Evaluation of the effects of an oral notch inhibitor, crenigacestat
(LY3039478), on QT interval, and bioavailability studies conducted
in healthy subjects
Eunice Yuen1  · Maria Posada2
· Claire Smith1
· Katharine Thorn1
· Dale Greenwood2
· Michelle Burgess2
Karim A. Benhadji2
· Demetrio Ortega2
· Louise Chinchen1
· Jeffrey Suico2
Received: 29 August 2018 / Accepted: 3 December 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
Purpose Crenigacestat (LY3039478) is a Notch inhibitor currently being investigated in advanced cancer patients. Conduct￾ing clinical pharmacology studies in healthy subjects avoids nonbeneficial drug exposures in cancer patients and mitigates
confounding effects of disease state and concomitant medications.
Methods Three studies were conducted in healthy subjects, assessing safety, pharmacokinetics, effect on QT interval, and
relative and absolute bioavailability of crenigacestat. Crenigacestat was administered as single 25, 50, or 75 mg oral doses or
as an intravenous dose of 350 µg 13C15N2
H-crenigacestat. Electrocardiogram measurements, and plasma and urine samples
were collected up to 48 h postdose, and safety assessments were conducted up to 14 days postdose.
Results and conclusions Exposures were dose proportional in the 25 to 75 mg dose range and mean elimination half-life was
approximately 5–6 h. The exposure achieved from the new formulated capsule was approximately 30% and 20% higher for
area under the plasma concentration time curve from time zero to infinity [AUC(0–∞)] and maximum plasma concentra￾tion (Cmax), respectively, compared to the reference drug in capsule formulation. The geometric least-squares mean [90%
confidence interval (CI)] absolute bioavailability of crenigacestat was 0.572 (0.532, 0.615). The regression slope (90% CI)
of placebo-adjusted QTcF against crenigacestat plasma concentration was −0.001 (−0.006, 0.003), suggesting no signifi￾cant linear association. Thirty-nine subjects completed the studies and the majority of adverse events were mild. Single oral
doses of 25 to 75 mg crenigacestat and an IV dose of 350 µg 13C15N2
H-crenigacestat were well tolerated in healthy subjects.
Keywords LY3039478 · Crenigacestat · Notch · QT interval · Bioavailability · Pharmacokinetics
Introduction
Notch signaling is an evolutionary conserved pathway that
plays an integral role in development and tissue homeosta￾sis [1]. Crenigacestat [LY3039478; (4,4,4-trifluoro-N-[(1S)-
2-[[(7S)-5-(2-hydroxyethyl)-6-oxo-7H-pyrido[2,3-d] [3]
benzazepin-7-yl]amino]-1-methyl-2-oxo-ethyl]butanamide)]
is an orally available, small molecule, potent Notch inhibitor
that prevents the release of the Notch intracellular domain,
thereby decreasing Notch signaling and its downstream
biologic effects. Crenigacestat has been shown to inhibit
Notch signaling in cell lines and xenograft models repre￾senting a number of tissues such as human ovary, colon, and
nonsmall-cell lung cancers [2]. In the first-in-human study,
patients with advanced or metastatic cancer were given cren￾igacestat over the dose range of 2.5–100 mg three times a
week (TIW), using a drug in capsule formulation. The most
common study drug-related toxicities included gastrointes￾tinal-related symptoms, asthenia, and hypophosphatemia
[3]. The recommended phase 2 dose of crenigacestat was
50 mg TIW. Crenigacestat was rapidly absorbed with peak
concentrations occurring within 1 to 2 h and an elimination
half-life of approximately 6 h, with little-to-no accumulation
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00280-018-3750-1) contains
supplementary material, which is available to authorized users.
* Eunice Yuen
[email protected]
1 Eli Lilly and Company, Erl Wood Manor,
Windlesham Surrey GU20 6PH, UK
2 Eli Lilly and Company, Lilly Corporate Center, Indianapolis,
IN 46285, USA
Cancer Chemotherapy and Pharmacology
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upon TIW dosing. Apparent clearance of crenigacestat was
approximately 15 L/h, and renal clearance contributed to
approximately 20% of the apparent plasma clearance. 80%
of maximal biomarker effects [plasma amyloid beta (Aβ)
inhibition and inhibition of notch-regulated genes] were
obtained after approximately 15–50 mg TIW doses of creni￾gacestat [4].
Since the release of International Conference on Har￾monisation (ICH) E14 in 2005, regulatory agencies have
required drug sponsors to conduct thorough QT (TQT) stud￾ies to inform the extent of electrocardiogram (ECG) moni￾toring in Phase 3 [5]. However, it has been suggested that
high-quality data collected in Phase 1 may be sufficient to
inform and potentially obviate the TQT study [6, 7]. Thus,
dense sampling with time-matched pharmacokinetics (PK)
and ECG collections in healthy subjects can inform the
potential risk for QT interval increase with crenigacestat
in a clinically relevant dose range that can be translated to
cancer patients. Conducting studies in healthy subjects miti￾gates the potential confounding effects of the disease state
and concomitant medications, and avoids nonbeneficial drug
exposures in cancer patients.
A change in the formulation of crenigacestat, from the
drug in capsule formulation to a formulated capsule, has
occurred since the start of the first-in-human study. This
is common in the drug development process, where non￾formulated drug products may be used in early clinical tri￾als, with subsequent changes to formulated products once
safety and other drug characteristics have been evaluated.
It was, therefore, relevant to conduct a pilot relative bio￾availability study to evaluate the differences between the two
formulations, and to inform if any dose adjustments were
required for future clinical trials when switching between
formulations.
Absolute bioavailability data are required by regula￾tory agencies in some geographical areas [8]. Knowledge
of absolute bioavailability is also helpful in the design of
future clinical studies and in the interpretation of PK data.
The conventional oral absolute bioavailability study design
requires 2 periods for separate administrations of oral and
IV drug formulations. In contrast, incorporation of stable
isotopically labelled drug to the IV formulation allows con￾current administration of oral and IV formulations in a sin￾gle period, which eliminates day-to-day PK variability and
ensures that the systemic clearance is equivalent for the IV
and oral doses [9].
Three clinical pharmacology studies were conducted in
healthy subjects: Study 1 assessed safety, tolerability, and
the effect of crenigacestat on QT interval following single
ascending doses. PK and pharmacodynamics (PD) were also
characterized as secondary and exploratory objectives in
this study. Study 2 was a pilot relative bioavailability study
of crenigacestat administered as drug in capsule versus
formulated capsule, and Study 3 was conducted to estimate
the absolute bioavailability of crenigacestat following a sin￾gle oral administration of crenigacestat and an intravenous
(IV) administration of 350 µg 13C15N2
H-crenigacestat (a
stable isotopically labelled crenigacestat).
Materials and methods
Study designs
In all studies, overtly healthy males and females not of
childbearing potential, aged 18–65 years, with a body mass
index of 18–32 kg/m2
, were eligible for entry into the stud￾ies. Subjects were admitted to the clinical research unit
(CRU) the day before the start of each period. Following
an overnight fast of at least 10 h, crenigacestat, placebo, or
13C15N2
H-crenigacestat was administered according to study
protocol. Subjects resided in the CRU for up to 48 h post￾dose, with additional blood sample collections and adverse
event (AE) assessments conducted 7 days and approximately
14 days after dosing. Where there was more than 1 period
in the study, washout between periods was at least 14 days.
Safety assessments performed during the studies included
the recording of AEs, clinical laboratory evaluations, vital
signs, 12-lead ECGs, and physical examinations. Sample
sizes used in all studies were typical of Phase 1 studies and
not intended to meet any priori statistical requirement. All
procedures performed in all studies were in accordance with
the ethical standards of the institutional and/or national
research committee, and with the 1964 Helsinki declaration
and its later amendments or comparable ethical standards.
Study 1 and 2 were performed at the same site.
Study 1 was a 3-period crossover, subject- and investiga￾tor-blind study conducted in a single center. Approximately
15 healthy subjects were enrolled in order that 12 complete
the study. Subjects were randomized to one of three treat￾ment sequences, and were administered placebo or single
oral doses of 25, 50, or 75 mg crenigacestat (formulated
capsules) in each period. Each subject received two of the
three crenigacestat doses and one dose of placebo over the
course of the study. Clinical laboratory sample collection
and collection of serial crenigacestat PK samples that were
time-matched to triplicate Holter ECG extractions were con￾ducted for up to 48 h postdose.
Study 2 was a 2-period, open-label, crossover design
study, which aimed to enroll approximately 14 healthy sub￾jects to ensure 12 completed. Subjects were randomized to
one of two treatment sequences. In each period, subjects
were administered a single oral dose of 50 mg crenigacestat
as a formulated capsule (test), or as drug in capsule (ref￾erence). The formulated capsule consisted of crenigacestat
blended with starch and silicone oil and filled into a gelatin
Cancer Chemotherapy and Pharmacology
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capsule, while the drug in capsule formulation consisted of
crenigacestat in a hydroxypropyl methylcellulose capsule.
Blood sample collection and safety observations were con￾ducted up to 48 h postdose, to characterize PK and safety
of crenigacestat.
Study 3 was a single-center, open-label, single-period
study. Up to 12 subjects were enrolled, so that at least 8
completed the study. Subjects received a single oral dose
of 75 mg crenigacestat (formulated capsule), and approxi￾mately 15 min later, an IV infusion of duration 45 min con￾taining approximately 350 µg 13C15N2
H-crenigacestat was
given. Blood and urine samples were collected up to 48 h
after the start of dosing to quantify the concentrations of
crenigacestat and 13C15N2
H-crenigacestat in plasma and of
crenigacestat in urine.
Bioanalytical methods
Human plasma samples were analysed for crenigacestat and
13C15N2
H-crenigacestat using two different validated liquid
chromatographies with tandem mass spectrometric (LC/MS/
MS) methods at Q2
Solutions (Ithaca, New York, USA). The
lower and upper limits of quantification were 0.1 ng/mL and
100 ng/mL, respectively, for crenigacestat and 0.005 ng/mL
and 5 ng/mL, respectively, for 13C15N2
H-crenigacestat. In
Studies 1 and 2, for crenigacestat, the inter-assay accuracy
(% relative error) during validation ranged from − 3.75
to 2.00%. The inter-assay precision (% relative standard
deviation) during validation was ≤7.14%. Crenigacestat
was stable for up to 634 days when stored at approximately
−20 °C and for up to 911 days when stored at approximately
−70 °C. In Study 3, the inter-assay accuracy (% relative
error) during validation ranged from −8.93 to 2.81% for
crenigacestat, and from −10.21 to −4.67% for 13C15N2
H-
crenigacestat. The inter-assay precision (% relative standard
deviation) during validation was ≤4.35% for crenigacestat
and ≤5.21% for 13C15N2
H-crenigacestat. Crenigacestat and
13C15N2
H-crenigacestat in plasma were stable for up to 61
days when stored at approximately −20 °C and for up to 61
days when stored at approximately −70 °C.
Plasma samples were also analysed for Aβ peptide con￾centrations using the INNO-BIA plasma Aβ forms assay at
Innogenetics (Ghent, Belgium). Further details of the assay
can be found elsewhere [10], but, briefly, the quantification
range was 7.6–1545 ng/L, with an inter-assay accuracy range
(% relative error) of -17 to 8% and inter-assay precision (%
coefficient of variation) of 5–17% during validation. Analyte
stability was demonstrated in plasma at 2–8 °C for up to 6 h
and frozen storage up to 12 months.
Urine samples, where collected, were analysed for
crenigacestat using a validated LC/MS/MS method. The
lower and upper limits of quantification were 1 ng/mL and
1000 ng/mL, respectively. In Studies 1 and 2, the inter-assay
accuracy (% relative error) during validation ranged from
3.59 to 6.00%. The inter-assay precision (% relative stand￾ard deviation) during validation was ≤7.55%. In Study 3,
the inter-assay accuracy (% relative error) during validation
ranged from −3.59 to 6.00%. The inter-assay precision (%
relative standard deviation) during validation was ≤7.55%
for crenigacestat. In all three studies, crenigacestat in urine
was stable for up to 336 days when stored at approximately
−20 °C, and for up to 1581 days when stored at approxi￾mately −70 °C.
Pharmacokinetic and pharmacodynamic analysis
methods
In all three studies, venous blood samples were collected
up to 48 h postdose to determine plasma concentrations of
crenigacestat and 13C15N2
H-crenigacestat (only in Study
3). Urine samples up to 48 h postdose were also collected
in Studies 1 and 3. PK analyses were conducted on sub￾jects who received at least one dose of crenigacestat and
had sufficient samples collected to allow the estimation of
PK parameters, which were determined using noncompart￾mental methods (Phoenix WinNonlin version 6.4; Certara,
Princeton, New Jersey, USA). Actual sampling times were
used in the analysis of PK parameters and predose samples
that were below the limit of quantification (BQL) were set to
zero, whilst postdose BQL samples were treated as missing.
Exploratory assessment of dose proportionality was
conducted in Study 1, based on the PK parameters of area
under the plasma concentration time curve from time zero
to infinity [AUC(0–∞)] and maximum plasma concentration
(Cmax). A power model [11] was used, fitting log (PK param￾eter) against log (dose) with a random effect for subject.
To delineate the effects of crenigacestat formulation, log￾transformed AUC(0–∞) and Cmax estimates were evaluated
in a mixed-effects model with fixed effects for formulation,
period, and a random effect for subject. Predose samples
were checked to confirm that there was no evidence of car￾ryover effects.
The absolute bioavailability of crenigacestat was cal￾culated using crenigacestat AUC(0–∞) estimates, after
oral dosing of crenigacestat and after IV administration of
13C15N2
H-crenigacestat, with adjustment for dose. A mixed￾effects analysis of variance model was applied to the log￾transformed dose-adjusted AUC(0–∞)s.
For PD analyses, venous blood samples were collected up
to 48 h postdose to determine plasma concentrations of Aβ.
The percentage (%) inhibition of plasma Aβ was calculated
at each time point, and plasma Aβ concentrations below the
limit of quantification were replaced with a value of half the
lower limit of quantification for calculation of % inhibition.
PD parameters for % inhibition of Aβ were determined using
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noncompartmental methods (Phoenix WinNonlin version
6.4; Certara, Princeton, New Jersey, USA).
Statistical analysis methods for TQT and safety
Triplicate QT corrected for heart rate using Fridericia’s
equation (QTcF) from Study 1 was averaged prior to analy￾sis. Time-matched placebo-adjusted QTcF (ΔQTcF) for each
time point was calculated by subtracting each subject’s time￾matched placebo QTcF from their QTcF results after receiv￾ing crenigacestat. The relationship between plasma concen￾trations of crenigacestat and ΔQTcF was evaluated using a
linear mixed-effects modeling approach. Crenigacestat was
judged not to cause clinically significant QTcF prolongation
if the upper bound of the two-sided 90% confidence interval
(CI) of the predicted mean ΔQTcF was below 10 ms [12]
at the highest clinically observed plasma concentrations of
crenigacestat.
Across all studies, safety parameters assessed included
safety laboratory, vital sign, and ECG parameters. The
frequencies of treatment-emergent adverse events (TEAEs)
were summarized by treatment.
Results
Subject disposition
Forty-one healthy subjects were enrolled across the three
studies, with 14, 13, and 12 subjects completed in Studies
1, 2, and 3, respectively. All subjects were males, with the
exception of two females each in Study 1 and 2. The average
age across all three studies was approximately 39–41 years,
and average weight of subjects was approximately 81 to
83 kg with body mass index of approximately 26–27 kg/m2
Pharmacokinetics
The plasma concentration versus time profiles in healthy
subjects after single 25, 50, or 75 mg crenigacestat doses
Table 1 Summary of pharmacokinetic and pharmacodynamic parameter estimates of crenigacestat following single oral doses of 25, 50, or
75 mg in healthy subjects in Study 1
AUC(0–∞) area under the concentration versus time curve from time zero to infinity, CL/F apparent total body clearance of drug calculated after
extravascular administration, CLr renal clearance, Cmax maximum observed drug concentration, CV coefficient of variation, Fe(0–48) fraction of
oral dose excreted unchanged in urine over 48 hours, N number of subjects, t1/2 half-life associated with the terminal rate constant, tmax time of
maximum observed drug concentration, Vz/F apparent volume of distribution during the terminal phase, AUEC area under the effect curve from
time zero up to 48 h postdose, Emax maximum observed effect, SD standard deviation, TEmax time of maximum observed effect
Geometric mean (range)
Pharmacokinetic parameter Geometric mean (CV%)
25 mg crenigacestat
Drug in capsule formulation
(N=10)
50 mg crenigacestat
Drug in capsule formulation
(N=9)
75 mg crenigacestat
Drug in capsule formulation
(N=10)
AUC(0–∞) (ng h/mL) 711 (39) 1400 (21) 2090 (41)
Cmax (ng/mL) 158 (43) 296 (31) 461 (44)
tmaxa
(h) 1.58 (0.55–2.10) 1.05 (1.05–4.10) 2.10 (0.55–2.10)
t1/2b
(h) 5.63 (4.67–7.06) 5.47 (4.48–6.44) 5.71 (5.03–6.60)
CL/F (L/h) 35.2 (39) 35.7 (21) 35.9 (41)
Vz/F (L) 285 (43) 282 (16) 296 (48)
CLr (L/h) 7.92 (30) 7.61 (17) 6.44 (28)
Fe(0–48) (%) 22.5 (24) 21.3 (13) 18.0 (30)
Pharmacodynamic param￾eter
Arithmetic mean (SD)
Placebo (N=14) 25 mg crenigacestat
Drug in capsule formulation
(N=10)
50 mg crenigacestat
Drug in capsule formulation
(N=9)
75 mg crenigaces￾tat
Drug in capsule
formulation
(N=10)
AUEC (h%) − 98.6 (491) 1240 (436) 1690 (465) 2110 (702)
Emax (%) 16.8 (10.2) 81.7 (16.5) 91.8 (2.79) 87.6 (10.6)
TEmaxa
(h) 24.1 (0.00–48.0) 6.21 (4.10–8.10) 6.10 (6.10–8.10) 6.10 (3.10–12.1)
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in Study 1 were characterized by a rapid absorption phase
and biphasic elimination. Median time at which maximum
plasma concentrations were reached (tmax) ranged from
approximately 1 to 2 h, and mean terminal elimination half￾life (t1/2) ranged from 5.5 to 5.7 h (Table 1). Dose propor￾tionality was assessed using the ratio of dose normalized
geometric means at the highest dose (75 mg) compared to
the lowest dose (25 mg). Both estimates, 0.95 [90% CI (0.86,
1.05)] for AUC(0–∞) and 0.96 [90% CI (0.82, 1.13)] for
Cmax, were close to the dose proportional value of one and
had 90% CIs entirely contained within the region (0.8, 1.25)
[11]. These findings support dose proportionality of expo￾sures for the specified dose range of 25–75 mg crenigacestat
in healthy subjects.
Intra-individual variation in AUC(0–∞) and Cmax ranged
between 11 and 18%, and inter-individual variation ranged
between 32 and 34%. The majority of drug excreted in the
urine was recovered within the first 6 h postdose. Mean renal
clearance (CLr) values ranged from 6.4 to 7.9 L/h, and rep￾resented approximately 20% of apparent plasma clearance
(CL/F).
Relative bioavailability
In Study 2, the estimated geometric means of AUC(0–∞)
and Cmax were higher for the formulated capsule (test) than
the drug in capsule (reference) (Table 2). The estimated
ratios of geometric mean exposures were greater than 1 (test/
ref); with values estimated at 1.31 (90% CI [1.23, 1.40])
for AUC(0–∞) and 1.20 (90% CI [1.07, 1.35]) for Cmax.
The exposure achieved from the new formulated capsule
was approximately 30% and 20% higher for AUC(0–∞) and
Cmax, respectively, compared to the reference drug in capsule
formulation.
Absolute bioavailability
Following oral administration of a single dose of 75 mg
crenigacestat in Study 3, median tmax of 1.5  h (range
1.0–3.5 h) was achieved (Table 3). After oral crenigacestat
administration, plasma concentrations appeared to decline
in a biphasic manner after tmax, and the resulting geometric
Table 2 Summary of PK parameter estimates following single oral
doses of 50  mg crenigacestat as drug in capsule formulation (refer￾ence) or formulated capsule (test) in healthy subjects (Study 2)
AUC(0–∞) area under the concentration versus time curve from zero
to infinity, CL/Fapparent total body clearance of drug calculated after
extravascular administration, Cmax maximum observed drug concen￾tration, CVcoefficient of variation, Nnumber of subjects, t1/2 half-life
associated with the terminal rate constant in noncompartmental anal￾ysis, tmax time of maximum observed drug concentration, Vz/Fappar￾ent volume of distribution during
Geometric mean (range)
Parameter Geometric mean (CV%)
50 mg crenigacestat
formulated capsule,
test
50 mg crenigacestat
drug in capsule,
reference
N 14 13
AUC(0–∞) (ng h/mL) 1750 (19) 1340 (22)
Cmax (ng/mL) 384 (21) 322 (25)
tmaxa
(h) 2.00 (1.00–4.00) 1.00 (1.00–3.00)
t1/2b
(h) 5.74 (5.16–7.85) 6.42 (5.66–7.86)
CL/F (L/h) 28.6 (19) 37.2 (22)
Vz/F (L) 237 (24) 344 (22)
Table 3 Summary of PK parameter estimates following a single
75 mg oral dose and 13C15N2
H-crenigacestat following a 350 µg IV
dose in healthy subjects in Study 3
AUC(0–∞) area under the concentration versus time curve from time
zero to infinity, AUC(0–∞)/dose dose-normalized area under the con￾centration versus time curve from time zero to infinity, CL total body
clearance of drug calculated after IV administration, CL/F apparent
total body clearance of drug calculated after extravascular administra￾tion, Cmax maximum observed concentration, CV coefficient of varia￾tion, F bioavailability of drug, N number of subjects studied, t1/2 half￾life associated with the terminal rate constant in noncompartmental
analysis, tmax time of maximum observed concentration, Vz volume
of distribution during the terminal phase after IV administration,
Vz/F apparent volume of distribution during the terminal phase after
extravascular administration
Equivalent parameter after IV administration
Parameter Geometric mean (CV%)
75 mg crenigacestat oral 350 µg
13C15N2H-cren￾igacestat IVa
N 12 12
AUC(0–∞) (ng h/mL) 1920 (23) 15.6 (16)
mean t1/2 based on the terminal elimination phase was 5.4 h.
For IV 13C15N2
H-crenigacestat, the geometric mean t1/2
appeared to be shorter (3.3 h); as concentrations were only
quantifiable until 24 h postdose, it is likely that the terminal
elimination phase for the IV dose had not been fully defined.
The geometric least-squares mean (90% CI) absolute bio￾availability of crenigacestat after oral administration was
0.572 (0.532, 0.615). For individual subjects, absolute bio￾availability ranged from 0.469 to 0.676.
Pharmacodynamics
Administration of single doses of crenigacestat across the
25–75 mg dose range resulted in rapid inhibition of Aβ con￾centrations, which was not observed following administra￾tion of placebo (Fig. 1).
Mean maximum observed effect (Emax) was similar across
the dose levels ranging from 81.7 to 91.8%. The median time
of maximum observed effect was also similar across the dose
levels and occurred at approximately 6 h postdose. Although
Emax was similar across the 25 to 75 mg dose range, the
mean area under the effect curve from time zero up to 48 h
postdose (AUEC) values increased with dose, from minimal
change after placebo (− 98.6 h%) to 1240 h% after 25 mg
crenigacestat dosing and 2110 h% after 75 mg crenigacestat
dosing.
TQT and safety
The analysis was conducted using time-matched placebo￾adjusted QTcF at post-treatment time points, across the
25–75 mg crenigacestat dose range in Study 1. All matched
PK and ECG samples were taken within 30 min of each
other. A scatter plot of placebo-adjusted QTcF against cren￾igacestat plasma concentration with the fitted regression
line and 90% confidence band overlaid is shown in Fig. 2.
The regression slope did not significantly differ from zero
[− 0.001 90% CI (− 0.006, 0.003)] suggesting no significant
linear association between placebo-adjusted QTcF and creni￾gacestat plasma concentration.
The upper 90% CI of the predicted mean placebo-adjusted
QTcF was below 10 msec even at the highest observed con￾centrations; therefore, there was no evidence of any signifi￾cant QTcF prolongation effect from crenigacestat dosing for
this plasma concentration range.
Of the 41 subjects across all three studies who received
one or more doses of placebo, crenigacestat, or 13C15N2
H-
crenigacestat, 18 treatment-emergent adverse events
Fig. 1 Mean plasma creni￾gacestat concentration and Aβ
inhibition versus time profiles
following single oral doses of
25, 50, or 75 mg crenigacestat
or placebo in healthy subjects
in Study 1
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(TEAEs) were reported by 12 subjects. Of these, eight
TEAEs, reported by eight subjects, were considered related
to study treatment as judged by the investigator (Table 4).
The majority of AEs were of mild severity, and none were
considered severe. There were no safety concerns in terms
of clinical laboratory evaluations, vital signs, and 12-lead
ECGs across all studies.
Discussion
Three clinical pharmacology studies were conducted in
healthy subjects using single oral doses of LY30397478
and one micro-dose of IV 13C15N2
H-crenigacestat. The
first study investigated the safety, tolerability, effect on
QT interval, and PK and PD effects of single 25, 50, and
75 mg oral doses of crenigacestat compared to placebo.
The second study was a pilot relative bioavailability study
using 50 mg oral doses of crenigacestat formulated as
drug in capsule or formulated capsule, and the third study
was an absolute bioavailability study with an oral dose of
75 mg crenigacestat followed by an IV infusion of 350 µg
13C15N2
H-crenigacestat in the same period.
These clinical pharmacology studies were conducted in
healthy subjects instead of cancer patients, to mitigate the
potential confounding effects of disease state and use of
concomitant medications, as well as to avoid nonbeneficial
drug exposures in cancer patients. In 1-month nonclinical
Fig. 2 Time-matched placebo-adjusted QTcF versus crenigacestat plasma concentrations following single oral doses of 25, 50, or 75 mg creni￾gacestat in healthy subjects in Study 1
Table 4 Frequency of treatment-emergent adverse events (related to study treatment)
AE adverse effect
Treatment group N Number of AEs [number of subjects with AEs]
Diarrhoea Oral Herpes Headache Arthralgia Dysgeusia Cough
Study 1
Placebo 14 1 [1]
25 mg crenigacestat 10 1 [1]
50 mg crenigacestat 9 1 [1]
75 mg crenigacestat 10
Study 2
50 mg crenigacestat drug in capsule 13
50 mg crenigacestat formulated capsule 14
Study 3
75 mg crenigacestat oral+350 µg 13C15N2
H-crenigacestat IV
12 1 [1] 2 [2] 1 [1] 1 [1]
Overall 41 2 [2] 1 [1] 1 [1] 2 [2] 1 [1] 1 [1]
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toxicology studies in rats and dogs using TIW dosing, the
primary target organ was the gastrointestinal (GI) tract,
with reversibility of mucoid enteropathy demonstrated in
the 1-month rat study (data on file). Mucoid enteropathy is
a known target-mediated toxicity of Notch inhibitors [13].
There was no expected risk of QT prolongation based on
human ether-à-go-go-related gene (hERG) in-vitro assay
results, and the most common TEAEs in the first-in-human
trial considered related to the study treatment were GI
events [3]. There were no reported TEAEs of Grade 3
severity or higher (regardless of relationship to study drug)
on the first day of treatment in patients receiving 75 mg of
crenigacestat (data on file). Taken together, the in-vitro,
nonclinical and initial clinical data supported the conduct
of single-dose (or single dose over repeated periods with
sufficient washout) crenigacestat clinical pharmacology
studies in healthy subjects.
Crenigacestat exposure following administration of the
formulated capsule was dose proportional in the 25–75 mg
dose range tested. The fraction of drug excreted unchanged
in the urine over the first 48 h across the same dose range
was approximately 20%, with the majority of drug recovered
in the urine within the first 6 h postdose. Renal clearance val￾ues represented approximately 20% of the apparently plasma
clearance following oral dosing and approximately 40% of
the total body clearance following IV dosing. Observed
apparent plasma clearances in healthy subjects after a dose
of 50 mg crenigacestat in Study 1 were approximately 2.5
times higher than those observed in patients with advanced
cancer [4] after receiving the same formulation, resulting in
lower exposures in healthy subjects. Clinical and prelimi￾nary in-vitro data have shown that crenigacestat is cleared by
a mixture of routes involving both the kidney and hydrolases
in the red blood cells (data on file). The differences seen in
CL/F between healthy subjects and patients with advanced
cancer may in part be explained by the differences in renal
and hematologic function between these two populations,
where patients with advanced cancer tend to have poorer
renal function and lower red blood cell counts due to disease
state and multiple pretreatment regimens. The requirements
for fasting around dosing were also less rigorous for patients
compared to HVs, which may have also contributed to differ￾ences in exposures. However, based on preliminary in-vitro
data, food is not expected to influence PK. Renal clearance
values also represented approximately 20% of the appar￾ent plasma clearance in patients, suggesting that elimina￾tion pathways did not differ between the two populations.
The inter-individual variabilities in exposure were lower in
healthy subjects (range 16–34%) compared to patients with
advanced cancer (up to 95%) [4].
Since crenigacestat prevents release of the Notch Intra￾cellular Domain (NICD) by inhibiting proteolytic activ￾ity of the gamma (γ)-secretase complex, and γ-secretase
is also responsible for the cleavage of amyloid precursor
proteins, plasma Aβ levels can be used as biomarkers for
PD effects of Notch activity. In Study 1, administration of
single 25–75 mg doses of crenigacestat, but not placebo, had
inhibitory effects on plasma Aβ concentrations. Emax was
achieved in the majority of subjects even at the lowest dose
of crenigacestat administered (25 mg); however, the area
under the effect curve increased with crenigacestat dose, as
a result of the response being sustained for longer as the
dose was increased. In patients with advanced cancer, 80%
inhibition of plasma Aβ occurred at approximate doses of 45
to 100 mg [3], which were slightly higher than that observed
in healthy subjects, where approximately 80% inhibition
occurred after doses of 25 mg (also exposures in healthy
subjects were lower than patients with advanced cancer at
similar doses). In addition to the differences in PK between
healthy subjects and patients as discussed, there may also be
differences in the PK–Aβ relationships between these two
populations. A population PK/PD model combining patient
and healthy subject data will help to identify the covariates
and to quantify the differences in PK and PD between the
two populations, but is outside the scope of this report.
Analysis of placebo-adjusted QTcF revealed no evi￾dence of significant prolongation in the crenigacestat
plasma concentration range achieved in healthy subjects in
the 25–75 mg dose range. These findings were supported
by those of the 1-month dog toxicology study in which no
drug-related effects were observed in heart rate, RR interval,
PR interval, QRS duration, QT, or corrected QT intervals
(data on file). High-quality data collected in Phase 1 studies
such as in Study 1 may be sufficient to inform the extent of
ECG monitoring in Phase 3 and potentially obviate the TQT
study [6, 7]. Although there have been PK differences noted
between patients and healthy subjects with the latter exhib￾iting faster drug clearances and, therefore, lower plasma
concentrations, the maximum tolerated dose in patients was
50 mg TIW [3], whereas the highest dose tested in Study 1
was 75 mg. PK and ECG data from the first-in-human study
may also be pooled with the healthy subject data to widen
the concentration range for further analyses.
Study 2 demonstrated that, in healthy subjects, admin￾istration of single 50 mg oral doses of crenigacestat as for￾mulated capsules resulted in approximately 30–20% higher
exposure, as measured by AUC(0–∞) and Cmax, respec￾tively, compared to crenigacestat as drug in capsule for￾mulation. Formulated capsules are easier to scale up and
are closer to the market-image formulation compared to the
drug in capsule formulation, and, thus, a relative bioavail￾ability study was conducted to ensure that PK profiles do
not change significantly and no change to dosing regimen
is required in future and current clinical trials when the for￾mulation is switched over. Although the formulated cap￾sules resulted in higher exposures, no changes to the dosing
Cancer Chemotherapy and Pharmacology
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regimen were instituted when formulations were switched,
since the observed patient variability was over 90% [4], and
no concentration threshold had been previously identified
for safety reasons from the patient data.
In Study 3, subjects received an oral dose of 75 mg creni￾gacestat followed 15 min later by an IV infusion of 350 µg
13C15N2
H-crenigacestat of duration 45 min to evaluate abso￾lute bioavailability. Maximum concentrations of crenigaces￾tat for the oral dose were achieved at a median time of 1.5 h
postdose, which was close to the end of the IV infusion of
13C15N2
H-crenigacestat. There are several advantages to
the use of this method to estimate bioavailability. First, by
administering an IV dose in the same period as an oral dose,
day-to-day variation of systemic clearances is avoided, and
thus, more accurate bioavailability readouts can be obtained.
Second, the use of a micro-dose allows the absolute bioavail￾ability study to be conducted without the need for a local
tolerability toxicity study, and avoids solubility issues for
compounds with lower solubility. In addition, the use of a
stable isotope instead of radioisotope labels eliminates the
safety concerns to the subject and staff involved with the
collection of radioactive material.
Single oral doses of crenigacestat and IV doses of 350 µg
13C15N2
H-crenigacestat were well tolerated by healthy sub￾jects across all three studies. Eight TEAEs that were consid￾ered related to study drug were reported by 8 out of a total of
41 subjects. Of the 8 TEAEs, there were only two incidences
related to the gastrointestinal tract, which nonclinical and
clinical studies suggest is the target organ for crenigacestat
toxicity. There were no safety concerns following adminis￾tration of oral doses of 25, 50, and 75 mg crenigacestat and
IV doses of 350 µg 13C15N2
H-crenigacestat based on clinical
laboratory evaluations, vital signs, and 12-lead ECG assess￾ments performed during the studies.
Conclusions
Following single oral doses of 25 to 75 mg crenigacestat
administered to healthy subjects, there was no evidence of
any QTcF prolongation. Single oral doses of 25 to 75 mg
crenigacestat and an IV dose of 350 µg 13C15N2
H-creni￾gacestat were generally well tolerated in healthy subjects.
There were no other safety concerns in terms of clinical lab￾oratory evaluations, vital signs, and 12 lead ECGs during the
studies. The AUC(0–∞) and Cmax of crenigacestat admin￾istered as the formulated capsule (test) was approximately
30% and 20% higher, respectively, than the drug in capsule
formulation (reference) in healthy subjects. The geometric
mean (90% CI) absolute bioavailability of a 75 mg oral dose
of crenigacestat was 0.572 (0.532, 0.615). Crenigacestat PK
was characterized by a rapid absorption phase and biphasic
elimination with an elimination half-life of approximately
6 h. Exposure to crenigacestat in healthy subjects was dose
proportional in the 25 to 75 mg range tested, and the cumula￾tive fraction of the dose excreted unchanged over 48 h post￾dose was approximately 20%. Administration of 25–75 mg
crenigacestat had an inhibitory effect on plasma Aβ con￾centrations, with the maximum inhibition achieved in the
majority of healthy subjects even at the lowest dose of 25 mg
crenigacestat; however, the inhibition of Aβ was sustained
for a longer period of time as the dose increased.
Funding This study was funded by Eli Lilly and Company.
Compliance with ethical standards
Conflict of interest All authors are employees of Eli Lilly and Com￾pany.
Ethical approval All procedures performed in studies involving human
participants were in accordance with the ethical standards of the insti￾tutional and/or national research committee and with the 1964 Helsinki
declaration and its later amendments or comparable ethical standards.
Informed consent Informed consent was obtained from all individual
participants included in the study.
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