All subjects provided informed consent and received investigational study drug on a National Heart, Lung and Blood Institute Institutional Review Board (IRB)-approved research protocol designed to study the safety and CD34+ cell mobilizing activity of escalating doses of s.c. plerixafor. The enrollment period was from June 14, 2006 until May 7, 2008. Volunteers ≥18 and ≤50 years of age who had normal renal, hepatic and haematological function were eligible. Exclusion criteria included cerebrovascular disease, cardiac disease, a Framingham 10-year coronary risk of >10%, positive human immunodeficiency virus status, a history of hepatitis B or C, a history of cancer in the past 5 years, or a history of autoimmune disease.
This three-cohort, dose-escalation, pilot study investigated doses of plerixafor up to 0·48 mg/kg in healthy volunteers and was designed so that each subject received two different doses of plerixafor. The two dose per-subject design allowed for the exploration of dose-response relationships for both safety and activity that minimized inter-subject variability because each subject served as their own control. The protocol mandated that further enrollment be prohibited (pending IRB and Data and Safety Monitoring Board review) for any occurrence of a Grade 4 treatment-related AE or any Grade 3 AE known to be related to plerixafor (for example, ≥Grade 3 thrombocytopenia, gastrointestinal symptoms and neuropathy).
Healthy volunteers (Fig 1 ) received the following s.c. doses of plerixafor: 0·24 and 0·32 mg/kg (Cohort 1); 0·32 and 0·40 mg/kg (Cohort 2); and 0·40 and 0·48 mg/kg (Cohort 3). Subjects received the higher dose at least 14 days after the first to allow adequate wash-out of plerixafor. The absolute number of circulating CD34+ cells was measured by standard flow cytometry techniques after each dose of plerixafor at the following times: 0, 2, 4, 6, 8, 10, 12, 14, 18 and 24 h. The samples were evaluated for CD34+ cell content using the International Society of Hematotherapy and Graft Engineering guidelines (Sutherland et al, 1996 (link)). Flourochromes used were CD45 APC-Cy7 and CD34 APC (BD Biosciences, San Jose, CA, USA), using the BD FACSCanto instrument (BD Biosciences). Analysis was performed using the FACSDiva software (BD Biosciences).
Adverse reactions were recorded, graded and summarized using National Cancer Institute Common Terminology Criteria for Adverse Events, version 3 (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf ). Each cohort consisted of a planned enrollment of six subjects. If a subject did not complete both doses of plerixafor and did not experience DLT, that subject was replaced. Dose escalation was allowed if protocol-specified stopping rules for toxicity were not met. In addition to inpatient monitoring during the first 24 h after each injection, subjects returned for an assessment 1 week after each dose. Complete blood counts and serum chemistry panels were obtained on all subjects after each dose of plerixafor and during each follow-up visit at 1 week. Subjects in the 0·40 and 0·48 mg/kg dose cohorts were monitored by telemetry for 24 h following each dose. Additionally, following a protocol amendment, subjects in Cohort 3 underwent electrocardiography (EKG) monitoring with QTc measurements at baseline, 8, 12 and 24 h after each dose.
To assess colony forming units (CFU), peripheral blood mononuclear cells were inoculated at 1 × 105 cells/ml in three different methylcellulose culture medias (MethoCult H4230; Stem Cell Technologies, Vancouver, Canada), supplemented with 5 u/ml of recombinant human (rHu) erythropoietin (Epo; Amgen, Thousand Oaks, CA, USA), 10 ng/ml rHu granulocyte-macrophage colony stimulating factor (GM-CSF; Sandoz, East Hanover, NJ, USA), 10 ng/ml rHu interleukin-3 and 100 ng/ml rHu stem cell factor (SCF; R&D Systems, Minneapolis, MN, USA). Plated cells were incubated at 37°C with 5% CO2 for 10–14 days. Colonies were then counted with pre-plerixafor dosing colonies compared to post-plerixafor dosing colonies.
Blood samples for pharmacokinetic (PK) analyses were collected for six subjects in Cohort 2 who received a dose of 0·40 mg/kg and six subjects in Cohort 3 who received a dose of 0·48 mg/kg. Samples for PK analysis were collected prior to dosing and at 0·083, 0·25, 0·5, 1, 2, 4, 6, 8, 10 and 24 h post-dose. Plasma plerixafor concentrations were determined using a validated liquid chromatography-mass spectrometry/mass spectrometry method (Eruofins AvTech, Kalamazoo, MI, USA). Pharmacokinetic parameters [performed by the Clinical Pharmacology Department at Genzyme Corporation using WinNonlin Professional, version 5·2 (Pharsight Corp., Mount View, CA, USA)] were determined from non-compartmental methods using nominal times of blood collection. Pre-dose concentrations below the lower limit of quantitation were set to zero for the purposes of the analysis.
The study was primarily designed to determine the safety of escalating doses of plerixafor. The safety analysis included data from all subjects. An exploratory analysis of CD34+ cell mobilization was conducted to evaluate whether preliminary data suggested higher doses of plerixafor increased CD34+ cell mobilization as a rationale for a potentially larger study. The analyses of CD34+ cell mobilization included data from subjects who received two successive doses of plerixafor. Summary statistics were analysed for demographics, peak CD34+ mobilization, intra-subject differences in CD34+ cell mobilization, CD34+ cell AUC and intra-subject differences in CD34+ cell AUC. For the primary analyses of CD34+ cell mobilization, peak CD34+ cell numbers and CD34+ cell area under the curve (AUC) values for 24 h following plerixafor injection (beginning at 2 h) were calculated for each subject at each dose level. The Kruskal–Wallis test statistic was used to compare the peak CD34+ cell numbers and AUCs at the three-first-dose levels and the three-second-dose levels. The Kruskal–Wallis test was used to compare the differences in peak CD34+ cell counts and AUC at the higher and lower dose levels in the successive cohorts. Linear regression was used to model peak CD34+ cell AUC at the second dose level as a function of peak CD34+ cell AUC at the first dose level, dose, age and gender.
This three-cohort, dose-escalation, pilot study investigated doses of plerixafor up to 0·48 mg/kg in healthy volunteers and was designed so that each subject received two different doses of plerixafor. The two dose per-subject design allowed for the exploration of dose-response relationships for both safety and activity that minimized inter-subject variability because each subject served as their own control. The protocol mandated that further enrollment be prohibited (pending IRB and Data and Safety Monitoring Board review) for any occurrence of a Grade 4 treatment-related AE or any Grade 3 AE known to be related to plerixafor (for example, ≥Grade 3 thrombocytopenia, gastrointestinal symptoms and neuropathy).
Healthy volunteers (
Adverse reactions were recorded, graded and summarized using National Cancer Institute Common Terminology Criteria for Adverse Events, version 3 (
To assess colony forming units (CFU), peripheral blood mononuclear cells were inoculated at 1 × 105 cells/ml in three different methylcellulose culture medias (MethoCult H4230; Stem Cell Technologies, Vancouver, Canada), supplemented with 5 u/ml of recombinant human (rHu) erythropoietin (Epo; Amgen, Thousand Oaks, CA, USA), 10 ng/ml rHu granulocyte-macrophage colony stimulating factor (GM-CSF; Sandoz, East Hanover, NJ, USA), 10 ng/ml rHu interleukin-3 and 100 ng/ml rHu stem cell factor (SCF; R&D Systems, Minneapolis, MN, USA). Plated cells were incubated at 37°C with 5% CO2 for 10–14 days. Colonies were then counted with pre-plerixafor dosing colonies compared to post-plerixafor dosing colonies.
Blood samples for pharmacokinetic (PK) analyses were collected for six subjects in Cohort 2 who received a dose of 0·40 mg/kg and six subjects in Cohort 3 who received a dose of 0·48 mg/kg. Samples for PK analysis were collected prior to dosing and at 0·083, 0·25, 0·5, 1, 2, 4, 6, 8, 10 and 24 h post-dose. Plasma plerixafor concentrations were determined using a validated liquid chromatography-mass spectrometry/mass spectrometry method (Eruofins AvTech, Kalamazoo, MI, USA). Pharmacokinetic parameters [performed by the Clinical Pharmacology Department at Genzyme Corporation using WinNonlin Professional, version 5·2 (Pharsight Corp., Mount View, CA, USA)] were determined from non-compartmental methods using nominal times of blood collection. Pre-dose concentrations below the lower limit of quantitation were set to zero for the purposes of the analysis.
The study was primarily designed to determine the safety of escalating doses of plerixafor. The safety analysis included data from all subjects. An exploratory analysis of CD34+ cell mobilization was conducted to evaluate whether preliminary data suggested higher doses of plerixafor increased CD34+ cell mobilization as a rationale for a potentially larger study. The analyses of CD34+ cell mobilization included data from subjects who received two successive doses of plerixafor. Summary statistics were analysed for demographics, peak CD34+ mobilization, intra-subject differences in CD34+ cell mobilization, CD34+ cell AUC and intra-subject differences in CD34+ cell AUC. For the primary analyses of CD34+ cell mobilization, peak CD34+ cell numbers and CD34+ cell area under the curve (AUC) values for 24 h following plerixafor injection (beginning at 2 h) were calculated for each subject at each dose level. The Kruskal–Wallis test statistic was used to compare the peak CD34+ cell numbers and AUCs at the three-first-dose levels and the three-second-dose levels. The Kruskal–Wallis test was used to compare the differences in peak CD34+ cell counts and AUC at the higher and lower dose levels in the successive cohorts. Linear regression was used to model peak CD34+ cell AUC at the second dose level as a function of peak CD34+ cell AUC at the first dose level, dose, age and gender.
