Embed Size (px)
Citation preview
Cardiotoxicity Associated with NicotinamidePhosphoribosyltransferase Inhibitors in Rodents and in Ratand Human-Derived Cells Lines
D. L. Misner1 • M. A. Kauss1 • J. Singh1 • H. Uppal1 • A. Bruening-Wright2 •
B. M. Liederer1 • T. Lin1 • B. McCray1 • N. La1 • T. Nguyen1 • D. Sampath1 •
P. S. Dragovich1 • T. O’Brien1 • T. S. Zabka1
� Springer Science+Business Media New York 2016
Abstract Nicotinamide phosphoribosyltransferase (NAMPT)
is a pleiotropic protein that functions as an enzyme, cytokine,
growth factor and hormone. As a target for oncology,
NAMPT is particularly attractive, because it catalyzes the
rate-limiting step in the salvage pathway to generate nicoti-
namide adenine dinucleotide (NAD), a universal energy- and
signal-carrying molecule involved in cellular energy metabo-
lism and many homeostatic functions. Inhibition of NAMPT
generally results in NAD depletion, followed by ATP reduc-
tion and loss of cell viability. Herein, we describe NAMPT
inhibitor (NAMPTi)-induced cardiac toxicity in rodents fol-
lowing short-term administration (2–7 days) of NAMPTi’s.
The cardiac toxicity was interpreted as a functional effect
leading to congestive heart failure, characterized by sudden
death, thoracic and abdominal effusion, and myocardial
degeneration. Based on exposures in the initial in vivo safety
rodent studies and cardiotoxicity observed, we conducted
studies in rat and human in vitro cardiomyocyte cell systems.
Based on those results, combined with human cell line
potency data, we demonstrated the toxicity is both on-target
and likely human relevant. This toxicity was mitigated in vitro
by co-administration of nicotinic acid (NA), which can enable
NAD production through the NAMPT-independent pathway;
however, this resulted in only partial mitigation in in vivo
studies. This work also highlights the usefulness and predic-
tivity of in vitro cardiomyocyte assays using human cells to
rank-order compounds against potency in cell-based phar-
macology assays. Lastly, this work strengthens the correlation
between cardiomyocyte cell viability and functionality, sug-
gesting that these assays together may enable early assessment
of cardiotoxicity in vitro prior to conduct of in vivo studies
and potentially reduce subsequent attrition due to
cardiotoxicity.
& D. L. Misner
M. A. Kauss
J. Singh
H. Uppal
A. Bruening-Wright
B. M. Liederer
T. Lin
B. McCray
N. La
T. Nguyen
D. Sampath
P. S. Dragovich
T. O’Brien
T. S. Zabka
1 Genentech, 1 DNA Way, M/S 59, South San Francisco,
CA 94080, USA
2 ChanTest, 14656 Neo Parkway, Cleveland, OH 44128, USA
123
Cardiovasc Toxicol
DOI 10.1007/s12012-016-9387-6
Keywords Heart � Tumor metabolism � Nicotinamide
adenine dinucleotide � Nicotinic acid mononucleotide �Pathology � Myocardial degeneration � Cardiomyocytes �Cardiotoxicity � Viability � Impedance
Abbreviations
NAMPT Nicotinamide phosphoribosyltransferase
NAPRT Nicotinic acid phosphoribosyltransferase
NAD Nicotinamide adenine dinucleotide
NA Nicotinic acid
NAMPTi NAMPT inhibitors
ESC Embryonic stem cell
iPSC Induced pluripotent stem cell
H&E Hematoxylin and eosin
ATP Adenosine triphosphate
Introduction
Nicotinamide phosphoribosyltransferase (NAMPT) is a
pleotropic protein that functions as an enzyme, cytokine,
growth factor, and hormone [2, 22, 24]. As an oncology
target, NAMPT is attractive, because it catalyzes the rate-
limiting step in one of two intracellular salvage pathways
[i.e., via NAMPT or nicotinic acid phosphoribosyltrans-
ferase (NAPRT)] that generate nicotinamide adenine din-
ucleotide (NAD), and cancer cells are highly dependent on
the NAD-driven biosynthetic and redox pathways for
proliferation and survival (for more detail on pathway and
biology, see [23]). Further, cancer cells rely on the
NAMPT-mediated salvage pathway, as they have inher-
ently low levels of nicotinic acid (NA) required for de novo
synthesis and in some cases even overexpress NAMPT
[2, 21, 24]. In cells that express NAPRT1, the addition of
NA can increase cellular levels of NAD via the NAPRT1-
mediated salvage pathway and thus protect against oxida-
tive stress [3, 20]. The addition NA to cell culture media
exposed to NAMPT inhibitors (NAMPTi) allows the syn-
thesis of NAD via the NAPRT1-mediated salvage pathway
and can mitigate NAMPT-induced cytotoxicity
[3, 4, 13, 17, 20]. However, in cancer cells that lack
NAPRT1, depletion of NAD following treatment with
NAMPTi results in decreased proliferation and cell death
should not be mitigated by addition of NA [3, 17, 20].
NAMPTi treatment of NAPRT1-negative subsets is there-
fore expected to be highly effective in killing those cancer
cells while efficacy is not affected by addition of exoge-
nous NA.
Administration of three NAMPTis of two structurally
distinct classes, APO866 (formerly FK866) [5] and GMX-
1778 (formerly CHS-828) and its inactive pro-drug
GMX1777 [6, 15, 16, 19] in clinical trials resulted in dose-
limiting platelet toxicity and gastrointestinal toxicity that
prevented achievement of efficacious doses. Thus, based on
the biology and safety profile, our efficacy and diagnostic
strategy were to select NAPRT1-deficient tumors for
treatment with NAMPTi and co-administration of NA to
enable the NAPRT1-mediated salvage pathway in normal
cells in order to improve the therapeutic window (separa-
tion between efficacious and toxic exposures) [17, 21]. This
strategy, however, may not be feasible as once hypothe-
sized, as O’Brien et al. [11] recently demonstrated that
efficacy of NAMPTi can be rescued in the presence of NA
in vivo in NAPRT1-negative mouse xenograft models due
to regeneration of NAD.
A repeat dose safety study in rats with oral administra-
tion of a potent NAMPTi, GNE-617 [25] was associated
with hematopoietic toxicity [18], retinal toxicity [23], and
cardiac toxicity (reported herein). Based on findings of
cardiac toxicity, we investigated the effects of a struc-
turally unrelated but potent NAMPTi as well as a low
potency analog in order to elucidate the mechanism of
cardiac toxicity and to determine whether effects were on-
target, could be mitigated by co-administration of NA, and
were potentially translatable to humans. We employed
several approaches to address these questions, including
additional in vivo rat toxicity studies, biochemical and
functional analysis of rat and human cardiomyocytes
in vitro, and measurement of NAD and adenosine
triphosphate (ATP) levels in NAMPTi-treated cardiomy-
ocytes in vitro. The utility of human pluripotent stem cell-
derived cardiomyocytes to assess cardiac risk in vitro has
been demonstrated previously across several different
testing platforms (for reviews, see [8, 9, 14]. We therefore
utilized these in vitro systems to compare effects across
species and employed in vitro to in vivo correlations in the
rat to establish relevance of the in vitro results and
potential translatability of effects to humans. Lastly, we
were able to use these in vitro assays to screen many
compounds and prioritize compounds to be tested further
in vivo, demonstrating the utility of such assays in the drug
discovery process.
Materials and Methods
Compounds
NAMPTi’s properties and structures were described pre-
viously (Zabka et al. this issue). Briefly, these compounds
include internally synthesized competitor compounds and
structurally distinct compounds with a range of cellular
potency across species and different physiochemical
properties (GNE-617: [12, 25]; GNE-643: [12]; GNE-875:
[1]; GNE-618: [21, 26]. The human cellular potency for
these compounds was similar across GNE-617, GNE-875,
Cardiovasc Toxicol
123
GNE-618, GMX-1778, and APO-866 and approximately
180-fold less for GNE-643 [12], which was a structural
analog of GNE-617. NA (Sigma-Aldrich, St. Louis, MO;
No. N0761-100G) was formulated in-house for co-admin-
istration. Cellular potencies were derived as previously
reported [23], and IC50 values at A2780 cells reported
herein (Table 2).
Animal Use
All animal care and experimental procedures complied
with IACUC, Animal Welfare act, AAALAC, and the NIH
Guide for the Care and Use of Animals and were approved
by the Institute’s Animal Care and Use Committee.
In Vivo Safety Studies
The specifics of dose selection, co-administration of NA,
animal sex, and study duration, which were sometimes
modified by early termination due to early euthanasia of
moribund animals and mortality, were described previously
[23]. Male and female naive Crl:CD Sprague–Dawley rats
(Hollister, CA) were used, with four animals/sex/group.
Each study included a vehicle control group, and for the first
study in which NA was co-administered, an NA-only dose
group was included to establish the lack of NA-associated
toxicity. Separate toxicokinetic groups were included to
generate exposure data to ensure dose linearity and
achievement of supra-efficacious exposure multiples. Ani-
mals were administered compound by oral gavage, except
for APO-866 that was administered by intraperitoneal
injection in order to achieve sufficient exposure levels.
Compounds administered orally were formulated as a solu-
tion in the vehicle of 60 % polyethylene glycol (PEG 400)/
10 % ethanol/30 % dextrose in water (D5 W), and NA was
formulated as a solution in water. APO-866 was formulated
in phosphate-buffered saline with 3 % hydroxypropyl-beta-
cyclodextrin and 48 % propylene glycol. Necropsy was
performed the day following the final dose, except where
early termination was noted. Tissues were collected in 10 %
formalin and processed routinely into 5-micron-thick
hematoxylin and eosin (HE)-stained slides for light micro-
scopic evaluation by the same pathologist, and peer review
was performed by one of two other pathologists. All studies
were conducted, and animals handled in accordance with
regulatory compliance for animal care use.
In Vitro Study Design
Primary rat neonatal cardiomyocytes (Lonza, Basel,
Switzerland) and human cardiomyocytes derived from stem
cells (embryonic stem cell (ESC) derived from GE
Healthcare, Buckinghamshire, UK and induced pluripotent
stem cell (iPSC) derived from Cellular Dynamics Inc.,
Madison, WI) were obtained and seeded at a density of 8000
cells/well in gelatin-coated 384-wells plates for cell viability
measurements. Cardiomyocytes were incubated in a humid-
ified atmosphere with 5 % CO2 at 37 �C. The medium, pro-
vided by each vendor, was changed every 3 days, except
when described elsewhere. 10 mM stocks of NAMPTi’s
(GNE-617, GNE-618, GMX-1778, APO-866, GNE-875, and
GNE-643) and NA were prepared in DMSO and were further
diluted in cell-specific medium as required. After overnight
plating, cells were treated with NAMPTi (n = 6 wells/con-
centration) at seven concentrations (3.7, 0.41, 0.045, 0.015,
0.005, 0.017, and 0.0002 lM), and the plates were incubated
for 3–7 days, depending on cell type. On the third or seventh
day (depending on cell type), plates were removed from the
incubator and cell viability was determined by the CellTiter-
Glo� Luminescent Cell Viability Assay kit (G7571, Pro-
mega, Madison, WI), as described in the manufacture’s pro-
tocol. Cell viability was also measured using the CyQuant�
nucleic acid stain (Life Technologies, Carlsbad, CA; data not
shown). All the data were plotted and analyzed using Spotfire
and IC50s calculated using a nonlinear regression analysis.
Because NAMPTi’s appeared to be less potent on rat neonatal
cardiomyocytes, concentrations were adjusted to capture a
full concentration range in each cell type. The cytotoxicity in
cardiomyocytes was repeatable (n = 6) in human iPSC-
derived or ESC-derived cardiomyocytes, but was more
variable in rat neonatal primary cardiomyocytes; therefore,
cytotoxicity assays were repeated across multiple donors for
rat neonatal cardiomyocytes (n C 3 donors). NA was co-
administered across a range of concentrations with two con-
centrations of GNE-617 in order to determine the most active
concentration required to completely prevent the toxicity
caused by NAMPTi to all cell types (100 % rescue). To
determine specificity, one concentration of staurosporine
inducing approximately 30 % inhibition was co-administered
with varying concentrations of NA.
In Vitro Impedance Assay Measurements
All measurements were taken at physiological temperature
in a tissue culture incubator (37 �C; 5 % CO2) using human
iPSC-derived cardiomyocytes. Overall impedance and
transient impedance signals were measured to establish
baseline responses. Only wells with stable baseline respon-
ses were used. After stable baseline responses were estab-
lished, test article, positive control, and vehicle additions
were made and recording resumed. No cell culture media
changes were performed over the course of the experiment.
Data acquisition was performed using RTCA Cardio Soft-
ware 1.0 (ACEA Biosciences, San Diego, CA). Recordings
(60 s sweeps; interpoint interval of 12.9 ms) were made
Cardiovasc Toxicol
123
every 30 min for the first 6 h after addition of compound,
then after 24, 48, 72, and 168 h. Data analysis was performed
using RTCA Cardio Software 1.0 and Microsoft Excel. The
following parameters were quantitated: cell index (overall
impedance and transient contraction), amplitude, rate,
kinetics, and irregular beating (if quantifiable). A cell index
of all the parameters was calculated, and data reported as
mean ± SEM. Pooled data were tabulated for the baseline
control and each test article concentration. Changes in
parameters were evaluated to determine whether the percent
change from baseline observed after equilibration for each
compound concentration at each time point was significantly
different (P\ 0.05) from that observed in vehicle control.
Analysis of In Vitro NAD Levels
Both rat neonatal primary and human ESC-derived and
iPSC-derived cardiomyocytes were treated with the
NAMPTi’s described above, and the NAD? levels were
determined by LC–MS/MS and then calculated from a
standard curve [10]. Additionally, ATP levels were con-
currently measured by CellTiter-Glo� following treatment
with either vehicle or NAMPTi’s in rat neonatal and human
ESC-derived cardiomyocytes on day 3 and in human iPSC-
derived cardiomyocytes on day 6.
Results
Rats were orally administered GNE-617, a potent and
selective NAMPTi, as part of an initial safety evaluation.
At doses of 30 mg/kg QD, early deaths or euthanasia
occurred in all animals on day 4 of the study, and those rats
co-administered NA reached the scheduled study termina-
tion on day 8. The cause of mortality and moribundity was
attributed to cardiac toxicity characterized by thoracic clear
to serosanguinous effusion on gross examination and
myocardial degeneration on microscopic examination. The
myocardial degeneration was a global injury characterized
by interstitial separation (interpreted as edema) and hem-
orrhage with areas of pallor corresponding to cardiomy-
ocyte vacuolation, sarcoplasmic fragmentation, and less
often, loss of distinct cross-striations (Fig. 1a, b). The
nature of the thoracic effusion and the disparity between
the severity of microscopic finding and manifestation of
mortality and/or thoracic effusion suggested a functional
insult that at least in some rats resulted in congestive heart
failure. These findings also were observed in rats co-ad-
ministered NA, but were of lesser incidence to suggest
partial mitigation of cardiac toxicity.
Due to the severity of the cardiac findings and partial
mitigation with NA co-administration, we next dosed rats
with structurally unrelated compounds that previously were
in clinical trials, GMX-1778 and APO-866, to begin to
understand whether the cardiac toxicity was related to an
on-target or off-target (i.e., structural) mechanism. Cardiac
toxicity was again observed with both compounds fol-
lowing a similar dosing duration of 5–7 days (Table 1) and
with similar potency as GNE-617 in killing human cancer
cells (Table 2). These results suggested that different
chemotypes were not responsible for cardiac toxicity, and
with APO-866, the thoracic effusion was identified as a
modified transudate, supporting its relationship to heart
failure. To confirm the latter findings, an additional com-
pound, GNE-875, that is structurally similar to GNE-617
and equipotent, was dosed for 5 days but with the addition
Fig. 1 Representative photomicrographic images from rat of
NAMPTi-induced heart toxicity of moderate severity (three out of
five; the most severe score noted among studies). a Heart toxicity was
characterized by global injury with obvious interstitial separation and
hemorrhage (arrow) as well as more subtle areas of cardiomyocyte
pallor (asterisk) that were consistent with findings observed at higher
magnification (hematoxylin and eosin (H&E) stain; 92). b Higher
magnification demonstrated cardiomyocyte vacuolation (thin arrow),
fragmentation of the sarcoplasm (asterisk), and loss of distinct cross-
striations (arrowhead); interstitial separation and hemorrhage (block
arrow). The loss of cross-striations in this more severe lesion
indicates eventual cell death of individual cells; however, this is not
the primary toxicity feature (H&E; 920)
Cardiovasc Toxicol
123
of pre-treatment of NA for 5 days prior to co-administra-
tion and a BID dosing regimen to ascertain whether this
could mitigate cardiac toxicity. Similar cardiac toxicity,
including only partial mitigation with NA pre- and co-ad-
ministration, was observed as with GNE-617, except
abdominal instead of thoracic effusion manifested on gross
examination (Table 1). Lastly, GNE-643, a compound that
is in a similar structural class as GNE-617 and GNE-875
but is 36- to 180-fold less potent (Table 2), was tested
following a similar dosing duration (5 days) in order to
establish the relationship between NAMPT potency (as
measured by enzyme potency and toxicity to cancer cells)
and cardiac toxicity findings. As hypothesized, GNE-643
did not induce any cardiac findings, despite achieving
similar free plasma exposures as those achieved with GNE-
617 (Table 1). Taken together, these in vivo rat studies
suggested that the cardiac toxicity was (1) related to the
mechanism of action and potency at the target and not the
scaffold; (2) tracked with efficacy in dose and duration (by
4 days) observed in mouse models [1, 11, 21, 25, 26]; (3)
associated with a functional effect leading to congestive
heart failure; and (4) was unlikely to be sufficiently
mitigated by pre- or co-administration of NA, of which the
maximum percent mitigation reached among studies in
which NA was co-administered was 75 %.
To further investigate the potential mechanism of car-
diac toxicity and assess the relevance of species, we
examined the effects of compounds tested in vivo in both
rat and human cardiomyocytes in vitro. Primary rat
neonatal cardiomyocytes from different donors (at least
3/experiment) were used, along with human ESC-derived
and iPSC-derived cardiomyocytes. An initial time course
study using each cell type was conducted with the most
potent NAMPTi, including GNE-617 and GMX-1778, to
determine a time frame whereby non-specific ATP loss and
cell death in DMSO-treated cells were minimal and effects
induced by NAMPTi treatment were maximal. For rat
primary and human ESC-derived cardiomyocytes, this
window was 3 days, and for iPSC-derived cardiomyocytes
was approximately 7 days. We then conducted experiments
using ATP levels as an end point as a surrogate measure of
toxicity. IC50 values for each cell type were calculated
across a range of concentrations; a summary of those val-
ues is presented in Table 2. Rat cardiomyocytes were less
Table 1 Summary of in vivo safety studies in rat with gross and microscopic findings in the heart and toxicokinetic results
Compound Dose level
(mg kg-1)
Dosing
duration
Gross finding Microscopic
finding
Heart tox incidence (NA
co-admin)dAUC
(lM h)
Free AUC
(lM h)
GNE-617 30 ± NA 4–7 days Thoracic
effusion
Myocardial
degeneration
100 % (25 %) 26 1.25
GNE-875 C30 ± NA 2–5 days Abdominal
effusionaMyocardial
degeneration
91 % (60 %) C89.3 C5.8
GMX-
1778
200 5–7 days None Myocardial
degeneration
60 % 230 1.3
APO-866 120 1–3 daysb Thoracic
effusioncMyocardial
degeneration
100 % 760 40.3
GNE-643 120 5 days None None 0 % 64.7 3.2
a Only in animals administered GNE-875 alone; b Study design was for 5 days of dosing, but early deaths occurred; c Clinical pathology analysis
demonstrated a modified transudate, which is consistent with heart failure. N.D. = not determined; d Total percent incidence of heart toxicity
across dose groups; heart toxicity defined by gross observation of cavitary effusion and/or microscopic myocardial degeneration
Table 2 Summary of cellular potency and inhibitory effects in rat and human cardiomyocytes
Compound Human A2780
(NAMPT potency)
Rat primary
cardiomyocytes
(ATP)
Human EPSC-derived
cardiomyocytes (ATP)
Human iPSC-derived
cardiomyocytes (ATP)
Human iPSC-derived
cardiomyocytes
(impedance)
GNE-617 2.0 22 5 2 18.7
GNE-875 10.2 256 13 22 N.D.
GNE-618 4.3 3592 4 5 28.4
GMX-1778 5.0 400 5 4.6 15.6
APO-866 1.0 454 2.4 2.6 5.1
GNE-643 360.0 [3700 1177 474 4981
Staurosporine N.D. 42 14.5 18 3.4
IC50: 50 % inhibitory concentration of cell viability (values reported in nanomolar units) after 3 days of compound administration. N.D. = not
determined
Cardiovasc Toxicol
123
sensitive to NAMPTi-mediated toxicity than human car-
diomyocytes (Table 2), indicating either a difference in
species sensitivity to cardiac effects or in the source and
types of cells employed and thus a lack of direct compa-
rability between the systems. Human ESC-derived car-
diomyocytes did not reach levels near 100 % inhibition
with NAMPTi, possibly due to the presence of fibroblasts,
which were not sensitive to NAMPTi, as confirmed when
fibroblasts were tested separately (IC50[ 10 lM; data not
shown). Human iPSC-derived cardiomyocytes showed the
most consistent results and also the best correlation with
NAMPTi cellular potency (Fig. 2); therefore, these cells
were used to characterize potentially translatable effects in
human cells. Across all three cell types, rank-order potency
of toxicity to cardiomyocytes and correlation to potency in
A2780 cancer cells (measure of NAMPT cellular potency)
were maintained, and the sensitivity of cells to NAMPTi-
induced cardiotoxicity was repeatable (iPSC-derive-
d[ESC-derived � rat primary cardiomyocytes; Fig. 2).
These results support those from the in vivo studies and
suggest the cardiac toxicity is relevant to humans, who may
even be more sensitive than rats.
We also co-treated human iPSC-derived cardiomyocytes
with varying concentrations of NA (representative IC50
curves are shown in Fig. 3a) and either a low concentration
of GNE-617 (20 nM; Fig. 3b) or a high concentration
(20 lM; Fig. 3c). Under both conditions, 5 lM NA fully
mitigated the toxicity induced by GNE-617 treatment in
cardiomyocytes. Therefore, co-administration of 5 lM NA
was used for all additional experiments and was able to
mitigate cardiotoxicity across all NAMPTi to a similar
level as GNE-617 (data not shown). To demonstrate
specificity, NA was co-treated with a concentration of
staurosporine that induced approximately 30 % toxicity;
co-treatment had no effect on staurosporine-induced toxi-
city up to 5 lM of NA (Fig. 3d). Lastly, because ATP
depletion has been associated with decreased NAD levels,
we confirmed that the decreased ATP corresponded to cell
death using a nucleic acid stain. Thus, rank-order potencies
compared to NAMPTi on human iPSC-derived
cardiomyocytes were maintained, indicating that the loss of
ATP was accompanied by cell death (data not shown).
While rat and/or human cardiomyocytes exhibited
NAMPT-induced toxicity as measured by microscopic
examination or biochemical endpoints, the effects on cardiac
function were still unknown. Functional-based assays using
human iPSC-derived cardiomyocytes have demonstrated
utility for assessing cardiac function (for review, see
[8, 9, 14]), and effects can be monitored repeatedly over an
extended period of time using impedance assays. We
therefore evaluated the effects of NAMPTi using the impe-
dance assay over a range of concentrations and compared
effects to the positive control staurosporine. While stau-
rosporine-induced changes in cardiac function began within
hours of treatment initiation, NAMPTi treatment did not
begin to affect cardiac function until day 4 and peaked
around day 7 in human iPSC-derived cardiomyocytes,
reflective of the time course of the change in cellular ATP
levels, where maximal effects occurred around day 7
(Fig. 4). We also evaluated impedance through day 9 and
determined the IC50 values of NAMPTi at this time point
(Fig. 5). Rank-order potency at cardiomyocytes versus
NAMPT potency was generally maintained at both time
points, where potent NAMPTi showed much more potent
IC50s in the impedance assay and the much less potent
NAMPTi GNE-643 showed very slight inhibition of car-
diomyocyte function at only the highest concentration tested
(Figs. 4, 5; Table 2). These results supported the interpre-
tation of in vivo findings, suggesting a primary functional
rather than organic (i.e., a change at the cellular level that is
morphologically manifested) effect on cardiomyocytes.
We finally examined the levels of NAD? across the
different types of cardiomyocytes, along with levels of
ATP, in order to confirm that NAD? levels were indeed
decreased in NAMPTi-treated cardiomyocytes (Fig. 6). In
all instances where effects were noted, levels of NAD?
were either lower or undetectable compared to ATP levels,
suggesting that the depletion of NAD? preceded the loss of
ATP and subsequent cell dysfunction and death. These
results supported an on-target effect related to the NAMPT
Fig. 2 Correlation between cellular potency and toxicity on day 3 in rat primary cardiomyocytes (a) and human ESC-derived (b),
cardiomyocytes and on day 7 in human iPSC-derived (c) cardiomyocytes
Cardiovasc Toxicol
123
pathway and suggested that additional stressors within the
in vivo system prevented full mitigation of cardiac toxicity
as observed in vitro with co-administration of NA. Further,
morphologic examination of cardiomyocytes, indicated
that most cells did not die by necrosis or apoptosis but
rather floated intact to the surface, suggesting they just
stopped beating due to ATP depletion. Additional analyses,
including viability assessment, of these floating cells were
not performed. This finding is consistent with our in vivo
observations, whereby the outcome of heart failure and
death was not reflected by the severity of myocardial
degeneration microscopically.
Discussion and Conclusions
Inhibition of NAMPT was associated with cardiac toxicity
in rats characterized by sudden death, thoracic and
abdominal transudative effusion grossly, and myocardial
degeneration microscopically. The nature of the findings
suggested a primary functional effect rather than organic
effect (i.e., a change at the cellular level that is morpho-
logically manifested) on cardiomyocytes resulting in
congestive heart failure. The onset of toxicity within
2–5 days corresponded with the onset of efficacy in human
xenograft models [11, 21, 25, 26] and occurred with
structurally distinct and equipotent compounds (GNE-617,
GNE-875 and GMX-1778) but did not occur with a
structurally similar but much less potent compound (GNE-
643), suggesting that the toxicity was both on-target and
tracked with efficacy. The toxicity was only partially mit-
igated by pre- and co-administration of NA at high doses,
which suggests that the NAMPT pathway is critical for
NAD and subsequent ATP production in cardiomyocytes
in vivo. Because cardiac toxicity was attributed to the
cause of the mortality and moribundity in the non-clinical
safety studies, we undertook additional in vitro studies to
confirm that the mechanism was on-target and relevant to
humans.
To investigate the mechanism and potential relevance of
the rat toxicity to humans, the same NAMPTi’s were tested
in vitro in rat neonatal primary and human stem cell-
derived cardiomyocyte cell systems. The human car-
diomyocytes were more sensitive than the rat, which is
consistent with efficacy data that demonstrated human cell
lines were more sensitive to NAMPTi than rat cell lines
Fig. 3 Effects of NAMPTi treatment on cardiomyocyte viability and
prevention of toxicity by co-treatment with NA in human iPSC-
derived cardiomyocytes. Percentage of control ATP following
NAMPTi and staurosporine treatment for 7 days, as well as the
positive control staurosporine, is shown a. Co-treatment of varying
concentrations of NA with 20 nM (b) or 20 lM (c) GNE-617
prevented cardiotoxicity, but had no effect on cardiotoxicity induced
by a sub-maximal concentration of staurosporine (d)
Cardiovasc Toxicol
123
[23]. Alternatively, this species-dependent sensitivity could
be attributed to differences in the cell composition and
origin of in vitro systems, where rat cultures were primary,
neonatal and composed of a mixture of atrial, ventricular,
and nodal cells while human cultures were stem cell
derived, of unknown maturity, and primarily ventricular.
Regardless, these results suggest that human patients could
potentially be even more sensitive than rodents to cardiac
toxicity. Additionally, the lack of toxicity following treat-
ment with a much less potent analog (GNE-643) and the
strong correlation between NAMPTi potency and inhibi-
tory effects at human iPSC-derived cardiomyocytes
(Fig. 3) further support the hypothesis that cardiac toxicity
may be more likely due to on-target effects. The toxicity of
Fig. 4 Time course of effects of NAMPTi treatment on cardiac function as measured in the impedance assay using iPSC-derived human
cardiomyocytes with a staurosporine, b GMX-1778, c GNE-617, d GNE-618, e APO-866, and f GNE-643
Cardiovasc Toxicol
123
NAMPTi was specific to certain cell types, as treatment of
cryopreserved rat and human hepatocytes and fibroblasts
did not result in decreased ATP levels or cytotoxicity when
tested up to 10 lM (data not shown). Toxicity with potent
NAMPTi has been observed in rapidly dividing cells [21],
bone marrow/megakaryocytes [18], retinal cells [23], and
beating cardiomyocytes described herein, indicating that
toxicity may occur in cells with a higher energy demand,
but not broadly across all cell types. This is further sup-
ported by the complete versus partial attenuation with co-
administration of NA in vivo and in vitro, respectively, as
cardiomyocytes in rodents may have additional stressors
(i.e., energy demands) not present in in vitro cell culture
systems. Finally, the functional in vitro assessment by
impedance demonstrated that decreased NAD? and ATP
levels and cell viability corresponded to impaired car-
diomyocyte function (including beat rate and amplitude),
with a similar time course to onset of effects (4 days post-
treatment) and correlated with cellular potency of the
NAMPTi tested. The onset of toxicity induced by NAMPTi
was delayed relative to the positive control and well-
established cardiotoxicant staurosporine (day 4 vs day 1),
suggesting that inhibition of NAMPT and cellular deple-
tion of NAD? and subsequent decrease of ATP precede the
loss of cell viability and impaired cardiac function. In
addition, examination of cell morphology suggested that
most cells did not die by necrosis or apoptosis but rather
floated intact to the surface, suggesting they just stopped
beating due to ATP depletion, although viability of these
floating cells was not assessed. The time course, sequence
of events, and cell morphology observed in the in vitro
cardiomyocyte system closely match the time course and
nature of effects observed in vivo, further supporting that
the mechanism of toxicity may be on-target and due to a
primary functional effect potentially leads to congestive
heart failure and death in the absence of a corresponding
morphologic severity observed in vivo.
The co-administration of NA with NAMPTi in rat and
human cells completely mitigated the cardiomyocyte tox-
icity, whereas the cardiac toxicity was only partially miti-
gated with NA co-treatment in vivo. It is possible that
in vivo, NA levels are not sufficiently sustained in every
animal to fully prevent cardiac toxicity, but in the in vitro
system, cells are continuously exposed to a constant level
of NA, resulting in an active rescue pathway. Indeed, rat
mRNA expression data [23] demonstrated that the heart,
relative to other organs examined, had the highest levels of
NAMPT and NMNAT1 expression, the second highest
level for NAPRT1 and NMNAT3, and the third highest
level of NMNAT2 expression. These expression data
suggest that the NAMPT-mediated pathway may be
essential for NAD generation in the heart, and that NAD
may be more critical for nuclear and mitochondrial func-
tion, as the cytoplasmic isoform, NMNAT2, was expressed
at much lower levels, relatively. This interpretation is
consistent with the downregulation of NAMPT causing
cytochrome c release in cardiac myocytes (although not
assessed in these experiments) and thus demonstrating a
mitochondrial component in the resulting cardiomyocyte
apoptosis in vitro [7]. The similar levels of expression for
NAMPT and NAPRT1 suggest that when enabled by NA
supplementation, the NAPRT1 pathway should be able to
mitigate cardiac toxicity. This interpretation was supported
by complete mitigation in the in vitro systems with co-
administration of NA; however, its only partial mitigation
in vivo suggests that additional stressors in rodents may
increase energy demand relative to physiological status or
alter NAMPT expression [7] that cannot be sufficiently
rescued by NA administration. Taken together, the
expression of key enzymes in the NAMPT-mediated sal-
vage pathway and the pharmacology support our hypoth-
esis that cardiac toxicity in rodents may be due to an on-
target mechanism. Due to the serious nature of our find-
ings, lack of monitorability, and failure to completely
prevent the toxicity with NA co-administration, it is unli-
kely that an acceptable safety margin between efficacy and
cardiac toxicity could be established.
In summary, we describe NAMPTi-induced cardiac tox-
icity in rodents. Using in vivo safety rodent studies, human
and mouse cell line potency data, human and rat in vitro
systems, and rat mRNA expression data, we demonstrate that
the toxicity is on-target, human relevant, associated with a
functional deficiency. These effects lead to congestive heart
failure, track with the onset of efficacy, and are only partially
mitigated by NA co-administration in vivo. The lack of
cardiac toxicity in human clinical trials with GMX-1778 and
APO-866 was likely due to the dose-limiting platelet toxicity
preventing drug administration to efficacious levels. Inhi-
bition of the NAMPT-mediated pathway and therefore the
production of NAD, an essential coenzyme for energy
Fig. 5 Overall effects of NAMPTi treatment on cardiac function in
the impedance assay after 9 days of treatment were similar to effects
observed in the cell viability assay in human iPSC-derived
cardiomyocytes
Cardiovasc Toxicol
123
Fig. 6 NAD? (gray bars) and
ATP (black bars) levels in rat
primary (a) and human EPSC-
derived (b) cardiomyocytes on
day 3 and human iPSC-derived
cardiomyocytes (c) on day 6
Cardiovasc Toxicol
123
metabolism and cellular homeostatic functions, resulted in
the rapid onset and progression of cardiac toxicity. Our
results are supported by the in vivo and in vitro results of Hsu
et al. [7] that demonstrated NAMPT is both necessary and
sufficient for the regulation of the intracellular level of
NAD? in cardiac myocytes and has a protective function in
the heart following stress of ischemia/reperfusion or pressure
overload in vivo and of treatment with a DNA alkylating
agent or glucose deprivation in vitro. This work also high-
lights the usefulness and predictivity of in vitro human
pluripotent stem cell-derived cardiomyocyte assays to rank-
order compounds against potency in cell-based assays, and to
probe mechanisms of toxicity and translation of non-clinical
safety concerns to humans. Lastly, this work strengthens the
correlation between cardiomyocyte cell viability and func-
tionality, suggesting that these assays together may be useful
to enable prioritization of compounds prior to conduct of
in vivo studies and potentially reduce attrition due to
cardiotoxicity.
Acknowledgments The authors thank Dolo Diaz, Donna Dambach,
and Jacqueline Tarrant for valuable discussions and input.
References
1. Bair, K., Baumeister, T. R., Dragovich, P., Gosselin, F., Yuen, P.
W., Zak, M., et al. (2013). Preparation of pyridinyl and pyrim-
idinyl sulfoxide and sulfone derivatives as NAMPT inhibitors.
WO2013127267.
2. Burgos, E. S. (2011). NAMPT in regulated NAD biosynthesis and
its pivotal role in human metabolism. Current Medicinal Chem-
istry, 18, 1947–1961.
3. Cerna, D., Li, H., Flaherty, S., Takebe, N., Coleman, C. N., &
Yoo, S. S. (2012). Inihibition of nicotinamide phosphoryltrans-
ferase (NAMPT) activity by small molecule GMX1778 regulates
reactive oxygen species (ROS)-mediated cytotoxicity in a p53-
and nicotinic acid phosphoribosyltransferase1 (NAPRT1)-de-
pendent manner. Journal of Biological Chemistry, 287,
22408–22417.
4. Hasmann, M., & Schemainda, I. (2003). FK866, a highly specific
noncompetitive inhibitor of nicotinamide phosphoryltransferase,
represents a novel mechanism for induction of tumor cell apop-
tosis. Cancer Research, 63, 7436–7442.
5. Holen, K., Saltz, L. B., Hollywood, E., Burk, K., & Hanauske, A.
R. (2008). The pharmacokinetics, toxicities, and biologic effects
of FK866, a nicotinamide adenine dinucleotide biosynthesis
inhibitor. Investigational New Drugs, 26, 45–51.
6. Hovstadius, P., Larsson, R., Jonsson, E., Skov, T., Kissmeyer, A.
M., Krasilnikoff, K., et al. (2002). A phase I study of CHS 828 in
patients with solid tumor malignancy. Clinical Cancer Research,
8, 2843–2850.
7. Hsu, C. P., Oka, S., Shao, D., Hariharan, N., & Sodoshima, J.
(2009). Nicotinamide phosphoribosyltransferase regulates cell
survival through NAD? synthesis in cardiac myocytes. Circula-
tion Research, 105, 481–491.
8. Kirsch, A. G., Obejero-Paz, C. A., & Bruening-Wright, A.
(2014). Functional characterization of human stem cell-derived
cardiomyocytes. In S. J. Enna, M. Williams, T. Kenakin, P.
McGonigle, & B. Ruggeri (Eds.), Current protocols in pharma-
cology (pp. 11.12.1–11.12.26). San Francisco, CA: Wiley.
9. Leyton-Mange, J. S., & Milan, D. J. (2014). Pluripotent stem cells
as a platform for cardiac arrhythmia drug screening. Current
Treatment Options in Cardiovascular Medicine, 16, 334–352.
10. Liang, X., Yang, L., Qin, A. R., Ly, J., Liederer, B. M., Messick,
K., et al. (2014). Measuring NAD? levels in mouse blood and
tissue samples via a surrogate matrix approach using LC-MS/MS.
Bioanalysis, 6, 1445–1457.
11. O’Brien, T., Oeh, J., Xiao, Y., Liang, X., Vanderbilt, A., Qin, A.,
et al. (2013). Supplementation of nicotonic acid with NAMPT
inhibitors results in loss of in vivo efficacy in NAPRT1-deficient
tumor models. Neoplasia, 15, 1314–1329.
12. Oh, A., Ho, Y. C., Zak, M., Yongbo, L., Chen, X., Yuen, P., et al.
(2014). Structural and biochemical analyses of the catalysis and
potency impact of inhibitor phosphoribosylation by human
nicotinamide phosphoribosyltransferase. ChemBioChem, 15,
1121–1131.
13. Olesen, U. H., Hastrup, N., Sehested, M. (2011). Expression
patterns of nicotinamide phosphoribosyltransferase and nicotinic
acid phosphoribosyltransferase in human malignant lymphomas.
APMIS, 119(4–5), 296–303.
14. Peters, M. F., Lamore, S. D., Guo, L., Scott, C. W., & Kolaja, K. L.
(2014). Human stem cell-derived cardiomyocytes in cellular
impedance assays: Bringing cardiotoxicity screening to the front
line. Cardiovascular Toxicology. doi:10.1007/s12012-014-9268-9.
15. Pishvaian, M. J., Marshall, J. L., Hwang, J. J., Malik, S., He, A.
R., Deeken, J. F., et al. (2009). A phase I trial of GMX1777, an
inhibitor of nicotinamide phosphoribosyl transferase (NAMPRT),
given as a 24-hour infusion. Journal of Clinical Oncology, 27,
A3581.
16. Ravaud, A., Cerny, T., Terret, C., Wanders, J., Nguyen Bui, B.,
Hess, D., et al. (2005). Phase I study and pharmacokinetic of
CHS-828, a guanidino-containing compound, administered orally
as a single dose every 3 weeks in solid tumours: an ECSG/
EORTC study. European Journal of Cancer, 41, 702–707.
17. Shames, D. S., Elkins, K., Walter, K., Holcomb, T., Du, P., Mohl,
D., et al. (2013). Loss of NAPRT1 expression by tumor-specific
promoter methylation provides a novel predictive biomarker for
NAMPT inhibitors. Clinical Cancer Research, 19, 6912–6923.
18. Singh, J., Zabka, T., Uppal, H., Diaz, D., Tarrant, J., Clarke, E.,
et al. (2013). Effects of nicotinamide phosphoribosyltransferase
inhibitors on platelet development. Toxicological Sciences,
52(Suppl), 2179.
19. von Heideman, A., Berglund, A., Larsson, R., & Nygren, P.
(2010). Safety and efficacy of NAD depleting cancer drugs:
results of a phase I clinical trial of CHS 828 and overview of
published data. Cancer Chemotherapy and Pharmacology, 65,
1165–1172.
20. Watson, M., Roulston, A., Belec, L., Billot, X., Marcellus, R.,
Bedard, D., et al. (2009). The small molecule GMX1778 is a
potent inhibitor of NAD? biosynthesis: Strategy for enhanced
therapy in nicotinic acid phosphoribosyltransferase 1-deficient
tumors. Molecular and Cellular Biology, 29, 5872–5888.
21. Xiao, Y., Elkins, K., Durieux, J. K., Lee, L., Oeh, J., Yang, L. X.,
et al. (2013). Dependence of tumor cell lines and patient-derived
tumors on the NAD salvage pathway renders them sensitive to
NAMPT inhibition with GNE-618. Neoplasia, 15, 1151–1160.
22. Ying, W. (2008). NAD?/NADH and NADP?/NADPH in cel-
lular functions and cell death: Regulation and biological conse-
quences. Antioxidants and Redox Signaling, 10, 179–206.
23. Zabka, T. S., Singh, J., Dhawan, P., Liederer, B. M., Oeh, J.,
Kauss, M. A., et al. (2015). Retinal toxicity, in vivo and in vitro,
associated with inhibition of nicotinamide phosphoribosyltrans-
ferase. Toxicological Sciences, 144(1), 163–172.
24. Zhang, L. Q., Heruth, D. P., & Ye, S. Q. (2011). Nicotinamide
phosphoribosylatransferase in human diseases. Journal of Bio-
analysis and Biomedicine, 3, 13–25.
Cardiovasc Toxicol
123
25. Zheng, X., Bauer, P., Baumeister, T., Buckmelter, A. J., Caligiuri,
M., Clodfelter, K. H., et al. (2013). Structure-based discovery of
novel amide-containing nicotinamide phosphoribosyltransferase
(nampt) inhibitors. Journal of Medicinal Chemistry, 56,
6413–6433.
26. Zheng, X., Bair, K. W., Bauer, P., Baumeister, T., Bowman, K. K.,
Buckmelter, A. J., et al. (2013). Identification of amides derived from
1H-Pyrazolo[3,4-b]pyridine-5-carboxylic acid as potent inhibitors of
human nicotinamide phosphoribosyltransferase (NAMPT). Bioor-
ganic and Medicinal Chemistry Letters, 23, 5488–5497.
Cardiovasc Toxicol
123
![Nuevas soluciones de las ecuaciones de Einstein para los modelos de universo ... · 2005. 8. 8. · ese tiempo, Misner [9], [15] realizó un estudio de este modelo de universo al](https://img.pdfslide.tips/doc/110x75/611550761a972c73943ee9c9/nuevas-soluciones-de-las-ecuaciones-de-einstein-para-los-modelos-de-universo-.jpg)
