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C13009 FINAL CLINICAL STUDY REPORT SYNOPSIS Study Title: A Phase 1 Single Dose Study to Determine the Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of a Lyophilized Formulation (Process C Drug Product) of MLN0002 in Healthy Subjects Investigators: Study Center: Charles River Clinical Services Northwest, Inc. (Tacoma, WA) Publication (reference): None Phase: 1 Initiation Date (first subject enrolled): 24 November 2008 Completion Date (last subject completed): 30 November 2009 Study Objectives: The objectives of this study were: To determine the pharmacokinetics (PK) and pharmacodynamics (PD) of a single intravenous (IV) 300 mg dose of the Process C drug product of MLN0002 To determine the PK and PD of a single IV 600 mg dose of the Process C drug product of MLN0002 relative to the Process B drug product of MLN0002 To assess the safety and tolerability of a single IV dose of the Process C drug product of MLN0002 To evaluate the effect of MLN0002 on cardiac repolarization METHODS Design: The study was comprised of 2 parts: Part 1, which was an open-label study of a single dose of IV 300 mg MLN0002; and Part 2, which was a randomized, placebo-controlled, double-blind, parallel-group study of a single dose of IV 600 mg MLN0002. In Part 1, subjects received IV 300 mg Process C drug product. In Part 2, subjects were randomized in a 1:1:1 ratio to receive IV 600 mg Process B drug product, 600 mg Process C drug product, or placebo. Number of Subjects (planned and analyzed): Up to 100 subjects were planned, such that evaluable data could be obtained from approximately 50 subjects who received MLN0002 and approximately 20 subjects who received placebo. A total of 87 subjects were enrolled: 62 subjects who received MLN0002 and 25 subjects who received placebo. Diagnosis and Main Criteria for Inclusion: Healthy male and female subjects between the ages of 18 and 45 years with body mass index (BMI) between 18 and 32 kg/m 2, inclusive. Test Product, Dose and Mode of Administration, Batch Number: Process B drug product (MLN0002 Solution for Infusion, 5 mg/ml), supplied as a frozen liquid dosage form in 10 ml vials; 600 mg; Batch number: IB004SA01. Process C drug product (MLN0002 Powder for Solution for Infusion, 180 mg), lyophilized solid formulation; 300 mg or 600 mg; Batch number: IC006LA01. Each product was administered by intravenous infusion in 250 ml solution of 0.9% sodium chloride over a 30-minute period, Duration of Treatment: The treatment consisted of a single IV dose of study drug. The total duration on study for each subject was up to 8 months, including screening and postdose observation. 1
Subjects were to be followed by telephone contact at 6-month intervals for 1.5 years after the last study assessment (for a total of 2 years from study drug dose). Reference Therapy, Dose and Mode of Administration, Batch Number: Sodium chloride solution, 0.9%; 250 ml; IV administered over 30 minutes. Batch numbers were not documented. Pharmacokinetic Assessments: PK blood samples were collected on Day 1 (prior to the start of study drug infusion, 5 minutes after the end of infusion, and 1, 2, and 12 hours after the start of infusion), and Days 2 (± 2 days), 8 (± 2 days), 29 (± 2 days), 57 (± 2 days), 85 (± 2 days), 113 (± 2 days), 141 (± 2 days), 169 (± 2 days), and 197 (± 2 days). Only PK samples from subjects randomized to active treatment were planned for analysis. MLN0002 concentrations in serum were measured by a validated direct MLN0002 capture PK assay, and the following MLN0002 PK parameters were estimated: C max (maximum drug concentration); t 1/2 (terminal half-life [if possible]); AUC 0-tlast (area under the drug concentration-time curve from administration time to the last measurement time point at which the concentration is above the lower limit of quantification); AUC 0-inf (area under the drug concentration-time curve, extrapolated to infinity [if possible]) ; CL (total clearance following the dose administered [if possible]); Vss (steady state volume of distribution); V z (volume of distribution based on the terminal phase [if possible]); and MRT (mean residence time [if possible]). Pharmacodynamic Assessments: PD blood samples were collected on Days 1 (prior to the start study drug infusion), 2, 8, 29, 57, 85, 113, 141, 169, and 197. Only PD samples from subjects randomized to active treatment were planned for analysis. PD blood samples were analyzed for the presence of MLN0002 on the surface of cells bearing the 4 7 integrin. Analysis of these samples consisted of 2 validated flow cytometric assays: (1) ACT-1 Binding Interference Assay and (2) MAdCAM-1-Fc Binding Interference Assay. Peripheral blood samples were also examined for the frequency of the MLN0002 target cell populations: CD4 + CD45RO +, and CD8 + CD45RO + cells expressing high levels of integrin 7 (beta 7 bright). Immunophenotyping Assessments: Blood samples for immunophenotyping (IP) were collected on Days 1 (prior to the start study drug infusion), 2, 8, 29, 113, 141, and 197. In order to evaluate the effect of MLN0002 on subsets of peripheral blood cells, an 8-color staining panel for flow cytometry was used to stain peripheral blood (CD3Alexa700, CD4 PacBlue, CD8 Pac Orange, CD25 Qdt605, CD34APC, CD45RO PerCp, CD103 FITC, 7 PE). The panel was designed to examine changes in peripheral cell populations expressing CD103 (E integrin), 7 expressing cells (using an antibody that only marginally was competed by MLN0002) and CD34 + lymphohemopoietic stem and progenitor cells. In addition, the activation marker CD25 (high affinity chain of interleukin 2 receptor) was included to subdivide the populations into active and quiescent. Safety Assessments: Safety evaluations were based on incidence, severity, and type of adverse events (AEs) (including clinically significant changes or abnormalities in the subject s physical examination), progressive multifocal leukoencephalopathy (PML) symptom checklist results, vital signs, electrocardiogram (ECG) results, and laboratory test results (including standard hematology, clinical chemistry, and urinalysis). Blood samples for human antihuman antibody (HAHA) assessment were obtained to evaluate the potential immunogenicity of MLN0002. ECG Assessments: ECG data were collected before (prior to the start of study drug infusion on Day 1 of both parts) and after (on Days 2 and 197 of Part 1, and Days 1, 2, 8, 29, 85, and 197 of Part 2) the administration of MLN0002. Postdose ECGs were time matched to the predose ECG to minimize any influence of diurnal variation on the QT interval. Post dose ECGs were collected during times of highest MLN0002 concentration as well as during washout (through Day 85 post administration) to fully characterize any potential acute or delayed effects of MLN0002 on cardiac repolarization. All ECGs were digitally processed and centrally analyzed in triplicate at each of the pre-specified time points by a blinded ECG laboratory. 2
Statistical Methods: Summary tabulations display the number of observations, mean, median, standard deviation (SD), minimum and maximum for continuous variables, and the number and percent per category for categorical data. Statistical analyses were primarily descriptive in nature. No formal statistical hypothesis testing was performed. The safety population, defined as all subjects receiving any amount of study drug, was used to evaluate the safety and tolerability (including clinical safety and immunogenicity) of MLN0002. The population for PK and PD analyses included all subjects in Part 1 and Part 2 who received the dose of active drug and had sufficient blood sampling to allow for PK and PD evaluation (as determined by the responsible pharmacologist), but excluded subjects with positive HAHA at any time point after study drug administration. There were 2 ECG populations defined for the clinical study report. The primary ECG population was defined as the safety population. The secondary ECG population was defined as the primary ECG population but with the exclusion of subjects who did not have PK profiles that were consistent with IV administration. Comparability of demographic and baseline characteristics was summarized and assessed among the 300-mg Process C MLN0002, 600-mg Process B MLN0002, and 600-mg Process C treatment groups and placebo group. The PK results were listed. Summary statistics by dose group were performed on PK results for each treatment (Process B and Process C). Measured concentrations of MLN0002 in serum were listed for each subject and each sampling time point. In addition, arithmetic means, arithmetic SD, coefficient of variation (CV) and number of data points (N) were given per sampling point and treatment group. For PK parameters, the geometric mean and the arithmetic mean together with their SD and CV were tabulated as well as the median, minimum, and maximum. In order to determine a single IV 600-mg dose of the Process C drug product of MLN0002 relative to the Process B drug product of MLN0002, the relative bioavailability of MLN0002 was calculated as the geometric mean ratio of AUC 0-inf (600-mg Process B versus 600-mg Process C). The associated 90% confidence intervals (CIs) of this ratio were also calculated. Measured PD markers and evaluated PD parameters were listed. Summary statistics were performed on PD markers and parameters for each treatment. In addition, arithmetic means, arithmetic SD, CV and number of evaluated subjects (N) were given per sampling point and treatment. For PD parameters, the geometric mean and the arithmetic mean together with their SD and CV were tabulated as well as the median, minimum, and maximum. IP data were listed and summary statistics by time point were performed by treatment group. Safety data were presented by Part 1 and by Part 2, as well as both parts combined. Descriptive statistics were calculated. The largest time-matched mean change from baseline in QT interval corrected for heart rate using Fridericia's formula (QTcF) was presented by treatment group along with 90% CIs for the differences. The analysis was based on an analysis of covariance (ANCOVA) model. ANCOVA model of change from baseline to largest time-matched difference included treatment group and baseline measurement. Baseline QTcF was used as a covariate in the model in order to adjust for possible treatment difference at baseline. HAHA assays were carried out on all subjects, and the frequency and titers of HAHA and neutralizing HAHA were summarized over time by treatment and by dose group. RESULTS Subject Disposition: A total of 87 subjects were enrolled in the study. In Part 1 (unblinded), 13 subjects were assigned to a single IV dose of 300-mg Process C MLN0002. In Part 2 (blinded, randomized), 23 subjects were randomized to a single IV dose of 600-mg Process B MLN0002, 3
26 subjects were randomized to a single IV dose of 600-mg Process C MLN0002, and 25 subjects were randomized to a single IV dose of placebo. All 87 subjects were included in the safety population, and 56 subjects (10, 22, and 24 subjects in the 300-mg Process C MLN0002, 600-mg Process B MLN0002, and 600-mg Process C treatment groups MLN0002, respectively) were included in the PK and PD analysis populations. Seventy-three subjects were included in the primary ECG population and 71 subjects were included in the secondary ECG population. A total of 66 (76%) subjects completed the study; 8 (9%) subjects withdrew their consent and 13 (15%) subjects were lost to follow-up. Demographic Results: The study enrolled 87 healthy subjects (47 male and 40 female) with a median age of 29.0 years (range: 19-45 years). Overall, the majority of subjects were white (64 subjects, 74%). Mean BMI and body weight were 25.4 kg/m 2 (range: 18.7-32.5 kg/m 2 ) and 74.97 kg (range: 75.0-113.2 kg), respectively. Demographic characteristics were similar among the 300-mg Process C MLN0002, 600-mg Process B MLN0002, and 600-mg Process C treatment groups. Pharmacokinetic Results: Following the 30-minute, IV infusion administration of MLN0002, after reaching peak concentration, serum concentration of MLN0002 fell in a biphasic manner beginning with a rapid distribution phase followed by a slower elimination phase. The mean concentration time profiles for 600-mg Process B and Process C MLN0002 are superimposable. As expected for IV infusion administration, C max was achieved in most subjects by the first time point, which was immediately following the end of infusion. C max was dose proportional across the doses tested in the study. Subjects receiving 300-mg Process C had a geometric mean C max of 115 g/ml. Subjects receiving 600-mg Process C MLN0002 (2-fold dose increase) had an approximately 2-fold higher C max (206 µg/ml) relative to the 300-mg treatment group. C max was similar between subjects receiving 600-mg Process B and Process C MLN0002 (205 µg/ml and 206 µg/ml, respectively). AUC 0-inf was dose proportional across the doses tested in the study. Subjects receiving 300-mg Process C MLN0002 had a geometric mean AUC 0-inf of 2000 d*µg/ml. Subjects receiving 600-mg Process C MLN0002 had an approximately 2-fold higher geometric mean AUC 0-inf (3890 d*µg/ml) relative to the 300-mg Process C MLN0002 treatment group. AUC 0-inf was similar between subjects receiving 600-mg Process B and Process C MLN0002 (4040 µg/ml and 3890 µg/ml, respectively). The calculated treatment mean ratios and associated 90% CIs for both the C max and AUC 0-inf parameters for the 600-mg Process B compared to 600-mg Process C treatment groups demonstrated that the 2 process groups were very similar. Furthermore, based on the Food and Drug Administration bioequivalence guidance criteria, these 2 process groups can be considered to be bioequivalent. Similar conclusions can be made when comparing the treatment groups based on AUC 0-tlast. The mean CL was similar across the treatment groups (151, 157, and 149 ml/day for the 300-mg Process C MLN0002, 600-mg Process C and Process B MLN0002 treatment groups, respectively). The mean t 1/2 was similar across the treatment groups (18.3, 21.0, and 19.4 days for the 300-mg Process C MLN0002, 600- mg Process C and Process B MLN0002 treatment groups, respectively). The mean Vss was similar across the treatment groups (4.53, 5.04, and 4.73 L for the 300-mg Process C MLN0002, 600-mg Process C and Process B MLN0002 treatment groups, respectively). Similarly, the mean Vz was 3.93, 4.74, and 4.12 L for the 300-mg Process C MLN0002, 600-mg Process C and Process B MLN0002 treatment groups, respectively. These estimates are consistent with the expected distribution volume of large protein therapeutics. 4
HAHA Results: Overall, 5 (8%) subjects were positive for HAHA, all of whom were receiving active treatment. For most of these subjects, HAHA were persistent (4 subjects) rather than transient (1 subject). Of the time points where a measurement was made, HAHA were most frequently observed on Day 29 and Day 197 (2 subjects each). A total of 4 (6%) subjects had neutralizing HAHA. Pharmacodynamic Results: Following the 30-minute IV infusion administration of MLN0002, near maximal inhibition of ACT-1 and MAdCAM-1-Fc was achieved by the time of the first PD sampling (24 hours after the end of the infusion). Near maximal inhibition of ACT-1 was achieved in all treatment groups. Based on graphical evaluation, the loss of near maximal inhibition (> 10% of baseline) of ACT-1 began by Day 144 in the 300-mg Process C MLN0002 treatment group, and by approximately Day 169 in the 600-mg Process C and Process B MLN0002 treatment groups. The time to loss of near maximal inhibition was similar in both of the 600-mg treatment groups. Near maximal inhibition of MAdCAM-1-Fc was achieved with all treatment groups. Based on graphical evaluation, the loss of near maximal inhibition (> 10% of baseline) occurred at Day 144 in the 300-mg Process C MLN0002 treatment group. In the 600-mg Process C and Process B MLN0002 treatment groups, loss of near maximal inhibition (> 10% of baseline) occurred by Day 169. The maximum effect (E max ) of MLN0002 as measured by the ACT-1 markers was approximately 99% for all treatment groups and approximately 98% based on MAdCAM-1-Fc for all treatment groups. No dose- or concentration-response relationship in regard to E max was detectable as all concentrations of MLN0002 following the 300- and the 600-mg dose maximally inhibited both ACT-1 and MAdCAM-1Fc. Following IV administration of MLN0002, the AUEC (0-tlast) for ACT-1 increased slightly with increasing dose from approximately 13100 %*day for the 300-mg treatment group to approximately 16100 %*day and approximately 13100 %*day in the 600-mg Process B and Process C MLN0002 treatment groups, respectively. Similar conclusions were obtained for the AUEC data for MAdCAM-1. Generally, serum concentrations of MLN0002 obtained after the infusion of 300-mg Process C MLN0002 or 600-mg Process B or C MLN0002 led to maximal PD effects as measured with ACT-1. There were only a limited number of observed concentrations that appeared to produce less than complete saturation of 4 7 receptors. Peak increases from mean baseline values were observed at Day 8 in only a small population ( 2% of total CD3 + cells) of CD4 +, CD45RO + bright, CD25 +, β 7 + bright cells, and to a lesser extent CD8 +, CD45RO + bright, CD25 +, β 7 + bright cells, in individuals treated with MLN0002 and not placebo. This small population of lymphocytes represents cells anticipated to be bound by MLN0002 and reflects the study drug's mechanism of action. The changes observed in the T cell subpopulations represent a small fraction of the total white blood cell compartment (< 1%) and did not affect the overall lymphocyte or leukocyte populations in that there were no increases in counts of circulating lymphocytes or leukocytes. No changes were observed in total or scatter gated CD34 + cells (hematopoietic progenitor cells), total CD3 + cells, CD103 + (E) cells, overall CD4 + cells, overall CD8 + cells, β 7 memory cells, or CD25 - T cells compared to baseline values. Consistent with previous research, there were no apparent changes from baseline in either the presumed B cells (CD3 - CD56 - lymphocyte gated) or NK cell (CD8 - CD56 + lymphocyte gated) populations. These results support the MLN0002 mechanism of action of being gut selective without overall systemic effects such as increasing circulating leukocytes or mobilization of bone marrow progenitor cells to the circulation. 5
Safety Results: Overall, 63 (72%) subjects experienced at least 1 treatment-emergent AE, with 36 (41%) subjects experiencing an AE that was considered to be drug-related. Forty-three (69%) of the 62 subjects receiving active treatment (300-mg Process C or 600-mg Process B or C MLN0002) versus 20 (80%) of the 25 subjects receiving placebo experienced a treatment-emergent AE; 24 (39%) of the 62 subjects receiving active treatment and 12 (48%) of the 25 subjects receiving placebo experienced a drug-related AE. The most common AEs were: headache experienced by 17 (27%) of the 62 subjects receiving active treatment and 6 (24%) of the 25 subjects receiving placebo, and upper respiratory tract infection experienced by 14 (23%) of the 62 subjects receiving active treatment and 1 (4%) of the 25 subjects receiving placebo. The most common drug-related AE was upper respiratory tract infection experienced by 7 (8%) subjects, all of whom were receiving active treatment (11% of that population). Two (4%) subjects in the combined 600-mg Process B and Process C MLN0002 treatment group experienced infusion site reactions; both were unrelated to study drug and resolved. There were no infusion site reactions reported for the 300-mg Process C MLN0002 treatment group. The only reported cardiac event in this study was atrial fibrillation, reported by 1 subject in the placebo treatment group, which resolved. Most AEs were mild or moderate in severity. Five (6%) subjects experienced a severe AE (ie, gastroesophageal reflux, syncope, cellulitis, headache, and comminuted fracture); 1 was reported as drug-related (cellulitis) and all but 1 resolved. There were no on-study deaths, SAEs, or AEs that resulted in discontinuation. Overall, no trends were noted for mean changes in any laboratory parameters in the MLN0002 versus placebo treatment groups across all time points. Evaluation of individual subject laboratory parameters showed that there were no clinically significant MLN0002-attributable increases or decreases in white blood cells, lymphocyte count, or neutrophil count across study time points. There was no association between clinically significant liver function test abnormalities and MLN0002 treatment; elevations in aspartate transaminase and/or alanine transaminase, all <5x upper limit of normal, were observed for 7 subjects in both the placebo and MLN0002 treatment groups, but these were generally at isolated time points. No subject had elevations above the normal range in total bilirubin on study, and creatinine levels were all within normal limits. Additionally, there were no lab abnormalities reported as AEs. There were no marked mean changes from baseline in blood pressure, heart rate, temperature, and respiratory rate in subjects in the MLN0002 treatment groups. For the primary ECG population, the largest time matched mean baseline adjusted difference of the MLN0002 and placebo was observed at Day 8. The mean change from baseline was -4.5 for placebo and 0.6 for MLN0002. The 1-sided 95% (or 2-sided 90%) upper confidence bound for the largest mean change adjusted for placebo was 9.3 observed on Day 8. The upper bound of 95% 1-sided confidence interval for the largest time-matched mean effect of MLN0002 on the QTc interval excluded 10 msec, which is the threshold for pharmacologic effect on cardiac repolarization, as detected by QT/QTc prolongation. Overall, no effect of MLN0002 on QTc was observed. The mean adjusted change from baseline at Day 1 post was -0.8 for placebo and 0.6 for MLN0002. The 1-sided 95% upper confidence bound of the adjusted mean change was 5.0 observed at Day 1 post. Overall, there were no marked mean changes in ECG parameters. Two (8%) subjects in the placebo treatment group and 10 (20%) subjects in the MLN0002 treatment group had maximum post baseline QTcF values of between 430 to 449 msec, and 7 (14%) subjects in the MLN0002 treatment group 6
had maximum post baseline QTcB values between 430 to 449 msec. Four (17%) subjects in the placebo treatment group and 4 (9%) subjects in the MLN0002 treatment group had a 30 msec change in QTcB, and 2 (4%) subjects in the MLN0002 treatment group had a 30 msec change in QTcF. No subjects had QTc > 450 msec or had 60 msec change in QTc from baseline. One (5%) subject in the placebo treatment group had abnormal ECG parameters on Day 85 that were considered clinically significant. Though some subjects in the MLN0002 treatment groups had abnormal ECG parameters during the study, none were considered clinically significant. For the secondary ECG population, the largest mean baseline adjusted difference of MLN0002 and placebo was observed at Day 8. The 1-sided 95% (or 2-sided 90%) upper confidence bound of 8.1 was observed for the secondary ECG population. ECG results for the secondary ECG population were similar to those of the primary ECG population. One subject in the placebo treatment group had a positive subjective finding on the PML checklist, while there were no abnormal findings on the objective checklist and no algorithm findings reported. Date of Report: 17 September 2010 7