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PHARMACOKINETICS AND PHARMACODYNAMICS |
From Eli Lilly and Company, Indianapolis, Indiana.
Address for correspondence: Pamela A. Welch, MD, PhD, Eli Lilly and Company, Lilly Corporate Center DC 0734, Indianapolis, IN 46285; e-mail: pamela_welch{at}lilly.com.
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Key Words: enzastaurin • protein kinase C • phase I • pharmacokinetics
Preliminary data suggested that enzastaurin is primarily metabolized by cytochrome P450 3A (CYP3A), leading to the formation of multiple metabolites, including the 2 active metabolites: a desmethylenepyrimidyl metabolite (LY326020) and a desmethyl metabolite (LY485912). These metabolites are pharmacologically active, inhibiting PKC-ß with similar potencies to enzastaurin (IC50 approximately 6 nM) (data on file).2
This article describes the first human dose study of enzastaurin, as well as a subsequent multiple-dose study, conducted in healthy subjects. The healthy subject population provided an assessment of safety parameters and adverse events, without the confounding factors of underlying disease state and background medications, as well as potentially less within-subject and between-subject variability in pharmacokinetics. In the first study, healthy subjects received a placebo dose followed by up to 3 single doses of enzastaurin. This was followed by a second study, in which healthy subjects received placebo for 7 days, followed by multiple doses of enzastaurin for 15 days. The primary objectives in both studies were to evaluate the safety and tolerability of enzastaurin and to characterize the pharmacokinetics of enzastaurin and its metabolites. Secondary objectives included evaluation of pharmacokinetic dose linearity and an assessment of the QT interval corrected (QTc) following enzastaurin administration.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Eligibility Criteria
Men or women of nonchildbearing potential, deemed healthy by medical history and physical examination, were eligible to participate. Subjects were required to be 18 to 60 years of age, with a body mass index (BMI) of 19 to 31 kg/m2, in the single-dose study and 18 to 65 years of age, with a BMI of 19 to 34 kg/m2, in the multiple-dose study. All subjects were required to give written informed consent and to abstain from alcohol during the studies.
Subjects with a history or evidence of active disease that, in the investigator's opinion, increased their risk of participation in the study were excluded. Cardiovascular exclusion criteria included baseline abnormalities such as arrhythmias, conduction abnormalities, ischemic changes, or baseline QT interval corrected using Bazett's method (QTcB) of >430 msec in men or >450 msec in women. Electrolyte disorders, including hypokalemia, hypocalcemia, or hypomagnesemia, were specifically excluded. Hematologic exclusion criteria included white blood cells <3000/mm3, platelets <100 000/mm3, or hemoglobin <10.0 g/dL. Evidence of active renal disease, calculated (Cockcroft-Gault) creatinine clearance <70 mL/min, or urinalysis with 1 or more protein or blood was not allowed. Other exclusions were the use of concomitant prescription or over-the-counter medications (except vitamin or mineral supplements), drug abuse as determined by urinary drug screening, or participation in a study involving another investigational agent in the previous 30 days. Positive serologic tests for HIV, hepatitis B, hepatitis C, or syphilis (Venereal Disease Research Laboratory [VDRL]) were also exclusionary.
Study Design
The first human dose study was a single-blind, placebo-controlled, within-subject, dose escalation design to examine the safety and tolerability of orally administered, single-dose enzastaurin. The placebo lead-in was designed to collect baseline safety data and to familiarize the subjects with study procedures that were used during the remainder of the study. Healthy subjects received 1 placebo dose, followed by up to 3 single doses of enzastaurin, with each dose being separated by a washout period of 1 week. For logistical purposes only, the study was conducted in 2 groups: group 1 (dose escalation ranging from 2-100 mg) and group 2 (ranging from 100-400 mg). Pharmacokinetic findings in the initial subjects (group 1) showed that the half-life for the enzastaurin metabolite LY326020 was longer than predicted from the preclinical studies; therefore, the washout period between doses was extended to 2 weeks for the remaining subjects (group 2). Comprehensive safety assessments, including safety laboratory studies, vital signs, and serial electrocardiograms, were performed during each dosing period and assessed prior to dose escalation. Dosing in several cohorts occurred in parallel but was temporally staggered, such that dose escalation to the next higher dose level did not occur until safety monitoring had been completed for at least 3 subjects at the previous dose level (2 subjects at the 2-mg dose level). All electrocardiograms (ECGs) through 72 hours postdose for at least 3 subjects were assessed by an outside independent reviewer prior to dose escalation. Continuous ECG telemetry was provided solely for real-time monitoring of any acute events. Telemetry readings were also recorded on a Holter monitor, although Holter monitor readings were not to be formally read unless there was a clinical indication for further evaluation. Each subject was admitted to the clinical research unit on the evening before placebo or enzastaurin dosing and was discharged 3 days after receiving the dose.
Table I details the dose levels for the single-dose study. Doses started at 2 mg (
0.03 mg/kg), with a diminishing incremental sequence up to a planned maximum dose of 400 mg (6 mg/kg). The 400-mg dose did not exceed the human equivalent (11 mg/kg/day) of the no-adverse-effect dose (75 mg/kg/day) for the appearance of reproductive tissue findings in the male rat study, nor did it exceed the human equivalent (7 mg/kg) of the no-adverse-effect dose (15 mg/kg/day) in the 1-month dog study (data on file). The protocol provided for termination of dose escalation and exploration of lower dose levels if dose-limiting safety concerns were observed.
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The second study was designed as a single-blind, placebo-controlled, between-subject, dose escalation study. A single-blind, placebo lead-in was used to collect baseline laboratory and ECG measurements and to familiarize the subjects with study procedures that were used during the rest of the study. Healthy subjects received placebo doses for 7 days, followed by a minimum of 15 daily enzastaurin doses, with no within-subject dose escalation. The duration of dosing was based on the time needed to achieve steady-state exposures for both enzastaurin and its metabolites, given that the aforementioned single-dose study had shown that the metabolite LY326020 had a longer half-life than enzastaurin. Comprehensive safety assessments, including safety laboratory studies, vital signs, and serial ECGs, were performed at each step in the dose escalation, but telemetry was not required. Laboratory and ECG measurements were collected at the same time points on day 1 of placebo and on days 1 and 15 of enzastaurin. Dose escalation from the 25-mg to 100-mg dose level was allowed after evaluations of safety labs and ECGs (through 5 days post-first dose) were completed for at least 3 subjects. Further dose escalations occurred after evaluation of safety labs (through 72 hours) after final dose and ECGs (through 24 hours) obtained after the final dose for at least 4 subjects in the previous group. After receiving the last dose, subjects were discharged after a 2-week washout period.
Daily doses of 25, 100, 200, and 400 mg were chosen to explore the safety and pharmacokinetics of multiple-dose administration across potential therapeutic doses, as well as to potentially evaluate biomarkers for future clinical trials. Given the expected accumulation of enzastaurin and its metabolite, the exposures (area under the curve [AUC] and maximum plasma concentration [Cmax]) following multiple doses of 200 mg/day were predicted to be similar to those observed following the single dose of 400 mg in the first study. If the 200-mg dose was achieved without significant toxicities, escalation was to proceed to 400 mg/day.
As these were exploratory phase I studies, the sample sizes were based on the study objectives and not on any statistical criteria. In the single-dose study, at least 3 subjects were required to complete each dosing interval, with the exception that only 2 subjects were required for the 2-mg dose. In the multiple-dose study, at least 5 subjects were required to complete each dose level.
Dose Administration
Each enzastaurin or placebo dose was administered orally and taken with water. Enzastaurin was supplied as 1-, 5-, 25-, and 100-mg capsules. A combination of 4 capsules, with active drug or placebo, was given for each dose to maintain the single blind.
Because preliminary data suggested that enzastaurin had low solubility, it was anticipated that food would enhance absorption. Therefore, enzastaurin was administered with food to generate safety data at the highest possible exposure for a given dose. Given that enzastaurin is being developed for cancer patients, who may have reduced appetites and food intake, a standardized breakfast was developed for these studies that contained fewer calories (approximately 500 calories) and less fat (approximately 19 grams) than the typical high-fat meal prescribed in food effect studies. Subjects in the single-dose study were asked to complete this standardized breakfast within approximately 30 minutes and to take their study drug within approximately 5 minutes of completing the meal. Subjects in the multiple-dose study were asked to take their study drug (including placebo) within approximately 30 minutes to 1 hour after completing their breakfast, with the standardized breakfast provided on the day of each inpatient dose (first placebo dose, first enzastaurin dose, and last enzastaurin dose) given in the clinical research unit. Grapefruit or grapefruit juice was not allowed during the studies.
Safety Assessments
Safety assessments included vital signs, physical exams, serial ECGs, telemetry (single-dose study only), clinical laboratory tests (hematology, clinical chemistry, and urinalysis), and adverse events monitoring. Adverse events in both studies were coded using the Medical Dictionary for Regulatory Activities (MedDRA).
Vital signs (heart rate, blood pressure, respiratory rate, and temperature) were obtained before dosing, at regular intervals after dosing (up to 48 hours), and during outpatient visits. Physical examinations were performed at screening, as clinically indicated, and before discharge.
In the single-dose study, a time course of ECGs was obtained at each dose level. These time points included predose, as well as the estimated times of the predicted Cmax of enzastaurin and its metabolite, based on the preclinical data. Because (with acute drug administration) there can be a lag between the time of maximum plasma drug concentration and the time of maximum drug effect, ECGs were also assessed at additional time points after the time of predicted Cmax of enzastaurin and its metabolite. Electrocardiograms were therefore obtained at predose and at 2, 6, 9, 24, 48, and 72 hours following each dose. Electrocardiogram time points were coordinated with pharmacokinetic sampling times so that the plasma concentration of enzastaurin and LY326020 was measured at the approximate time of each ECG. A comparable time course of ECGs was obtained during the placebo period. For consistency, given the potential effect of meals on QTc measurements, predose ECGs were obtained before breakfast in both studies.
Electrocardiograms were monitored during the study for any clinically significant changes, including prolongation of the QTcB interval >50 msec, QTcB >450 msec in men or >470 msec in women, or QRS duration >120 msec. An independent cardiologist evaluated the ECGs for 3 subjects before dose escalation occurred. Telemetry was monitored during the placebo and enzastaurin dosing periods, beginning before each dose and continuing for 48 hours after each dose. Holter readings were formally read if clinically indicated.
In the multiple-dose study, the time course of ECGs was based on the predicted times of the Cmax of enzastaurin and its metabolite, as determined from preliminary analyses of the pharmacokinetic data from the single-dose study. Electrocardiograms were obtained at 0 hours (predose) and at 4, 8, 12, and 24 hours on day 1 of placebo dosing and on day 1 (first dose) and day 15 (predicted steady state) of enzastaurin administration. An ECG was also obtained predose on the sixth day of enzastaurin dosing and at discharge. An independent cardiologist evaluated the ECGs before dose escalation occurred.
In both studies, safety laboratory tests were assessed during the placebo and enzastaurin dosing periods. Safety labs were collected predose; at 1, 2, and 5 days after each dose; and at discharge in the single-dose study. During the multiple-dose study, safety labs were collected predose on days 1 and 2 of the placebo lead-in period; predose on days 1, 2, 6, and 15 of enzastaurin dosing; 3 days after the final enzastaurin dose; and at discharge.
Although these studies were conducted in healthy subjects, in light of the intended oncology target population, dose-limiting safety concerns were evaluated within the context of the National Cancer Institute's Common Toxicity Criteria (CTC) Version 2 (Revised 23 March 1998). Given the healthy subject population, the lowest grade of toxicity served as an objective reference point for the evaluation of laboratory results and/or clinical signs and symptoms. For example, if a subject developed persistent laboratory abnormalities or clinical signs or symptoms that fell within grade 1 toxicity after administration of enzastaurin or developed clinical evidence of grade 2 toxicity, the subject was to be evaluated for discontinuation from the study. The final determination to discontinue a subject from the study was based on the grade 1/2 toxicity criteria, with consideration of the presence of probable cause other than enzastaurin and the clinical judgment of the investigator.
QTc Interval Evaluation
QTcB intervals were used to define eligibility criteria and for safety monitoring during the study. In the single-dose study, clinically significant changes after the enzastaurin dose were defined as prolongation of the QTcB interval by more than 50 msec, absolute QTcB interval >450 msec in men or >470 msec in women, or QRS duration >120 msec. Clinically significant changes in QTc postdosing were defined in the multiple-dose study protocol as an absolute QTc interval >450 msec in men or >470 msec in women or prolongation of the QTc interval by more than 40 to 59 msec.
Despite the use of the Bazett's correction in clinical studies, in general, it overcorrects at elevated heart rates and undercorrects at heart rates below 60 beats per minute.12 Fridericia's correction (QTcF) may be more accurate than QTcB in subjects with such altered heart rates; therefore, it is more appropriate to use QTcF in any statistical analysis. The International Conference on Harmonization (ICH) guidance E14 recommends that thorough QT studies include QT/QTc interval analyses with an analysis of central tendency (eg, means and medians) and categorical analyses.12 The categorical analysis groups those subjects whose absolute QTcF intervals exceed 450, 480, and 500 msec and also groups those subjects whose change from baseline exceeds 30 and 60 msec.
The single- and multiple-dose studies were not designed as thorough QT studies and were carried out before this ICH E14 guidance was published. For the categorical analysis, intervals for both the protocol predefined criteria (QTcB) and subsequently the criteria suggested in ICH E14 (QTcF) were evaluated.
QTcF and QTcB intervals for each subject were examined with respect to maximum absolute intervals and time-matched maximum changes from placebo at each collection point. In both studies, time-matched differences were formed by subtracting the reading taken at the same nominal time during the placebo dosing period from the reading taken on either day 1 of each dosing period in the single-dose study, or on days 1 (first dose) and 15 (predicted steady state) in the multiple-dose study.
A mixed effects model was fitted to QTcF data with subject as a random effect and time within study day as a repeated measure for each study. Least squares means of time-matched changes from baseline were estimated together with their 90% confidence limits.
Pharmacokinetic Evaluations
Blood samples were collected in heparin-containing tubes and centrifuged, and plasma was stored at -70°C. In the single-dose study, plasma samples were collected at various times up to 120 hours after dose administration. In the multiple-dose study, plasma samples were collected at various times up to 24 hours (predose on day 2) following the first dose. Predose blood samples were also collected on days 5, 9, 14, and 15. Blood sampling following the last dose was conducted at scheduled time points until 312 hours following the dose.
Plasma samples were analyzed for concentrations of enzastaurin and LY326020 in the single-dose study and for concentrations of enzastaurin, LY326020, and LY485912 in the multiple-dose study, using validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods capable of quantitating parent and 1 or more metabolites in a single specimen. The plasma samples were analyzed by Advion BioSciences, Inc (Ithaca, New York).
Stable labeled analogs (13C2H3) of enzastaurin and LY326020 were included as internal standards. Transition ions (m/z) monitored for quantitation were 516.3 to 175.1, 425.2 to 84.1, and 502.3 to 175.1 for enzastaurin, LY326020, and LY485912, respectively. The limits of quantitation for enzastaurin, LY326020, and LY485912 assay were 0.5 to 150 ng/mL. Concentrations exceeding the upper limit of quantitation were diluted to within the 0.5- to 150-ng/mL range. The intra-assay and interassay precision data (relative standard deviation [RSD]) were 
6.68% for enzastaurin, 8.17% for LY326020, and 6.24% for LY485912. The interassay accuracy ranged from -7.10% to 4.21% for enzastaurin, from -11.07% to 2.77% for LY326020, and from -8.13% to 1.45% for LY485912. Analyte concentrations were provided in molar units for the pharmacokinetics analysis.
Noncompartmental pharmacokinetic analyses were performed for enzastaurin and the metabolite(s) using WinNonlin Pro 3.1 (Pharsight Corporation, Mountain View, California). An accumulation ratio after multiple oral dosing was calculated based on the observed data. The observed accumulation ratio (Robs) was assessed as Robs = AUC
,ss/AUC(0-24), where AUC(0-24) corresponds to the area after the first dose. A metabolic ratio at steady state (MRss) was calculated as metabolite area under the concentration curve at steady state (AUC,ss) to parent AUC,ss.
Dose proportionality was assessed for enzastaurin and metabolites, across all doses, for the single-dose study and at steady state for the multiple-dose study. A power model was fitted to both Cmax and AUC (AUC0-
for single-dose study and AUC,ss for multiple-dose study) of each analyte against the enzastaurin dose to estimate the dose proportionality ratio ("high" dose/"low" dose). This dose proportionality ratio is defined as the upper limit that ensures that the 90% confidence interval (CI) of the ratio of the dose-normalized least squares geometric means falls within the limits (0.8-1.25).13 Using this method, dose proportionality can be inferred for any dose ratio up to the dose proportionality ratio, even if dose proportionality cannot be inferred across the entire range of doses tested in the study.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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In the single-dose study, 19 of the 27 subjects received enzastaurin and completed the study. Four subjects withdrew from the study due to the subject's decision. Three of these subjects withdrew prior to any dosing; 1 subject withdrew after receiving 1 dose of placebo. Another 4 subjects were discontinued due to investigator decision. Three of these subjects were discontinued after receiving a single dose of enzastaurin. One subject developed a pericardial rub 7 days after receiving a single dose (100 mg) of enzastaurin. The normal chest x-ray and ECG, the absence of clinically significant findings on 2-D echo, and serologies consistent with recent infection with influenza type B were consistent with pericarditis (most likely viral). Findings resolved prior to discharge from the study. A second subject had mild elevations of uric acid during screening, placebo period, and following a single dose (150 mg) of enzastaurin. Although these elevations were not clinically significant or significantly different from the subject's baseline, in the strictest sense, they met the protocol's discontinuation criteria. A third subject who had received a single dose (200 mg) of enzastaurin was discontinued for a positive drug screen prior to the next dosing period. None of these discontinuations appeared to be related to enzastaurin. One subject developed intermittent hematuria/hemoglobinuria, without any clinical symptoms or other associated laboratory abnormalities, after receiving 2 doses of placebo (cohort E). All 27 subjects in the single-dose study were included in the safety analysis, and the 22 subjects who received at least 1 dose of enzastaurin were included in the pharmacokinetic analysis.
In the multiple-dose study, 21 of the 25 subjects completed the study. Three subjects withdrew from the study due to physician decision, and 1 subject withdrew due to sponsor decision. Three subjects were discontinued after receiving enzastaurin. One subject had an increase in liver function tests (aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase), associated with a normal bilirubin, which began prior to starting dosing with enzastaurin. These persisted, and enzastaurin dosing was stopped after 3 (100-mg) doses. Weight gain (6.5 pounds) was thought to be the most likely etiology of the changes in liver function. Serology results for hepatitis (A, B, and C), cytomegalovirus, and Epstein-Barr virus were unremarkable. Follow-up revealed decreased weight and normal liver function tests, consistent with original liver function changes having been secondary to weight gain. A second subject, who had received 8 (25-mg) doses of enzastaurin, reported twisting his thigh muscles after slipping and falling on a wet floor at a local store and was discontinued after appropriate follow-up. A third subject developed an upper respiratory infection requiring an antibiotic during the placebo lead-in period, received only 1 dose (400 mg) of enzastaurin, and had normal safety labs and ECGs on follow-up. None of these discontinuations appeared to be related to enzastaurin. A fourth subject had elevated liver function tests (alanine aminotransferase) immediately prior to placebo dose; alanine aminotransferase and aspartate amino-transferase continued to rise, but bilirubin and alkaline phosphatase remained normal. Placebo was stopped after 3 doses, and follow-up showed stable to improving liver function tests. All 25 subjects in the multiple-dose study were included in the safety analysis, and 24 subjects were included in the pharmacokinetic analysis.
Safety Assessments
In the single-dose study, no serious adverse events or deaths were reported. Adverse events possibly related to enzastaurin were observed in 15 of the 22 subjects who received at least 1 dose of enzastaurin. Sleepiness (10 subjects) and headache (6 subjects) were the most common adverse events. Nausea was reported by 4 subjects, and diarrhea or abdominal pain was reported by 3 subjects each. Other effects noted by 2 subjects each were vomiting, flatulence, epistaxis (minor), and rhinitis. No dose-limiting toxicities were observed, and the planned maximum dose (400 mg) was safely administered. No clinically significant alterations in hematology, serum chemistry, or urinalyses were noted.
Among the 25 subjects in the multiple-dose study, 62 adverse events were observed in 17 subjects; of these events, 27 were deemed possibly related to enzastaurin by the investigator. Headache (7 subjects) was the most common adverse event possibly related to enzastaurin, followed by dizziness (3 subjects) and fatigue (2 subjects). Abdominal pain and diarrhea were reported by only 1 subject each in this study. No clinically significant alterations in hematology, serum chemistry, or urinalyses results were noted. Inspection of vital sign tabulations revealed no clinically significant or dose-dependent changes in the vital signs; formal statistical analyses were not planned or performed for these parameters.
Chest pain, occurring in 1 individual in each study, was apparently not related to the heart and resolved spontaneously. For the subject in the single-dose study, no clinically significant changes were noted on telemetry, and serial cardiac enzymes and serial ECGs were normal. Symptoms did not recur when the subject received higher doses (10 mg, 25 mg) of enzastaurin during subsequent dosing periods. In the multiple-dose study, the transient episode of right-sided chest pain occurred approximately 72 hours after the last dose of enzastaurin. Because of the location of the chest pain, the worsening with inspiration, and the lack of associated symptoms (eg, shortness of breath or palpitations), the investigator did not think that the pain was cardiac in origin.
All subjects in the single-dose study were placed on continuous ECG telemetry to provide real-time safety monitoring immediately before (approximately 30 minutes) and for approximately 48 hours after dosing. Telemetry readings were also recorded on a Holter monitor, but Holter recordings were not formally read unless there was a clinical indication for further evaluation. Holter readings (all dosing periods) for 6 subjects were reviewed by the cardiologist, with no clinically significant changes noted.
QTc Interval Evaluations
No clinically significant changes in absolute QTcB intervals or changes in QTcB from baseline were apparent, based on the protocol-defined criteria in either study. There were also no clinically significant prolongations (>120 msec) of the QRS interval, with maximal intervals of 100 msec and 112 msec observed in the single- and multiple-dose studies, respectively.
In the single-dose study, no subject experienced QTcF >450 msec. Only 1 male experienced a QTcF change from baseline >30 msec (32.1 msec) in the single-dose study, and this was before his first enzastaurin dose (25 mg). The results from the mixed effects analysis showed no evidence of a dose effect on QTcF intervals.
In the multiple-dose study, only 1 subject (female) experienced a QTcF interval >450 msec, at predose (QTcF = 451 msec) prior to the sixth dose of enzastaurin. Overall, 5 subjects accounted for the 8 occurrences of QTcF changes from baseline >30 msec: in the 25-mg group, 1 male subject at 24 hours (36.0 msec) after the final dose of enzastaurin; in the 100-mg group, a female subject at 8 hours (30.1 msec) after the first dose of enzastaurin; and a male subject at predose (44.2 msec), prior to the sixth dose of enzastaurin. In the 200-mg group, 1 subject (female) had 3 occurrences of QTcF >30 msec following the administration of enzastaurin: at 8 hours (33.3 msec) and 24 hours (35.3 msec), after the first dose of enzastaurin, and predose (40.6 msec), prior to the sixth dose of enzastaurin. It should be noted that this subject also had a 39.4-msec increase in QTcF prior to the first dose of enzastaurin compared to the QTcF prior to the placebo dose. Also in the 200-mg group, 1 male subject had an increase (37.9 msec) occurring at 12 hours after the final enzastaurin dose.
Table II presents the least squares means and 90% confidence intervals (CIs) of time-matched changes from placebo. It is notable that the data from the female subject in the 200-mg dose group, who experienced 4 occurrences where her change from baseline was >30 msec, greatly influenced the results of the analysis. Table III presents the results from the analysis excluding this subject. Although there was no evidence of a dose-related effect in QTcF, it would appear that QTcF intervals were consistently raised on the last day of enzastaurin dosing in the 400-mg dose group. This increase, averaged over the day, was small (5.7 msec [90% CI: 1.5-9.9]).
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Pharmacokinetic Evaluations
The single-dose study pharmacokinetic data for enzastaurin and metabolite LY326020 are summarized in Table IV. Mean plasma concentration versus time profiles at each of the 10 dose levels are shown in Figure 1. Enzastaurin concentration-time profile data showed maximum concentration being achieved between 2 and 4 hours after administration followed by a biphasic decline. The 2-mg dose did not provide measurable concentrations for accurate characterizations. For 5- to 400-mg doses, the half-life in individual subjects ranged from approximately 3 to 56 hours for enzastaurin, whereas LY326020 half-life ranged from 25 to 88 hours. The median half-life was approximately 11 hours for enzastaurin and 40 hours for LY326020. No apparent difference in half-life was observed across doses of 5 to 400 mg. Apparent clearance (CL/F) was variable across doses, with mean values from 63.0 to 127 L/h. Despite this variability, there was no trend from 1 dose to the next and no statistical difference with dose (P = .921). The Vss/F was large at all doses, indicating a high degree of extravascular distribution, incomplete bioavailability, or both. The maximum plasma concentrations observed for LY326020 were lower than enzastaurin at each dose level. LY326020, however, had a greater overall exposure (AUC0-) than enzastaurin at doses of 5 to 400 mg.
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For the multiple-dose study, the pharmacokinetics of enzastaurin, LY326020, and LY485912 are presented in Table V. The concentration-time profiles following the first dose and the last dose after daily administration for 15 to 17 days are shown in Figure 2. For enzastaurin and its metabolites, Cmax and AUC0-24,ss increased with each dose increase. The time to reach maximum concentration for enzastaurin was approximately 2 to 4 hours, whereas the metabolites reached maximum concentration in 3 to 6 hours. Individual enzastaurin, LY326020, and LY485912 trough concentrations were assessed on days 14 and 15 and 24 hours after the last dose was administered on day 15 and indicated that steady-state was achieved by day 14 (data not shown). This is consistent with the half-lives of enzastaurin and its metabolites. The half-life in individual subjects ranged from 7 to 34 hours for enzastaurin, 30 to 73 hours for LY326020, and 4 to 33 hours for LY485912. The CL/F of enzastaurin was variable across doses, with mean values ranging from 78.1 to 177 L/h. Enzastaurin exposures had high intersubject variability (coefficient of variation [CV] >50%) at each dose. With its longer half-life, LY326020 accumulated more than enzastaurin or LY485912. The accumulation ratio predicted based on the half-life is approximately 1.5 for enzastaurin and approximately 3 for LY326020. The Robs, based on the ratio of exposures on day 15 to day 1 for enzastaurin and LY326020, gave similar results (Table V) as those predicted from their half-lives and dosing interval. The metabolic ratio for LY326020 was higher (1.22-1.78) than that of LY485912 (0.177-0.234), indicating that LY326020 circulated at higher concentrations than LY485912. For both metabolites, metabolic ratio was constant across all doses.
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The mean concentration-time data from the single-dose study were used for predicting the exposures in the multiple-dose study using superposition principles.14 The mean data predicted the mean exposure following multiple dosing in a reasonable manner for both enzastaurin and LY326020 (Figure 3). Thus, the pharmacokinetics of enzastaurin and LY326020 are constant over time.
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) was demonstrated over any 7.3-fold range. With multiple dosing at steady state, dose proportionality could be concluded for dose ratios of approximately 3, within the dose range of 25 to 400 mg for both the Cmax and AUC,ss. Exposures for the 200- and 400-mg doses of enzastaurin were similar in the multiple-dose study.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Single and multiple doses of enzastaurin were well tolerated in healthy subjects, with headache and sleepiness being the most common adverse events possibly or probably related to enzastaurin, followed by diarrhea and nausea. No subject was discontinued due to an adverse event that was considered by the investigator to be related to enzastaurin. No clinically significant changes in the ECGs, particularly the QTc interval, were noted in either study. The planned maximum dose of 400 mg was achieved in both studies, and there were no dose-limiting toxicities observed that would preclude administering doses higher than 400 mg to healthy subjects. In a phase I multiple-dose study of enzastaurin in cancer patients, doses up to 700 mg were administered without reaching the maximum tolerated dose.17 A 500-mg/day dose is being assessed in ongoing clinical trials in cancer patients.
In the single-dose study, LY326020 exposures (AUC0-) were similar to enzastaurin (metabolic ratio was approximately 1.1). The longer half-life of LY326020 compared to enzastaurin indicated that LY326020 is elimination-rate limited. The long half-life of this metabolite required a dosing period of 2 weeks for the multiple-dose study to allow achievement of steady state. Based on its longer half-life, LY326020 was expected to accumulate at a greater extent than enzastaurin following multiple dosing. An additional metabolite, LY485912, was measured in the multiple-dose study and was seen to have a half-life similar to enzastaurin, indicating that it is formation-rate limited. The observed accumulation of enzastaurin and its metabolites during once-daily dosing (approximately 1.5-fold for enzastaurin and LY485912 and approximately 3-fold for LY326020) is essentially similar to the expected value given by 1/(1 - e-k) for a 24-hour dosing interval (k represents the elimination rate constant). Although both these metabolites are active and may contribute to the activity and efficacy of enzastaurin, LY326020, with its longer half-life and similar exposure (AUC0-) to enzastaurin, is of greater importance than LY485912.
The exposures following the multiple-dose study were also well predicted from the single-dose study data, which indicated that the pharmacokinetics of enzastaurin and its metabolites are not altered over time. Estimates of half-life, CL/F, and Vss/F were similar following single and multiple doses, indicating that enzastaurin neither induces nor inhibits its own metabolism. Within-subject and between-subject variability for exposures of enzastaurin and its metabolites were high.
Although dose proportionality cannot be concluded for the entire dose range tested in either the single- or multiple-dose study (200-fold and 16-fold, respectively), the range of doses over which dose proportionality could be concluded was reasonable to guide the future clinical development given the predicted safety margin and exposures required for pharmacological activity. It was also noted that exposures in the multiple-dose study were similar for the highest 2 doses, but given the high variability, relatively small sample size, and only having 1 dose higher than 200 mg, an assessment of whether the relationship of exposure with dose was changed at the 400-mg dose cannot be made.
Enzastaurin and LY326020 are primarily metabolized by CYP3A, an enzyme with high variability (4- to 5-fold) in the general population.18 This variability may have contributed to the observed intersubject differences in the pharmacokinetics of enzastaurin. Given that enzastaurin is a low-solubility molecule, food was anticipated to cause an increase in bioavailability. This was tested in the single-dose study, where 3 subjects received a 25-mg dose of enzastaurin with and without food. No significant difference was seen in the exposures of enzastaurin when administered with or without food. However, no conclusion regarding food effect on enzastaurin bioavailability could be made due to the small sample size (n = 3), high variability, and low dose of enzastaurin that was studied for food effect. Further studies are ongoing to determine the effect of food on enzastaurin bioavailability.
In conclusion, enzastaurin was well tolerated by healthy subjects when orally administered as single and multiple doses up to 400 mg, and pharmacokinetic steady state was reached after 2 weeks of once-daily dosing.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Financial disclosure: This study was funded by Eli Lilly and Company, Indianapolis, Indiana.
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