Pharmacokinetics and pharmacodynamics of liposomal mifamurtide in adult volunteers with mild or moderate renal impairment

Abstract

Aims

To evaluate the pharmacokinetics and pharmacodynamics following a single dose of liposomal mifamurtide (L-MTP-PE, MEPACT®) in adult subjects with mild (calculated creatinine clearance [CL_cr] of 50–80 ml min⁻¹) or moderate (CL_cr 30–50 ml min⁻¹) renal impairment in comparison with age-, weight- and gender-matched healthy subjects with normal renal function (CL_cr >80 ml min⁻¹).

Methods

Subjects received a 4 mg dose of liposomal mifamurtide via 1 h intravenous infusion. Blood samples were collected over 72 h for analysis of plasma pharmacokinetics of total and non-liposome-associated (free) mifamurtide and assessment of pharmacodynamics (changes in serum interleukin-6 [IL-6], tumour necrosis factor-α [TNF-α], C-reactive protein [CRP]).

Results

Thirty-three subjects were enrolled: nine with mild renal impairment, eight with moderate renal impairment and 16 healthy subjects. Geometric mean (%CV) AUC_inf for total mifamurtide was 89.5 (58.1), 94.8 (27.8), 85.1 (29.0), 95.4 (18.1) nm h in the mild renal impairment, mild-matched healthy subject, moderate renal impairment and moderate-matched healthy subject groups, respectively. Mifamurtide clearance was not correlated with CL_cr, estimated glomerular filtration rate or iohexol clearance (all r² < 0.01). AUC_inf of free mifamurtide was similar across the renal function groups. There were no readily apparent differences in serum pharmacodynamic effect parameters (baseline-adjusted AUEC_last for IL-6 and TNF-α and E_max for CRP) between the renal function groups. No subjects reported grade ≥3 or serious adverse events.

Conclusions

Mild or moderate renal impairment does not alter the clinical pharmacokinetics or pharmacodynamics of mifamurtide. No dose modifications appear necessary for these patients based on clinical pharmacologic considerations.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Liposomal mifamurtide is an immunotherapeutic agent that is indicated for treating high grade, resectable, non-metastatic osteosarcoma in combination with postoperative, multi-agent chemotherapy in children, adolescents and young adults.

• The contribution of renal clearance to mifamurtide clearance and the impact of renal impairment on its clinical pharmacokinetics have not been characterized.

• This phase 1 study was therefore performed to characterize the pharmacokinetics and pharmacodynamics of liposomal mifamurtide in adult volunteers with mild or moderate renal impairment, to enable development of scientifically guided dosing recommendations for these special patient populations.

WHAT THIS STUDY ADDS

• No dose modifications of liposomal mifamurtide are needed in patients with mild or moderate renal impairment based on the lack of an effect on its clinical pharmacokinetics or pharmacodynamics, as demonstrated in this study.

• The findings from this study have resulted in an update to the EU Summary of Product Characteristics for MEPACT® (liposomal mifamurtide).

Introduction

Mifamurtide (muramyl tripeptide phosphatidyl-ethanolamine; MTP-PE) a synthetic derivative of muramyl dipeptide, a naturally occurring component of bacterial cell walls, is an immunotherapeutic agent that potently stimulates macrophages and monocytes to elicit tumouricidal effects 1,2. It is delivered as a liposome-encapsulated formulation (liposomal mifamurtide; L-MTP-PE; MEPACT®). Liposomal mifamurtide is approved in the European Union 3, Switzerland and various other countries for treating high grade, resectable, non-metastatic osteosarcoma in combination with postoperative multi-agent chemotherapy in children, adolescents and young adults who have undergone macroscopically complete surgical resection.

A phase 3 trial (INT-0133) in patients with newly diagnosed, non-metastatic, high grade osteosarcoma demonstrated that addition of mifamurtide to chemotherapy resulted in a statistically significant and clinically meaningful decrease in the risk of death without compromising safety 4. An updated analysis in 2007, which led to approval in the European Union, showed a relative reduction in the risk of death of 28% (P = 0.0313, HR = 0.72, 95% CI 0.53, 0.97) with the addition of mifamurtide compared with chemotherapy alone 3. The adverse events (AEs) associated with mifamurtide were generally mild to moderate in severity 4. A recent report of a patient access study of mifamurtide in relapsed/recurrent osteosarcoma demonstrated a 1 year overall survival (OS) rate of 70%; this rate was 75% for patients who enrolled more than 9 months after diagnosis and received mifamurtide in combination with chemotherapy 5. Mifamurtide was reported to be well tolerated in this high risk patient population, with a safety profile consistent with previous reports. Common infusion-related AEs included chills, headache, fatigue, nausea and pyrexia 5.

The specific mechanisms and pathways of mifamurtide clearance in humans have not yet been fully elucidated. A recent study has characterized the pharmacokinetic and pharmacodynamic profile of total mifamurtide following a single 4 mg dose in healthy adult volunteers, to examine the clinical pharmacology of mifamurtide independently of the effects of underlying conditions or concomitant chemotherapy 6. The results of this study indicated that single 4 mg doses of mifamurtide can be safely administered to healthy adult volunteers for the purposes of pharmacokinetic and pharmacodynamic characterization, and that variability in mifamurtide pharmacokinetics is low (the coefficient of variation [%CV] in both the area under the curve [AUC] and the maximum concentration [C_max] was less than 30%) 6. However, the effect of renal impairment on mifamurtide pharmacokinetics and pharmacodynamics is unknown.

Osteosarcoma primarily occurs in children, adolescents and young adults 7, an age group not normally associated with renal impairment. However, patients with osteosarcoma typically receive high dose multi-agent chemotherapy 7,8, including agents such as ifosfamide and cisplatin, which have the potential for renal toxicity. The effect of renal impairment on mifamurtide pharmacokinetics and pharmacodynamics is currently unknown.

This study evaluated the pharmacokinetics and pharmacodynamics following a single infused dose of liposomal mifamurtide in subjects with mild (calculated creatinine clearance [CL_cr] of 50–80 ml min⁻¹) or moderate (CL_cr 30–50 ml min⁻¹) renal impairment, in comparison with matched healthy subjects with normal renal function (CL_cr >80 ml min⁻¹). The selection of an otherwise healthy volunteer population of subjects for this renal impairment study was supported by the similarity in pharmacokinetics of mifamurtide in healthy adult volunteers and the target osteosarcoma patient population 9 (data on file, Millennium: The Takeda Oncology Company). Evaluation of single dose pharmacokinetics was supported by a short half-life of mifamurtide of approximately 2 h 6 and the previously demonstrated lack of accumulation following twice weekly repeat dose administration 10. Mild and moderate impairment were selected to represent the levels of renal toxicity that may develop in patients receiving concomitant high dose chemotherapy with mifamurtide, in order to enable development of scientifically guided dosing recommendations for liposomal mifamurtide in patients with renal impairment.

Methods

Subjects

All subjects had to be 18–75 years old and to have a body mass index (BMI) of 18–35 kg m⁻². At least 50% of subjects were required to have a BMI of 18–30 kg m⁻². Subjects with mild or moderate chronic renal impairment required a calculated CL_cr (Cockcroft–Gault equation 11) of 50–80 ml min⁻¹ or 30–50 ml min⁻¹, respectively, and healthy subjects required normal renal function (calculated CL_cr >80 ml min⁻¹). All subjects were required to have stable renal function for 1 month prior to screening, as demonstrated by two values of calculated CL_cr obtained within 2 weeks before dosing varying by <25%. The average of these two values was used to determine eligibility for enrolment. For subjects with renal impairment, a history of cardiovascular events, diabetes mellitus, high blood pressure and/or hypercholesterolaemia was allowed, provided that this did not pose a significant safety risk that precluded participation and that any of these conditions were stable and well controlled. Healthy subjects were identified as healthy by medical history, physical examination and clinical laboratory evaluations, and were required to have a urine cotinine concentration of ≤300 ng ml⁻¹. Healthy subjects were matched to subjects in the mild or moderate renal impairment groups by age (± 10 years), gender (± 2 per gender) and body weight (± 10 kg).

Key exclusion criteria included poorly controlled type 1 or 2 diabetes or a current diabetic ulcer, a requirement for undergoing dialysis at the time of the study, a history of organ transplantation or immunosuppressant therapy, active autoimmune or inflammatory disease, acute infections and any cardiovascular events in the preceding 6 months. Subjects with a history of shellfish allergy, allergic/adverse responses to contrast media, known sensitivity to iodine or known clinical hypersensitivity precluding the use of iohexol were excluded. Subjects with malignancy in the past 5 years (including leukaemia and lymphoma, but with the exception of adequately treated non-melanoma or basal cell carcinoma) were also excluded.

All subjects provided written informed consent. The study protocol and informed consent documentation was approved by the Institutional Review Boards at each of the investigative sites. The study was conducted in compliance with the ethical principles originating in or derived from the Declaration of Helsinki.

Study design

This phase 1, open label, single dose, parallel group study was conducted at two investigative sites in the USA. The primary objective was to evaluate the pharmacokinetics and safety of total mifamurtide following a single dose of liposomal mifamurtide in subjects with mild or moderate chronic renal impairment, compared with matched healthy subjects. Additional objectives were to evaluate the pharmacokinetics of free (non-liposome-associated) mifamurtide, to evaluate the urinary excretion of mifamurtide and to evaluate the pharmacodynamics of a single dose of liposomal mifamurtide, as assessed by measurements of changes in serum IL-6, TNF-α and C-reactive protein (CRP).

The study was conducted over a period of 4 days, with follow-up at 7 days post-mifamurtide infusion. Subjects were administered a single 4 mg dose of liposomal mifamurtide via intravenous infusion over 1 h. The 4 mg dose was selected to equate approximately to the 2 mg m⁻² of body surface area (BSA) approved clinical dose of mifamurtide 3 and has been previously evaluated safely in a single dose clinical pharmacology study in healthy adult volunteers 6.

Assessments

Blood samples (10 ml) for plasma pharmacokinetics were collected before the start of infusion, at 15, 30 and 45 min after the start of infusion, at 1 h (at the end of infusion), at 5, 15, 30 min, 1, 2, 3, 4, 6, 8 and 11 h after the end of infusion and at 24, 36, 48 and 72 h after the start of infusion. Urine samples were collected for pharmacokinetic analysis for 24 h before infusion and during 0–12, 12–24, 24–48 and 48–72 h post-dose. Plasma concentrations of total and free (non-liposome-associated) mifamurtide and urine concentrations of mifamurtide were quantified using validated liquid chromatography/tandem mass spectrometry (LC-MS/MS) methods.

Plasma samples (prepared with K₂EDTA and stored at −70°C) were either used directly for analyte extraction and bioanalysis of total MTP-PE concentrations or were processed first by ultrafiltration followed by analyte extraction and bioanalysis for measurement of free (non-liposome-associated) concentrations of MTP-PE in the ultrafiltrate. Ultrafiltration of 1 ml plasma aliquots was performed in Millipore 0.22 μm PVDF 2.0 ml filtration units by centrifugation at 2000 g for 10 min at room temperature. Internal standard (15 ng ¹³C₃,¹⁵N-MTP-PE) was added to plasma (150 μl) or ultrafiltrate (100 μl) samples and analytes were extracted by protein precipitation with 0.5% acetic acid in methanol, followed by centrifugation, evaporation of the supernatant under nitrogen at ∼45°C and reconstitution in a 50:50 v/v mixture of 20 mm ammonium formate with 0.1% formic acid and isopropanol. Bioanalysis was performed by high performance liquid chromatography (HPLC) with column switching and tandem mass (MS/MS) detection using negative ion electrospray. Chromatography consisted of a Load and Elution Column: Phenomenex Gemini C18, 5 μ, 2.0 × 30 mm and 50 mm (Phenomenex, Torrance, CA, USA) respectively using mobile phase A, 20 mm ammonium formate with 0.1% formic acid in water and mobile phase B, 20 mm ammonium formate in 98% acetonitrile/methanol (50/50) with 0.1% formic acid. The load programme was 40% B for 0.5 min, at a flow rate of 500 μl min⁻¹, 40% B to 100% B over 4 min, 100% B flow rate increased to 1000 μl min⁻¹ over 2.4 min, 100% B reduced to 40% B over 1.1 min with a reduction in flow rate to 500 μl min⁻¹. The elution programme was 100% B for 3 min at a flow rate of 300 μl min⁻¹, 100% B flow rate increased to 800 μl min⁻¹ for 3 min, 100% B reduced to 50% B for 2 min. The retention time for MTP-PE and the internal standard was 5.1 min. Mass spectrometric detection was performed using a Sciex API 5000, Triple Quadrupole LC/MS/MS mass spectrometer operated in an electrospray negative ion MRM mode (AB Sciex, Framingham, MA, USA). The mass transitions monitored for total MTP-PE and MTP-PE-d₄ were 1235.7→1032.7 and 1239.8→1036.6, respectively. The dynamic range of the assay was 0.1 to 20 nm MTP-PE and the standard curve was analyzed by linear regression with 1/concentration weighting. Precision and accuracy were evaluated by analyzing quality control pools prepared at 0.100, 0.210, 0.500, 1.50, 4.00, 15.0 and 40.0 nm. The observed range of inter-assay precision (%CV 3.56–9.92), intra-assay precision (%CV 2.0–10.9), inter-assay accuracy (absolute % difference from theoretical ≤14.3%) and intra-assay accuracy (absolute % difference from theoretical ≤14%) were within the pre-specified performance criteria for method validation.

Pharmacokinetic parameters of total and free mifamurtide were calculated by non-compartmental analysis (WinNonlin® [Pharsight, St Louis, MO, USA] Version 6.1). These included C_max, area under the plasma concentration–time curve from time 0 to time of last quantifiable concentration (AUC_last) and to infinity (AUC_inf), clearance (CL, for total mifamurtide only), volume of distribution at steady-state (V_ss, for total mifamurtide only) and terminal half-life (t_1/2).

Blood samples (5 ml) were collected for measurements of IL-6, TNF-α and CRP serum concentrations: before infusion (IL-6, TNF-α and CRP), at the end of infusion (sample drawn immediately before switching off the infusion pump for IL-6 and TNF-α) and at 1, 2, 3, 6 and 8 h following the end of infusion (IL-6, TNF-α) and at 24 h (IL-6, TNF-α, and CRP) and 72 h (CRP) following the start of infusion. Cytokine concentrations were determined using a standardized sandwich antibody assay approach with the Meso Scale Discovery (MSD) Sector Imager 2400 instrument, at Viracor IBT Laboratories, Inc. (Lee's Summit, MO, USA). The calibration curves in these assays were anchored to the National Institute for Biological Standards and Control reference material. Pharmacodynamic parameters calculated by non-compartmental analysis (WinNonlin® Version 6.1) included maximum effect (E_max) for change from baseline in serum IL-6, TNF-α and CRP, time of first occurrence of maximum effect (TE_max) and area under the effect–time curve from time 0 to time of the last point of quantifiable effect (AUEC_last) for change from baseline in serum IL-6 and TNF-α.

CL_cr was calculated using the Cockcroft–Gault equation 11, shown below for male subjects (for female subjects, multiply by 0.85):

or

Estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease (MDRD) formula 12:

Baseline iohexol clearance (a measure of GFR) was determined the day before mifamurtide was administered. An intravenous bolus injection of iohexol (10 ml of Omnipaque™, 300 mg I/ml, Amersham, Princeton, NJ, USA) was administered, followed by blood sampling at 120, 180, 240 and 300 min post-dose for measurement of plasma iohexol concentrations. Iohexol clearance (CL_iohexol, ml min⁻¹) was determined from plasma iohexol concentration–time data using the multiple sampling approach and the Bröchner–Mortenson method of correction for non-immediate mixing of the tracer 13:

CL₀ in ml min⁻¹ is calculated from iohexol serum concentration–time data using the following equation:

where b is the terminal disappearance rate constant of iohexol (min⁻¹) calculated as the negative of the slope of the terminal log-linear segment of the serum concentration–time curve and c1 is the corresponding intercept on the y-axis.

Serum concentrations of β₂-microglubulin were measured in samples collected on the day before mifamurtide was administered. The pre-dose 24 h urine collections performed on the day before mifamurtide administration were used for measurement of baseline urinary excretion of β₂-microglobulin, Kidney Injury Molecule-1 (KIM-1), and creatinine. The baseline ratios of urinary β₂-microglobulin : urinary creatinine and urinary β₂-microglobulin : serum β₂-microglobulin were calculated as measures of baseline renal proximal tubular function 14–16, and the urinary KIM-1 : urinary creatinine excretion ratio was calculated as a marker of baseline acute renal proximal tubular injury 17,18.

Sitting vital signs (including blood pressure and pulse rate) were evaluated prior to mifamurtide infusion, at the end of infusion, and at 4, 8, 12, 24, 48 and 72 h after initiation of infusion. Incidences of adverse events (AEs), including serious AEs (SAEs), were recorded and coded using the Medical Dictionary for Regulatory Activities (MedDRA) Version 14, and the severity was graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v4.0. Treatment-emergent AEs were defined as any AEs that occurred between start of infusion and follow-up on day 8, any drug-related AE or any AE present at baseline that worsened after baseline or was subsequently considered drug-related.

Statistical analysis

The pharmacokinetic and pharmacodynamic populations were defined as enrolled subjects who received the full infusion of liposomal mifamurtide and provided adequate samples for determination of pharmacokinetic and pharmacodynamic parameters by non-compartmental analysis. The safety population was defined as all enrolled subjects who received any amount of study medication.

A sample size of approximately eight subjects in the mild and moderate renal impairment groups and 16 subjects in the healthy subject groups was determined based on typically utilized sample sizes in pharmacokinetic studies of investigational drugs in patients with renal impairment 19. The selection of this sample size was supported by the relatively low variability in mifamurtide pharmacokinetics reported previously 6 and was not based on statistical considerations. Descriptive statistics were used to summarize all parameters by renal function group. Linear regression analyses were used to estimate potential relationships between mifamurtide CL and baseline measures of renal function (CL_cr, eGFR or CL_iohexol) using SAS® Version 9.1 (SAS Institute, Inc., Cary, NC, USA).

Results

Subjects

Between 30 August 2010 and 17 June 2011, 33 subjects were enrolled, received study medication and were included in the pharmacokinetic, pharmacodynamic and safety populations. Nine subjects had mild renal impairment, eight had moderate renal impairment, and eight subjects were enrolled to each of the corresponding matched healthy subject groups. Thirty-two subjects (97%) completed the study. One subject in the mild renal impairment group experienced an AE of urinary incontinence 3.25 h after the end of infusion that lasted for 3 days. Therefore, he could not provide urine samples for pharmacokinetic analysis and was discontinued from the study.

Subject demographics are summarized in Table 1 and baseline renal function characteristics for the pharmacokinetic population are summarized in Table 2. The median CL_cr values decreased with the level of renal impairment, reflecting the protocol eligibility criteria for each renal function group. In the moderate renal impairment group, median values of the urinary β₂-microglobulin : serum β₂-microglobulin ratio (an index of β₂-microglobulin renal clearance) and the urinary β₂-microglobulin : urinary creatinine excretion ratio were increased approximately 3.3-fold and 12-fold, respectively, in comparison with the respective medians in the combined group of all healthy subjects, indicating decreased renal tubular function in the moderate renal impairment group. There were no readily apparent differences in the urinary KIM-1 : urinary creatinine excretion ratio between the groups, consistent with chronic kidney disease as opposed to acute tubular injury in the subjects with mild or moderate renal insufficiency.

Table 1.

Subject demographics in the safety population, by renal function group

	Mild RI (n = 9)	Healthy match, mild RI (n = 8)	Moderate RI (n = 8)	Healthy match, moderate RI (n = 8)
Median age, years (range)	62.0 (53–75)	61.0 (51–71)	60.0 (41–70)	65.5 (42–68)
Male gender, n (%)	6 (67)	5 (63)	4 (50)	4 (50)
Race, n (%)
White	6 (67)	5 (63)	3 (38)	5 (63)
Black or African American	3 (33)	2 (25)	3 (38)	2 (25)
American Indian or Alaska Native	0	1 (13)	1 (13)	0
Other	0	0	1 (13)	1 (13)
Median BSA, m2 (range)	1.87 (1.68–2.02)	1.84 (1.75–2.05)	1.92 (1.79–2.17)	1.95 (1.74–2.12)
Median weight, kg (range)	76.4 (58.8–92.1)	74.4 (66.7–90.4)	80.1 (71.2–104)	79.7 (64.9–98.6)

BSA, body surface area; RI, renal impairment.

Table 2.

Baseline renal function characteristics in the pharmacokinetic population, by renal function group

	Mild RI (n = 9)	Healthy match, mild RI (n = 8)	Moderate RI (n = 8)	Healthy match, moderate RI (n = 8)	All healthy (n = 16)
Median CLcr, ml min−1 (range) (local laboratory)*	60.6 (51.6–75.1)	89.6 (81.1–106.6)	39.9 (30.3–47.7)	102.3 (83.9–133.6)	93.9 (81.1–133.6)
Median CLcr, ml min−1 (range) (central laboratory)*	54.2 (32.8–62.8)	74.7 (53.9–91.4)	38.2 (30.3–47.5)	97.1 (65.7–150)	82.5 (53.9–150)
Median iohexol clearance, ml min−1 (range)	38.8 (28.2–47.8)	44.2 (37.8–51.9)	17.1 (13.6–22.9)	52.1 (43.0–66.4)	46.2 (37.8–66.4)
Median eGFR, ml min−1 (range)	53.5 (32.9–70.0)	62.4 (48.9–102)	33.8 (22.5–49.5)	86.7 (65.5–142)	79.6 (48.9–142)
Median urinary β2-microglobulin : urinary creatinine ratio, μg mmol−1 (range)	7.39 (4.24–47.0)	7.09 (2.26–12.2)	86.2 (12.8–235)	7.06 (2.48–8.08)	7.06 (2.26–12.2)
Median urinary β2-microglobulin : serum β2-microglobulin ratio (range)	0.021 (0.01–0.14)	0.027 (0.02–0.05)	0.089 (0.01–0.32)	0.029 (0.00–0.06)	0.027 (0.00–0.06)
Median urinary KIM-1 : urinary creatinine ratio, μg mmol−1 (range)	0.086 (0.03–0.11)	0.048 (0.03–0.14)	0.062 (0.05–0.12)*	0.086 (0.04–0.51)†	0.071 (0.03–0.51)‡

^*Average of two baseline values calculated using Cockcroft–Gault equation;

^†n = 7;

^‡n = 15.

Pharmacokinetics

Mean plasma concentration–time profiles of total mifamurtide in the renal function groups are shown in Figure 1. In all groups, plasma concentrations of total mifamurtide increased during the 1 h infusion and then declined over time in a biphasic manner. A rapid initial distribution phase was characterized by a steep decline in plasma concentrations (>90% decrease from peak concentrations within 30 min of infusion cessation). This was followed by a slower decline in plasma concentrations in the terminal phase, with plasma concentrations falling below quantifiable limits by 24 h in most subjects. The profiles across the renal function groups were superimposable.

Figure 1.

Total mifamurtide concentrations by renal function group. (A) Mean plasma concentration–time profiles in subjects with () mild or () moderate renal impairment and () healthy all (normal renal function); and (B) (○) individual, () mean and () geometric mean values of AUC_inf by renal function group. (‘healthy all’ = all subjects in the two healthy matched subject groups with normal renal function)

Pharmacokinetic parameters for total mifamurtide by renal function group are summarized in Table 3. Geometric mean values of all pharmacokinetic parameters were similar between subjects with mild or moderate renal impairment and subjects in the respective matched healthy subject groups. The terminal half-life of total mifamurtide was short, with mean values ranging from 2.03 to 2.27 h across renal function groups. Consistent with the observed mean plasma concentration–time profiles and pharmacokinetic parameters, the distribution of individual AUC_inf values for total mifamurtide was generally similar, with substantial overlap across all renal function groups (Figure 1B). Pharmacokinetic variability was low. The CVs for AUC_inf and C_max were <30% across most renal function groups. The only exception was in the mild renal impairment group, in which the CV for AUC_inf was 58.1%, due to a single subject having higher exposure relative to the other subjects in this group (Figure 1B).

Table 3.

Geometric mean (%CV) of plasma pharmacokinetic parameters for total and free mifamurtide by renal function group

Parameter, geometric mean (%CV)	Mild RI (n = 9)	Healthy match, mild RI (n = 8)	Moderate RI (n = 8)	Healthy match, moderate RI (n = 8)
Total mifamurtide
AUCinf, nm h	89.5 (58.1)*	94.8 (27.8)	85.1 (29.0)	95.4 (18.1)
Cmax, nm	83.2 (52.3)	92.0 (22.7)	80.8 (29.8)	98.0 (21.1)
t1/2, h, mean (SD)	2.03 (0.508)	2.26 (0.345)	2.27 (0.708)	2.17 (0.188)
CL, ml min−1	602 (32.6)*	569 (30.7)	634 (22.9)	565 (17.5)
Vss, l	26.6 (22.9)*	28.7 (32.2)	33.5 (51.1)	25.0 (35.8)
Free mifamurtide
AUCinf, nm h	19.9 (101)*	20.4 (34)	22.0 (25)	18.6 (31)
Cmax, nm	7.34 (56)	7.44 (32)	10.1 (136)	6.84 (37)
tmax, h, median (range)	1.00 (0.98–1.00)	0.88 (0.75–1.00)	1.00 (0.75–1.00)	1.00 (0.75–1.00)
t1/2, h, mean (SD)	2.15 (0.645)*	2.23 (0.385)	2.24 (0.582)	2.12 (0.195)

AUC_inf, area under the curve from time 0 extrapolated to infinity; C_max, maximum serum concentration; CL, clearance; CV, coefficient of variation; RI, renal impairment; t_1/2, terminal half-life; t_max, time of first occurrence of maximum concentration; V_ss, volume of distribution at steady-state.

^*n = 8.

Scatter plots and linear regression analyses of total mifamurtide CL vs. baseline CL_cr, eGFR, and CL_iohexol showed no discernible associations (Figure 2). In all three analyses, 95% confidence intervals (CI) of the estimated slope included zero and there were no correlations between the baseline measures of renal function and mifamurtide CL (r² < 0.01).

Figure 2.

Scatter plots and regression analyses of mifamurtide clearance (CL) vs. the baseline renal function measurements of CL_cr (A), eGFR (B) and CL_iohexol (C). , individual; , regression; , 95% CI

Mean plasma concentration–time profiles of free mifamurtide in the renal function groups are shown in Figure 3. Mean plasma concentrations of free mifamurtide increased during the period of intravenous infusion of liposomal mifamurtide, reaching a peak at the end of infusion. During and immediately after infusion, mean plasma concentrations of free mifamurtide were considerably lower than the corresponding total plasma mifamurtide concentrations. After 0.5 h from the end of infusion, mean plasma concentrations of free mifamurtide were only slightly lower than those of total mifamurtide, and declined in parallel with total mifamurtide concentrations in a log-linear manner. Similar to observations for total mifamurtide, plasma concentration–time profiles of free mifamurtide were superimposable across the renal function groups. As observed for total mifamurtide, the distribution of individual AUC_inf values for free mifamurtide was generally similar with substantial overlap across renal function groups (Figure 3B).

Figure 3.

Free (non-liposome-associated) mifamurtide concentrations by renal function group. (A) Mean plasma concentration–time profiles in subjects with () mild or () moderate renal impairment and () healthy all (normal renal function); and (B) (○) individual, () mean and () geometric mean values of AUC_inf by renal function group. (‘healthy all’ = all subjects in the two healthy matched subject groups)

Pharmacokinetic parameters for free mifamurtide are summarized in Table 3. The terminal half-life of free mifamurtide was similar to that for total mifamurtide and the systemic exposure (AUC_inf) of free mifamurtide was one-fifth to one-quarter of that for total mifamurtide. Mean values of AUC_inf, C_max and terminal half-life of free mifamurtide were similar between subjects with mild or moderate renal impairment and the matched healthy subjects.

In all urine samples, mifamurtide concentrations were below the limit of assay quantification. Therefore, mifamurtide renal excretion and renal CL could not be reported and were inferred to be negligible.

Pharmacodynamics

Following mifamurtide infusion, serum concentrations of IL-6 (median TE_max 3–4 h after initiation of infusion) and TNF-α (median TE_max 3 h after initiation of infusion) increased, with a return towards baseline values at 7 h after initiation of infusion for both cytokines. Variability in the baseline-adjusted pharmacodynamic parameters was high (Table 4), with 5- to 578-fold and 5- to 17-fold ranges in individual values of AUEC_last observed with IL-6 and TNF-α, respectively. The time courses of median change from baseline in serum concentrations of IL-6 and TNF-α by renal function group are shown in Figures 4 and 5, respectively, and individual, mean and median values of AUEC_last are shown in Figures 4 and 5 for IL-6 and TNF-α, respectively. The baseline-adjusted values of AUEC_last for IL-6 and TNF-α overlapped substantially across the renal function groups and were generally similar across the groups when viewed in the context of the observed pharmacodynamic variability (Table 4, Figures 4 and 5).

Table 4.

Baseline-adjusted pharmacodynamic parameters for IL-6, TNF-α, and CRP by renal function group

Parameter	Mild RI (n = 9)	Healthy match, mild RI (n = 8)	Moderate RI (n = 8)	Healthy match, moderate RI (n = 8)
Median AUEClast, pg ml−1 h (range)
IL-6	5 150 (178–103 000)	5 850 (1 410–36 000)	7 080 (3 960–21 400)	4 850 (2 620–91 200)
TNF-α	5 820 (663–11 200)	6 250 (2 530–14 000)	14 600 (2 480–28 200)	6 820 (2 880–14 100)
Median TEmax, h (range)
IL-6	4.00 (3.00–7.02)	4.00 (3.00–4.00)	4.00 (3.02–4.03)	3.03 (3.00–4.00)
TNF-α	3.00 (3.00–3.07)	3.00 (3.00–3.07)	3.00 (3.00–3.20)	3.02 (3.00–3.07)
Median Emax (range)
IL-6, pg ml−1	2,360 (72.9–19,400)	1,870 (504–13,200)	2,440 (1,110–6,940)	1,590 (672–31,200)
TNF-α, pg ml−1	2,330 (117–43,800)	2,650 (590–8,700)	4,540 (822–13,100)	2,520 (912–6,270)
CRP, mg dl−1	4.59 (1.66–12.4)*	6.53 (3.09–11.2)	6.56 (2.52–12.4)	6.04 (2.06–11.9)

AUEC_last, area under the effect–time curve from time 0 to time of the last point of quantifiable effect; E_max, maximum effect; RI, renal impairment.

^*n = 8.

Figure 4.

Change from baseline in serum IL-6 by renal function group. (A) Time course of median change in subjects with () mild or () moderate renal impairment, and () healthy all (normal renal function); and (B) (○) individual, () mean and () median values of AUEC_last by renal function group. (‘healthy all’ = all subjects in the two healthy matched subject groups)

Figure 5.

Change from baseline in serum TNF-α by renal function group. (A) Time course of median change in subjects with () mild or () moderate renal impairment and () healthy all (normal renal function); and (B) (○) individual, () mean and () median values of AUEC_last by renal function group. (‘healthy all’ = all subjects in the two healthy matched subject groups)

Serum concentrations of CRP were elevated in all renal function groups at 24 h post-mifamurtide infusion (median TE_max 24 h after initiation of infusion). Median changes from baseline in serum concentrations of CRP over time are shown in Figure 6. Descriptive statistics of pharmacodynamic CRP parameters are shown in Table 4, with individual and median values of E_max graphically depicted by hepatic function group in Figure 6. The baseline-adjusted E_max values for serum CRP were generally similar across renal function groups.

Figure 6.

Change from baseline in serum CRP by renal function group. (A) Time course of median change in subjects with () mild or () moderate renal impairment and () healthy all (normal renal function); and (B) (○) individual, () mean and () median values of E_max by renal function group. (‘healthy all’ = all subjects in the two healthy matched subject groups)

Safety

All enrolled subjects received the protocol specified 4 mg dose of liposomal mifamurtide. All 33 subjects had at least one AE that was considered drug-related. No subjects reported grade ≥3 AEs or SAEs, and there were no on-study deaths. The most common drug-related AEs (≥25% of subjects) were chills (70%), headache (58%), hypotension (48%), vomiting (42%), pyrexia, tachycardia (39%), nausea (36%) and orthostatic hypotension (30%). No notable differences were observed between renal function groups (Table 5).

Table 5.

Most common all-grade, treatment-emergent, drug-related adverse events (≥10% subjects in total)

n (%)	Mild RI (n = 9)	Healthy match, mild RI (n = 8)	Moderate RI (n = 8)	Healthy match, moderate RI (n = 8)	Total (n = 33)
Chills	6 (67)	7 (88)	6 (75)	4 (50)	23 (70)
Headache	2 (22)	6 (75)	6 (75)	5 (63)	19 (58)
Hypotension	5 (56)	2 (25)	3 (38)	6 (75)	16 (48)
Vomiting	3 (33)	5 (63)	3 (38)	3 (38)	14 (42)
Pyrexia	4 (44)	5 (63)	3 (38)	1 (13)	13 (39)
Tachycardia	3 (33)	1 (13)	4 (50)	5 (63)	13 (39)
Nausea	4 (44)	3 (38)	1 (13)	4 (50)	12 (36)
Orthostatic hypotension	1 (11)	4 (50)	3 (38)	2 (25)	10 (30)
Asthenia	1 (11)	2 (25)	1 (13)	2 (25)	6 (18)
Dizziness	4 (44)	2 (25)	0	0	6 (18)
Influenza-like illness	0	0	3 (38)	3 (38)	6 (18)
Diarrhoea	1 (11)	1 (13)	1 (13)	2 (25)	5 (15)
Pain	1 (11)	0	1 (13)	2 (25)	4 (12)
Phlebitis	0	0	2 (25)	2 (25)	4 (12)

RI, renal impairment.

Discussion

This study was designed to evaluate the pharmacokinetics, pharmacodynamics and safety of liposomal mifamurtide in adult subjects with mild (CL_cr 50–80 ml min⁻¹) or moderate (CL_cr 30–50 ml min⁻¹) chronic renal impairment in comparison with age-, weight-, and gender-matched healthy subjects with normal renal function (CL_cr >80 ml min⁻¹). As this study was performed in an adult population, a fixed 4 mg dose of liposomal mifamurtide was administered to all subjects via intravenous infusion over 1 h. This dose was selected to approximate the approved clinical dose of 2 mg m⁻² that is administered to paediatric, adolescent and adult patients with osteosarcoma 3. The use of otherwise healthy subjects with mild or moderate renal impairment and groups of matched healthy subjects with normal renal function in this study was supported by prior clinical experience with liposomal mifamurtide in healthy adult volunteers 6. Importantly, data from studies in paediatric, adolescent and adult patients with osteosarcoma 5,9 and healthy adult volunteers 6 indicate similar pharmacokinetic properties, specifically BSA-normalized clearance of mifamurtide, in osteosarcoma patients and in healthy adults. These data supported the evaluation of the effect of renal insufficiency on the pharmacokinetics of mifamurtide in an adult volunteer population in this study to inform dosing of osteosarcoma patients with mild or moderate renal impairment.

This study characterized the plasma pharmacokinetics of both total (sum of liposome-associated and non-liposome-associated) mifamurtide and free (non-liposome-associated) mifamurtide. Total mifamurtide displayed biphasic disposition kinetics. Free mifamurtide concentrations were substantially lower than total mifamurtide concentrations during the infusion and immediate post-infusion periods, although the differences in concentrations between total and free mifamurtide were less pronounced in the terminal disposition phase, in which both total and free mifamurtide concentrations declined with similar half-lives of approximately 2 h. The mean overall exposure (AUC_inf) of free mifamurtide was approximately one-fifth to one-fourth of that of total mifamurtide. In addition to characterization of plasma pharmacokinetics, the urinary excretion of mifamurtide was evaluated in this study. There was no quantifiable urinary excretion of mifamurtide, indicating that renal clearance is not expected to contribute to the total systemic clearance of mifamurtide.

Results from this study showed that mild or moderate renal impairment did not produce any discernible effects on the pharmacokinetics of total or free mifamurtide, when compared with age-, gender- and weight-matched healthy subjects with normal renal function. The mean concentration–time profiles of total mifamurtide across the renal function groups were super-imposable, and there were no correlations identified between baseline measures of renal function (CL_cr, eGFR, CL_iohexol) and mifamurtide plasma clearance (r² < 0.01) in this study population of subjects with normal renal function and mild or moderate renal impairment. These observations are consistent with the observed lack of quantifiable renal excretion of mifamurtide in this study.

The expected pharmacodynamic effects of mifamurtide were observed in this study, characterized by post-infusion increases in serum concentrations of the pro-inflammatory cytokines IL-6 and TNF-α, and CRP, consistent with the pharmacologic mechanism of action of mifamurtide and with findings in the previous studies 5,6,9. Consistent with previously noted observations 6, the variability in these pharmacodynamic effects and associated parameters (AUEC_last and E_max) was greater than the variability in pharmacokinetic effects. When viewed in the context of observed pharmacodynamic variability, there were no consistent or meaningful effects of mild or moderate renal impairment on the pharmacodynamic effect parameters of mifamurtide, with substantial overlap between the renal function groups in the ranges of the pharmacodynamic effect parameters for serum IL-6, TNF-α and CRP. These observations are consistent with the observed lack of effect of mild or moderate renal impairment on the pharmacokinetics of total or free mifamurtide.

The AE profile observed in this study was generally consistent with the pharmacologic mechanism of action of mifamurtide and previous clinical experience with liposomal mifamurtide in an adult volunteer population 6. The most common drug-related AEs (≥25% incidence) in this study were chills, headache, hypotension, vomiting, pyrexia, tachycardia, nausea and orthostatic hypotension. These effects were consistent with the immunostimulatory pharmacodynamic effects of mifamurtide and the observed increases in serum concentrations of pro-inflammatory cytokines IL-6 and TNF-α. No grade 3 or higher AEs were observed in any of the renal function groups following administration of a 4 mg dose of mifamurtide. The observation of all drug-related AEs being of ≤grade 2 in severity is consistent with the relatively wide therapeutic range of liposomal mifamurtide and the dose level employed in this study (4 mg, approximating the 2 mg m⁻² clinical dose) being a biologically active dose despite being 2–3 times below the maximum tolerated dose of 4–6 mg m⁻² established in phase 1 studies in cancer patients 20,21. No notable differences were seen among treatment groups for these common AEs.

The results of this study collectively support the conclusion that mild or moderate renal insufficiency does not alter the clinical pharmacokinetics or pharmacodynamics of mifamurtide. These findings indicate that no dose modifications appear necessary for patients with mild or moderate renal insufficiency based on clinical pharmacologic considerations. The findings from this study have resulted in an update to the EU Summary of Product Characteristics for MEPACT (liposomal mifamurtide) 3.

Competing Interests

All authors have completed the Unified Competing Interest form at http://www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare KV, YL, DN, JM, MB, and AM are employees of Takeda Pharmaceuticals International Company, CO is an employee of Takeda Global Research and Development Europe and TM and KF have no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years.