30 post-transplant.
Statistical Design
The maximum tolerated dose (MTD) was determined using a conventional Phase I study design with cohorts of 3 to 6 patients each. The MTD was defined as the dose level immediately below the level at which 2 or more patients out of a cohort of 3-6 patients experienced a DLT. If no patient experienced a DLT at dose level 1 and 2, then a total of 6 patients were treated at level 3.
Patients were enrolled in the study between August 2010 and December 2012. All patients received the doses of plerixafor as scheduled. No patient was lost to follow up. The characteristics of patients are summarized using frequencies for categorical variables and mean, median, and range for continuous variables. SAS version 9.2 (SAS Institute, Cary, NC) was used for statistical analysis.
Results
A total of 12 patients were enrolled in the study. Patient characteristics are outlined in Table 1. Of the 12 patients, 8 were in complete remission (CR) and 4 were in morphological relapse at the time of second transplant. One of them had blasts in the peripheral blood. Five patients received TBI-based conditioning for the first transplant. The median interval between the first and the second transplant was 11 (range 3-24) months. For the second transplant 10 donors were matched-unrelated and 2 were matched-siblings. Two patients did not engraft before they died, both on day +14 due to infection. For the remaining 10 patients the mean time to neutrophil and platelet engraftment was 22 (range, 13-27) days and 34 (range, 17-64) days, respectively. The mean time to neutrophil and platelet engraftment for 10 historical controls at our center with the same conditioning regimen without plerixafor, during the same period was 19 (range, 14-23) days and 22 (range, 14-42) days, respectively. Criteria for primary graft failure was not met in this phase I trial. There were no effects of plerixafor administration on chimerism which was full in both lymphoid and myeloid compartments post-transplant. The mean CD34+ cell dose was 7.6 × 106 (range 2.2-25.6 × 106) cells/kg. Grade II-IV GVHD was seen in 3 patients, no patient had chronic GVHD.
Table 1. Patient characteristics.
| Characteristic | n = 12 |
|---|---|
| Median age years (range) | 9 (6-15) |
| Male sex | 8 |
| Diagnosis | |
| AML | 8 |
| ALL | 4 |
| CR2 | 4 |
| CR3 | 4 |
| Bone marrow blasts | |
| 5-25% | 2 |
| >90% | 2 |
Abbreviations: n, number of patients; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; CR, complete remission.
With a mean duration of follow up of 332 (range 14-754) days, 4 of the 12 patients (33%) are surviving at the time of this report, 2 (17%) without progression of disease. One of these patients was on dose level 2 and the other on dose level 3. All four patients were in CR prior to transplant. Four patients died of progression of underlying disease on day +754 (dose level 1), day +295 (dose level 1), day +94 (dose level 2), and day +511 (dose level 2). Three patients died due to infection: one each due to parainfluenza virus type 3 on day +14 (dose level 1), disseminated Fusarium oxysporum on day +14 (dose level 3), and Epstein-Barr virus induced lymphoproliferative disease (EBV-PTLD) on day +100 (dose level 3). One patient died of pleomorphic sarcoma of the chest wall as a second malignancy on day +129 (dose level 3). There were 2 patients who relapsed on day +260 (dose level 3) and day +134 (dose level 3), but both are currently in complete remission after further chemotherapy, and a third allogeneic HSCT, respectively.
Toxicity
Plerixafor at all 3 levels was well tolerated, with none of the 12 patients experiencing a DLT. Of the 12 patients, 7 had at least one adverse event possibly related to the drug, of NCI grade 1 (4 patients) or 2 (3 patients) in severity. Nausea and abdominal pain were the most common adverse events seen in 3 patients each, and observed between day -4 to day -1. One patient was on dose level 2, and the others on dose level 3. The abdominal pain resolved in 1-3 days, while the nausea lasted for 2 days in one patient and up 4-6 weeks in the other 2 patients. One patient on dose level 1 complained of headache, NCI grade 2 in severity, and another patient on dose level 3 had diarrhea, NCI grade 1 in severity, both on day -1 which resolved in 2 days. No patient experienced a severe adverse event (SAE) related to plerixafor.
Of the 12 patients on study, 9 had SAE unrelated to the use of plerixafor. Three patients had fever with neutropenia, one patient on dose level 1 had veno-occlusive disease on day +8, one had pericardial effusion related to EBV-PTLD on day +54, one had a sympathetic pericardial/pleural effusion from a second malignancy on day +78, one had a pleural effusion related to relapsed leukemia on day +58, and 2 patients with respiratory failure due to parainfluenza virus and disseminated Fusarium respectively, succumbed to the infection.
Pharmacokinetic profile
The pharmacokinetics of plerixafor, stratified by dose level, are summarized in Table 2. The highest dose cohort reached a median peak concentration of 801 (range, 693-860) ng/mL (Table 2). Plerixafor clearance increased linearly with body weight (P < 0.01, Figure 1). The clearance decreased (0.15, 0.10, 0.07 L/hr/kg for dose level 1, 2, and 3, respectively), and the elimination half-life increased (2.19, 2.42, 2.77 hours for dose level 1, 2, and 3, respectively) as the dose escalated from level 1 to 3 (P < 0.001; Table 2). The inclusion of this change in clearance, relative to the dose level, decreased the inter-individual variability coefficient of variation of clearance from 17.4% to 8.5%. The exposure or AUC to plerixafor over the first 24 hours accounted for 95%, 49%, and 33% of its total exposure, at dose level 1, 2 and 3, respectively. In addition we observed that weight normalized clearance of plerixafor was 50% higher in males (P < 0.05), and increased with GFR (P < 0.01; Figure 2). The sole patient with mild renal impairment and a GFR of 67mL/min/1.73 m2 at dose level 3 had a plerixafor clearance of 0.04 L/hr/kg, approximately 57% lower than the median clearance for that cohort. No adverse events related to plerixafor were observed in this patient. Clearance was not affected by age.
Table 2.
Summary pharmacokinetics of plerixafor at the 3 dose levels. The results are the posthoc median (range) of each variable.
| Dose Level | n | Clearance L/hr/kg | AUC0-24 (ng*hr/mL) | Cmax (ng/mL) | Cmin (ng/mL) | Half-life (hours) |
|---|---|---|---|---|---|---|
| 1 | 3 | 0.15 (0.08-0.17) | 1480 (1477-3009) | 548 (429-63) | 3.80 (1.31-8.84) | 2.19 (1.68-3.07) |
| 2 | 3 | 0.10 (0.08-0.15) | 4593 (3333-6058) | 641 (526- 710)) | 3.96 (3.06- 7.25 | 2.42 (2.10-2.86) |
| 3 | 6 | 0.07 (0.04-0.09) | 9628 (8366-18083) | 801 (693-860) | 7.15 (1.29-56.38) | 2.77 (2.19-6.44) |
Abbreviations: n, number of patients; AUC0-24, area under the concentration-time curve; Cmax, maximum concentration; Cmin minimum concentration. Plerixafor 0.24 mg/kg/day was given intravenously at 3 dose levels (1 dose; day -4; level 1), (2 doses; days -4 and -3; level 2), (3 doses; days -4, -3 and -2; level 3).
Figure 1.
Clearance of plerixafor (L/hr) with respect to body weight (kg).
Figure 2.
Clearance of plerixafor (L/hr/kg) with respect to the glomerular filtration rate (GFR; mL/min/1.73m2)
CXCR4 expression on leukemic cells and lymphocytes
Increase in the percentage of CXCR4-positive blasts and lymphocytes was documented in both the bone marrow (2 patients) and peripheral blood (3 patients) by flow cytometry after treatment with plerixafor (Figure 3). Of the 2 patients with adequate marrow samples, bone marrow blasts remained unchanged with 90% blasts pre-and post-plerixafor in the first patient and decreased from 24% to 18% post-plerixafor in the second patient, both on dose level 1. Peripheral blood blasts were not detected by morphologic review in the first patient, decreased from 10% to <1% post-plerixafor in the second patient, and was not detected in the third patient on dose level 2. Lymphocytes in the peripheral blood were scant by morphologic review pre-and post-plerixafor in all 3 patients. The increased proportion of CXCR4+ blasts detected by flow cytometry did not result in more leukemia blasts as detected by bone marrow morphology in the 2 patients, or in the peripheral blood in 3 patients.
Figure 3.
Percentages of CXCR4-positive blasts (4A, 4B) and lymphocytes (4C, 4D) as detected by flow cytometry (anti-CD184, clone ID9) in bone marrow (4A, 4C), and peripheral blood (4B, 4D) pre-plerixafor (gray), and post-plerixafor (black). Patient #3 did not have a satisfactory bone marrow sample for CXCR4 analysis.
The third patient who was in CR2 did not have a satisfactory bone marrow sample for CXCR4 analysis. Two other patients with morphological disease pre-transplant (90% and 6% blasts in bone marrow) were not able to undergo bone marrow tests pre-and post-plerixafor due to unstable respiratory status.
Discussion
Plerixafor given intravenously for 3 consecutive days at a dose of 0.24 mg/kg/day with fludarabine, thiotepa, melphalan and rATG was well tolerated in patients undergoing a second allogeneic HSCT for recurrent hematologic malignancy. The safety of this drug in healthy human volunteers [17], patients with myeloma and lymphoma for stem cell mobilization [18], and patients with acute leukemia [15], has been established. The most common adverse events attributable to plerixafor in this study were mild and transient gastrointestinal effects similar to that observed in other studies [15, 17, 18]. None of our patients had an increase in blasts in the peripheral blood after plerixafor. Symptomatic leukostasis was not observed after plerixafor in adult patients with acute leukemia [15]. Prolonged cytopenias, a theoretical concern of the use of CXCR4 antagonists with chemotherapy, were not observed in our HSCT setting. Use of fludarabine prior to plerixafor did not result in neurotoxicity. The cardiac complications were secondary in origin and no patient had premature ventricular contractions as reported previously in 2 patients with a history of cardiac disease [19].
Plerixafor exhibited linear pharmacokinetics in children, and most of the drug was eliminated within 24-48 hours of administration. There are no published pharmacokinetic parameters to compare with in either children or adults after intravenous administration. After subcutaneous administration of 0.24 mg/kg of plerixafor in 13 adults with hematologic malignancies, the median AUC0-24, was 4299 (range 2678-5287) hr* ng/mL, with a median terminal half-life of 4.6 (range 2.7-11.7) hours [20]. This contrasts with a median AUC0-24 of 1480 (range 1477-3009) hr* ng/mL, with a median terminal half-life of 2.19 (range 1.68-3.07) hours, after intravenous administration of 0.24 mg/kg in our study. Subcutaneous concentration-time profiles, when compared to intravenous profiles, after administration of 0.08 mg/kg showed median peak concentration equivalent to 48%, followed by identical AUC and elimination rates [17]. Hence the increased clearance in children cannot be attributed to the route of administration alone. The relationship between clearance of plerixafor and body weight shown in this study supports dosing by body weight. Increased clearance in males is a novel observation. There was no relationship between clearance and age after adjustment for body weight. Clearance decreased after repeated administration of the drug, with increase in the elimination half-life. The AUC in dose level 2 was comparable to that achieved in adults after single dose administration. This supports using level 3 dosing for children in future studies both from safety and pharmacokinetic considerations. Plerixafor clearance was reduced in the child with renal impairment, and positively correlated with GFR as noted in adults with renal impairment [21]. Dose reduction in patients with mild renal impairment (60-89 mL/min/1.73 m2) may result in exposure similar to that in patients with normal renal function.
Previous studies have demonstrated an increase in CXCR4-positive blasts in the peripheral blood after plerixafor administration [15]. Because serial marrow samples were not obtained concurrently, it was unclear whether the change was due to mobilization of CXCR4-expressing blasts from the marrow or due to up-regulation of CXCR4 surface expression. Herein, we provide concurrent blood and marrow data for the first time and show that the percentage of CXCR4-positive blasts increased simultaneously in both compartments after plerixafor treatment. Thus, these data argue against the notion of migration of CXCR4+ blasts from the marrow to the blood, but rather support the hypothesis of up-regulation of CXCR4 surface expression. This hypothesis was further supported by our data demonstrating an increase in CXCR4+ lymphocytes concurrent with the increase in CXCR4+ blasts in both compartments. Mechanistically, the increase in surface expression may be due to inhibition of SDF-1 induced internalization of CXCR4 [22], as hematopoietic cells are known to contain large intracellular stores of CXCR4 [23].
Without concurrent chemotherapy, this phenotype of increase circulating CXCR4+ blasts would be expected to enhance leukemia dissemination; however when given with conditioning chemotherapy, no increase in absolute number of blasts was observed in the peripheral blood of our patients. The increase in CXCR4 expression in leukemia cells and recipient lymphocytes during conditioning may improve leukemia control and reduce graft rejection, as augmented CXCR4 signaling may induce apoptosis in AML cells via regulation of the Bcl-2 family members Bcl-XL, Noxa, and Bak [24]. Future studies may examine their in-vitro susceptibility to chemotherapeutic agents.
In this phase I study where plerixafor was used in the novel setting of a second allogeneic HSCT, we showed that it was well tolerated, has unique pharmacokinetics in children, and can pharmacodynamically increase CXCR4 expression on leukemic blasts in the bone marrow and peripheral blood. Engraftment was robust even with a reduced-intensity conditioning regimen. Despite a very poor expected survival in such a group of patients, a third of the patients are alive, half of them without evidence of disease. Future studies using plerixafor in children with leukemia undergoing a first allogeneic HSCT are warranted.
Acknowledgments
The authors thank Traci-Stewart Saltwell, Tiffany Johns and Melanie Decker from the Clinical Research Office, Bone Marrow Transplantation and Cellular Therapy, at St. Jude Children's Research Hospital, Memphis, TN, for assistance in data collection.
This work was supported by research funding from Sanofi, US, National Cancer Institute Cancer Center CORE Support Grant P30 CA 21765 and by the American Lebanese Syrian Associated Charities.
Footnotes
The authors have no conflicts of interest to disclose.
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